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Secretory IgA and intestinal epithelial cells at the interface between homeostasis regulation and protection against infection
Thèse de doctorat ès science de la vie (PhD) Conduite au sein du département de médecine, service d’Immunologie et d’Allergie
Centre Hospitalier Universitaire Vaudois (CHUV)
Présentée à la Faculté de Biologie et de Médecine de l’Université de Lausanne Par
Amandine MATHIAS Diplômée en Agronomie et en Sciences pour les Industries Biologiques et Alimentaires
AgroParisTech (ex-cursus INA-PG, Paris, France)
Jury Prof. Alexander SO, Président
Dr. Blaise CORTHESY, Directeur de thèse Dr. Isabelle SCHWARTZ-CORNIL, Experte
Prof. François SPERTINI, Expert
Lausanne, 2011
TABLE OF CONTENTS
REMERCIEMENTS ............................................................................................................................ 4
RESUME DESTINE A UN LARGE PUBLIC ........................................................................................... 5
RESUME .......................................................................................................................................... 6
ABSTRACT ....................................................................................................................................... 7
ABBREVIATIONS.............................................................................................................................. 8
INTRODUCTION............................................................................................................................. 13
1 THE ANATOMY OF THE GASTROINTESTINAL TRACT.............................................................. 13
1.1 Generalities ................................................................................................................... 13
1.2 The gastrointestinal tract between absorption of nutrients and chemical barrier ........... 13
2 INTESTINAL MUCOSA: CELL TYPES, ORGANIZATION AND FUNCTIONS.................................. 15
2.1 General organization ..................................................................................................... 15
2.2 Histology of the small intestine supporting the intestinal barrier .................................... 17
2.3 Intestinal epithelial cells: a physical barrier .................................................................... 19
3 THE GUT-ASSOCIATED LYMPHOID TISSUES........................................................................... 21
3.1 Mesenteric lymph nodes ................................................................................................ 21
3.2 Peyer’s Patches.............................................................................................................. 23
3.3 Isolated lymphoid follicles and cryptopatches ................................................................ 23
3.4 Activation of immune responses at mucosal surfaces ..................................................... 25
4 MUCOSAL CHALLENGE BY THE GUT MICROBIOTA ................................................................ 27
4.1 Phylogenetic description ................................................................................................ 27
4.2 Recognition and immunomodulatory properties of the microbiota................................ . 29
4.3 Commensal-IEC crosstalk ............................................................................................... 31
4.4 Physiological roles of the microbiota.............................................................................. 33
5 FUNCTIONS OF SIGA IN THE GUT........................................................................................... 35
5.1 Structural features of SIgA ............................................................................................. 35
5.2 Protective role in pathogenesis ...................................................................................... 37
5.3 SIgA and mucosal homeostasis: interaction with commensals........................................ 45
5.4 SIgA receptors: between pro- and anti-inflammatory responses..................................... 47
AIMS OF THIS WORK..................................................................................................................... 52
1
2
PART I: N-GLYCANS ON SECRETORY COMPONENT: MEDIATORS OF THE INTERACTION BETWEEN
SIGA AND GRAM-POSITIVE COMMENSALS SUSTAINING INTESTINAL HOMEOSTASIS........... 53
1 OVERVIEW OF THIS PART...................................................................................................... 53
2 EXPERIMENTAL PROCEDURES, RESULTS AND DISCUSSION................................................... 54
2.1 Potentiation of polarized intestinal Caco-2 cell responsiveness to probiotics complexed
with secretory IgA. .................................................................................................................... 55
2.2 Recognition of Gram-positive intestinal bacteria by hybridoma- and colostrum-derived
secretory immunoglobulin A is mediated by carbohydrates. ...................................................... 64
2.3 N-glycans on Secretory Component: mediators of the interaction between SIgA and
Gram-positive commensals sustaining intestinal homeostasis. .................................................. 74
PART II: SECRETORY IGA-MEDIATED PROTECTION OF THE INTESTINAL EPITHELIAL CELL INTEGRITY
DURING INFECTION CAUSED BY SHIGELLA FLEXNERI................................................................ ..93
1 AIM OF THIS PART ................................................................................................................ 93
2 EXPERIMENTAL PROCEDURES, RESULTS AND DISCUSSION................................................... 93
PART III: RETROTRANSPORT OF SIGA THROUGH INTESTINAL EPITHELIAL CELL, A NEW GATE OF
RE-ENTRY FOR THE ANTIBODY ........................................................................................... 121
1 AIM OF THIS PART .............................................................................................................. 121
2 EXPERIMENTAL PROCEDURES............................................................................................. 122
2.1 Caco-2 cell culture and measurements of transepithelial electrical resistance .............. 122
2.2 Cell lines and protein production.................................................................................. 122
2.3 Flow cytometry ............................................................................................................ 123
2.4 Exposure of Caco-2 cell monolayers to pIgA and SIgA................................................... 123
2.5 Laser scanning confocal microscopy observation of Caco-2 cell monolayers ................. 124
2.6 Enzyme-linked immunosorbent assay (ELISA) ............................................................... 125
2.7 Statistical analysis ....................................................................................................... 125
3 RESULTS.............................................................................................................................. 127
3.1 SIgA retrotransport across Caco-2 cell monolayers....................................................... 127
3.2 SIgA retrotransport involves early endosome vesicles................................................... 131
3.3 Pathways of internalization induced during SIgA retrotransport................................. .. 131
3.4 Receptor(s) involved in the retrotransport of IgA.......................................................... 135
4 DISCUSSION........................................................................................................................ 141
CONCLUDING REMARKS / OUTLOOKS ........................................................................................ 145
REFERENCES ................................................................................................................................ 148
3
REMERCIEMENTS
Je tiens avant tout à remercier mon directeur de thèse, le Dr. Blaise CORTHESY de
m’avoir accueillie au sein de son laboratoire et ainsi de m’avoir permis de réaliser mon
travail de thèse. Je tiens tout particulièrement à le remercier pour son encadrement, sa
disponibilité sans faille, ses précieux conseils et un optimisme à toute épreuve qui m’a
permis d’avancer de façon sereine tout au long de ce travail.
Je tiens également à remercier les membres du jury : le Dr. Isabelle
SCHWARTZ-CORNIL et le Prof. François SPERTINI pour avoir pris de leur précieux temps pour
lire ce manuscrit ainsi que pour leur participation active au processus de validation de mon
travail de thèse. Un remerciement particulier au Prof. Alexander SO pour avoir accepté la
présidence du mon jury et le travail qui lui incombe.
Je veux également remercier le Dr. Jean-Yves CHATTON et Yannick KREMPP, directeur
et assistant technique de la plateforme de microscopie confocale de l’UNIL, pour leurs
précieux conseils et leur disponibilité, m’aillant éclairé sur le chemin complexe de la
microscopie confocale.
Un grand merci va également aux membres présents et passés du laboratoire :
Régine AUDRAN, Nathalie BARBIER, Gilles BIOLEY, Mélanie DUC, Stéphanie LONGET, Nicolas
ROL et Anne-Christine THIERRY pour avoir largement contribué au bon déroulement de mon
travail de thèse en créant une ambiance de travail détendue sans quoi tout ce temps passé
au laboratoire n’aurait pas eu la même saveur. Merci pour toutes ces discussions plus ou
moins scientifiques qui ont su mêler avec brio goût des épices et autres sujets qui nous
collent à la peau… Merci à vous !
Je clos enfin ces remerciements en dédiant cette thèse de doctorat à mes parents qui
m’ont donné l’envie d’aller toujours plus loin et qui m'ont soutenue tout au long de ces
longues années de travail. Merci Maman! Merci Papa! Un grand merci à mon compagnon,
Kévin, qui me soutient au quotidien, qui a su et qui sait surtout toujours me faire rire et me
faire passer en douceur ces moments de grands chamboulements.
4
RESUME DESTINE A UN LARGE PUBLIC
L’intestin est le siège d’intenses agressions de la part de l’ensemble des aliments ingérés,
de bactéries agressives dites pathogènes mais également de bactéries dites commensales
peuplant naturellement les surfaces intestinales muqueuses. Pour faire face, notre organisme
arbore de nombreux niveaux de protections tant physiques, chimiques, mécaniques mais aussi
immunitaires. La présence d’un type particulier de cellules, les cellules épithéliales (IEC) assurant
une protection physique, ainsi que la production d’anticorps spécialisés par le système
immunitaire appelés immunoglobulines sécrétoires A (SIgA) servent conjointement de première
ligne de défense contre ces agressions externes. Néanmoins, comment le dialogue s’articule
entre ces deux partenaires reste incomplet.
Nous avons donc décidé de mimer ces interactions en modélisant les surfaces muqueuses
par une monocouche de cellules différenciées en laboratoire. Des souches bactériennes isolées
de l’intestin humain seules ou associées à des SIgA non-spécifiques ont été mises au contact de
ce modèle cellulaire nous permettant de conclure quant à la présence effective d’une
modulation du dialogue bactérie/IEC impliquant une activation de la réponse cellulaire vers un
état de tolérance mutuelle. De façon surprenante, nous avons par ailleurs mis en évidence un
type d’interaction nouveau entre ces anticorps et ces bactéries. Une étude biochimique nous a
permis de détailler un nouveau rôle des SIgA médié par les sucres présents à leur surface dans le
maintien d’une relation pacifique avec les commensaux perpétuellement présents, relations
qualifiées d’homésostase intestinale.
Le rôle protecteur des SIgA a par ailleurs été abordé pour avoir une meilleure
appréhension de leur impact au niveau cellulaire lors d’infection par Shigella flexneri, bactérie
causant la Shigellose, diarrhée sanglante responsable de la mort de plus d’un million de
personnes chaque année. Basée sur le même modèle cellulaire, cette étude nous a permis de
démontrer une nouvelle entrée de ce pathogène directement via les IEC. La présence d’anticorps
spécifiques à la surface des bactéries restreint leur champs d’action contre les cibles
intracellulaires identifiées que sont les filaments soutenant le squelette de la cellule, les fibres
d’actine ainsi que les jonctions serrées, réseaux de protéines clés des interactions entre cellules.
Cette ouverture au niveau cellulaire apporte un nouvel élan quant à la compréhension du rôle
protecteur des SIgA lors d’attaques de l’intestin, protection semblant dépendante d’une
agrégation des bactéries.
Pour finir, nous avons mis en évidence la détection directe par les cellules de la présence
d’anticorps libres dans l’intestin ajoutant une nouvelle réplique dans le dialogue complexe entre
ces deux piliers de l’équilibre intestinal que sont les SIgA et les cellules épithéliales.
5
RESUME
La muqueuse intestinale est dotée d’un réseau complexe de protections physico-
chimiques, mécaniques ou immunologiques. Associées à un système immunitaire omniprésent,
les cellules épithéliales intestinales (IEC) bordant la lumière intestinale ont la double tâche de
protéger l’intérieur de l’organisme stérile contre l'invasion et la dissémination d'agents
pathogènes, et de maintenir une relation pacifique avec la flore intestinale, rôles également
joués par les immunoglobulines sécrétoires A (SIgA), anticorps les plus abondamment présents à
la surface des muqueuses. Tant les IEC que les SIgA sont ainsi décrites comme convergeant vers
le même objectif ; néanmoins, les rouages de leurs interactions restent largement inconnus.
Pour répondre à cette question, des monocouches épithéliales reconstituées in vitro ont
été incubées avec des souches commensales telles que des Lactobacillus ou des Bifodobacteria,
seules ou complexées avec des SIgA non-spécifiques, nous permettant de décrypter l’influence
des SIgA sur la détection des bactéries par les IEC, favorisant l'adhésion bactérienne et la
cohésion cellulaire, augmentant l’activation de la voie NF-κB ainsi que la sécrétion de la cytokine
thymic stromal lymphopoietin contrairement à celle de médiateurs pro-inflammatoires qui reste
inchangée. Par ailleurs, une interaction Fab-indépendante est suggérée dans l’interaction
SIgA/bactéries. Comme une interaction de faible affinité a été décrite comme prenant
naturellement place au niveau de l’intestin, nous avons donc disséqué les mécanismes sous-
jacents en utilisant un large spectre de bactérie associés à des protéines soit recombinantes soit
isolées à partir de colostrum, mettant en évidence un rôle crucial des N-glycanes présents sur la
pièce sécrétoire et soulignant une nouvelle propriété des SIgA dans l'homéostase intestinale.
Intrinsèquement liés aux caractéristiques des SIgA, nous nous sommes également
focalisés sur leur rôle protecteur lors d’infection par l’enteropathogène Shigella flexneri
reproduites in vitro sur des monocouches polarisées. Nous avons tout d'abord démontré une
nouvelle porte d'entrée pour ce pathogène directement via les IEC. L’agrégation des bactéries
par les SIgA confère aux cellules une meilleure résistance à l’infection, retardant croissance
bactérienne et entrée cellulaire, affectant par ailleurs leur capacité à cibler le cytosquelette et
les jonctions serrées. La formation de tels cargos détectés de façon biaisée par les IEC apparait
comme une explication plausible au maintien de la cohésion cellulaire médiée par les SIgA.
Enfin, le retrotransport des SIgA à travers les IEC a été abordé soulignant une
participation active de ces cellules dans la détection de l'environnement extérieur, les impliquant
possiblement dans l'activation d’un état muqueux stable.
Conjointement, ces résultats indiquent que les SIgA représentent l'un des éléments-clés à
la surface de la muqueuse et soulignent la complexité du dialogue établi avec l'épithélium en vue
du maintien d’un fragile équilibre intestinal.
6
ABSTRACT
The intestinal mucosa is endowed with a complex protective network melting
physiochemical, mechanical and immunological features. Beyond the ubiquitous intestinal
immune system, intestinal epithelial cells (IEC) lying the mucosal surfaces have also the dual task
to protect the sterile core against invasion and dissemination of pathogens, and maintain a
peaceful relationship with commensal microorganisms, aims also achieved by the presence of
high amounts of secretory immunoglobulins A (SIgA), the most abundant immunoglobulin
present at mucosal surfaces. Both IEC and SIgA are thus described to converge toward the same
goal but how their interplay is orchestrated is largely unknown.
To address this question, in vitro reconstituted IEC monolayers were first apically
incubated with commensal bacteria such as Lactobacillus or Bifodobacteria strains either alone
or in complexes with non-specific SIgA. Favoring the bacterial adhesion and cellular cohesion,
SIgA impacts on the cellular sensing of bacteria, increasing NF-κB activation, and leading to
cytokine releases restricted to the thymic stromal lymphopoietin and unaffected expression of
pro-inflammatory mediators. Of main interest, bacterial recognition by SIgA suggested a
Fab-independent interaction. As this low affinity, called natural coating occurs in the intestine,
we further dissected the underlying mechanisms using a larger spectrum of commensal strains
associated with recombinant as well as colostrum-derived proteins and pinpointed a crucial role
of N-glycans of the secretory component, emphasizing an underestimated role of carbohydrates
and another properties of SIgA in mediating intestinal homeostasis.
As mucosal protection is also anchored in SIgA and IEC features, we focused on the
cellular role of SIgA. Using IEC apical infection by the enteropathogen Shigella flexneri, we have
first demonstrated a new gate of entry for this pathogen directly via IEC. Specific SIgA bacterial
aggregation conferred to the cells a better resistance to infection, delaying bacterial growth and
cellular entry, affecting their ability to damage both the cytoskeleton and the tight junctions.
Formation of such big cargos differentially detected by IEC appears as a plausible explanation
sustaining at the cellular level the antibody-mediated mucosal protection.
Finally, SIgA retrotransport across IEC has been tackled stressing an active IEC sensing of
the external environment possibly involved in the steady-state mucosal activation.
All together, these results indicate that SIgA represents one of the pivotal elements at
mucosal surfaces highlighting the complexity of the dialogue established with the epithelium
sustaining the fragile intestinal balance.
7
ABBREVIATIONS
Ab Antibody
Ag Antigen
APC Antigen Presenting Cell
APRIL A Proliferation-Inducing Ligand
aSf avirulent strain of Shigella flexneri, BS176
BAFF B-cell Activating Factor of the TNF Family
BHI broth Brain Heart Infusion broth
BL Bifidobacterium lactis BL818
BSA Bovine Serum Albumin
Bt Bacteroides thetaitaomicron DSM 2079
CAMP Cationic Antimicrobial Peptide
CCR-9 CC-chemokine Receptor 9
CD Cluster of Differentiation
CFU Colony Forming Unit
CP Cryptopatch
CSR Class-Switch Recombination
Cy3 Indocarbocyanine
Cy5 Cyanine-5
DC Dendritic Cell
DIC Differential Interference Contrast
DMEM Dulbecco's Modified Eagle's Medium
EEA-1 Early Endosome Antigen-1
ELISA Enzyme-Linked immunosorbent Assay
EMSA Electrophoretic Mobility Shift Assay
FAE Follicular Associated Epithelium
FCS Fetal Calf Serum
FITC
GALT
Fluorescein Isothiocyanate
Gut Associated Lymphoid Tissue
GI Gastrointestinal
8
GFP Green Fluorescent Protein
HCl Hydrochloric acid
HEV High Endothelial Venule
HRP Horseradish Peroxidase
hSC human SC
IBD Intestinal Bowel Disease
IC Immune Complex
IEC Intestinal Epithelial Cell
IFR Interfollicular Region
Ig Immunoglobulin
IgA Immunoglobulin A
IL Interleukin
IL-7Rα IL-7 receptor α-chain
ILF Isolated Lymphoid Follicle
Ipa Invasion Plasmid Antigen
J chain Joining chain
LAMP-1 Lysosomal-Associated Membrane Protein 1
LB broth Luria-Bertani broth
LPR Lactobacillus rhamnosus strain LPR
LPS Lipopolysaccharide
LSCM Laser Scanning Confocal Microscopy
LTA Lipoteichoic Acid
M cell Microfold cell
MFI Mean of the Fluorescence Intensity
MLN Mesenteric Lymph Nodes
MRS broth Man-Rogosa Sharpe broth
mSC mouse SC
NLR NOD-like receptor
NOD Nucleotide-binding Oligodimerization Domain
OD Optical Density
PBS Phosphate Buffered Saline
9
P-DMEM Plain DMEM
PFA Paraformaldehyde
pIgA polymeric IgA
pIgR polymeric Immunoglobulin Receptor
PMN Polymorphonuclear
PNG Peptidoglycan
PP Peyer's Patch
PPAR-γ Peroxisome Proliferator-Activated Receptor-γ
RA Retinoic Acid
SC Secretory Component
SCcol Colostrum-derived SC
SCFA Short Chain Fatty Acid
SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
SED Subepithelial Dome
SEM Standard Error of the Mean
Sf Shigella flexneri
SIgA Secretory IgA
SIgAcol colostrum-derived SIgA
ST11 Lactobacillus paracasei ST11
T3SS Type-Three Secretion System
TA Teichoic Acid
TER Transelectrical Epithelial Resistance
TGF-β Transcription Growth Factor-β
TJ Tight Junction
TLR Toll-Like Receptor
TNF Tumor Necrosis Factor
TfR Transferrin Receptor
TSLP Thymic Stromal Lymphopoietin
XXXdg various deglycosylated proteins
ZO Zonula Occludens
10
UNITS
°C degree Celsius
μg microgram
μl microliter
μm micrometer
Da Dalton
g gram
h hour
kDa KiloDalton
l liter
mg milligram
min minute
ml milliliter
mM milliMolar
ng nanogram
% percent
pg picogram
RT Room Temperature
11
Major physiological processes
Secretion of acid (HCl)Start digestion of proteinspH2
Complete digestionAbsorption of monosaccharides, amino acids, fatty acids and waterpH4-5
Absorption of water, vitamins and electrolytespH7
esophagus
stomach
duodenum
ileum
jejunum
colon
cecum
rectumanus
liver
gallbladder
pancreas
Smal
l int
estin
eLa
rge
inte
stin
e
OrgansAccessory Organs
Figure 1. The general anatomy and the principal functions of the GI tract.
The upper part composed of the mouth, esophagus and stomach starts the digestion processes which are
completed in the lower part, the small intestine (the duodenum, the jejunum and the ileum) and the large
intestine (the cecum, the colon, the rectum and the anus). Basic scheme obtained from
http://www.sciencephoto.com/media/310982/enlarge.
12
INTRODUCTION
The gastrointestinal (GI) tract and the conjugated immune system have co-evolved to
bring the best benefits to the body: it should be an open barrier allowing efficient digestion
and absorption of nutrients, a “watch-dog” preventing the entry of harmful antigens (Ag)
and microorganisms and a sentinel for maintenance of the intestinal microbiota.
1 THE ANATOMY OF THE GASTROINTESTINAL TRACT
1.1 Generalities
Human mucosal surfaces comprising the mouth, respiratory, digestive and urogenital
tracts represent approximately 400 m2. The GI tract itself represents almost 75% of total
mucosal interfaces, is about 8 meter long in an adult subject, begins at the mouse and ends
with the rectum.1
The primary functions of the GI tract are digestion and absorption of
nutrients and elimination of the wastes. Two distinct parts compose the GI tract: the upper
part composed of the oral cavity, the esophagus and the stomach and the lower part
comprising the small intestine, the large intestine and finally the rectum. A pH gradient
sustains proper food digestion starting at low pH in the stomach to reach physiological,
neutral pH at the rectum. Various accessory organs also actively participate in the digestion
of nutrients such as the salivary glands, liver, pancreas and gallbladder (Figure 1).
1.2 The gastrointestinal tract between absorption of nutrients and chemical barrier
Food uptake by the mouth is first reduced at small particles, lubricated by mucus
secreted by salivary glands and transits by peristaltic movements through the esophagus to
reach the stomach. Diverse cells composed the stomachal wall: the goblet cell, cells
secreting the protective mucus layer; the parietal cells allowing stomachal acidification by
the production of high amount of hydrochloric acid (HCl); the chief cells, secreting
pepsinogen which, once clipped into pepsin by the action of HCl is involved in the primary
steps of protein digestion. Both enzymatic secretions and a highly acidic environment
contribute to initiate digestion and elimination of microorganisms potentially ingested with
the food intake, actively participating in mucosal protection. The stomach allows further
13
Figure 2. The general organization of the GI tract.
A. Despite differences in functions, a basic organization of the GI tract can be drawn: four concentric layers
are superimposed, the inner mucosa, the submucosa, the muscularis externa and the outer serosa.
(Adapted from http://coloncancer.about.com/) B. The intestinal mucosa is a complex tissue characterized
by the presence of the lamina propria underlying a single layer of epithelial cell. This complex tissue is
composed of non-immune cells such as enterocytes or Paneth cells and immune cells organized in inductive
sites (PP, ILF) and effector sites. Taken together, these cells allow to confer physical, chemical and immune
barriers to protect efficiently the GI tract. (Adapted from Magalhaes et al., Semin. Immunol., 2007,
19:106-115.)11
SIgA
Anti-microbialpeptides
Lymphatic duct
A
B
Lumen
14
hydration and storage of the resulting bolus to prepare it to complete digestion into the
intestine. The duodenum, the jejunum and the ileum are the three parts constituting the
small intestine where occurs the main part of the digestion processes. Pancreatic enzymatic
secretions together with bile secreted by the gallbladder orchestrate the main steps in the
regulation of the digestion in the small intestine: the secretin stimulates bicarbonate
secretion which neutralizes harmful stomachal pH; the amylase is involved in carbohydrate
digestion into absorbable monosaccharides such as glucose, fructose and galactose; the
tryspin ensures protein digestion into amino acids or small polypeptides able to cross the
intestinal barrier; the lipase and the bile are finally engaged into lipid digestion into free fatty
acids and monoglycerides.
Specialized enzymes present at the brush border of the epithelium such as the
dextrinase and the glucoamylase which are involved for the final degradation into simple
oligosaccharides, achieve nutrients digestion allowing their transport into the blood and
lymphatic system.
90% of the digestion is then accomplished; the large intestine comprising the cecum,
the colon and the rectum makes an end point of these mechanisms of nutrients and energy
absorption by reabsorption of water, vitamins and electrolytes. Finally, the undigested
leftovers are expulsed through the rectum and the anus. Beside host contribution, another
sizeable partner in digestion processes of otherwise indigestible components such as
polysaccharides is the colonizing microbiota which is described in further details in
Introduction section 4.4.
2 INTESTINAL MUCOSA: CELL TYPES, ORGANIZATION AND FUNCTIONS
2.1 General organization
Despite differences due to highly specialized functions, a general scheme of the
organization sustaining the GI tract can be drawn (Figure 2A). Four concentric layers are
superimposed: the inner mucosa, the submucosa, the muscularis externa and finally the
outer serosa. The mucosa is the innermost layer, that is, the layer nearest to the lumen of
the tube, and exhibits high variations throughout the tract. The mucosa is divided into three
parts: a single cell epithelial monolayer facing the inner lumen, the underlying lamina
15
16
propria (connective tissues) and a supporting layer of smooth muscles called muscularis
mucosae. The submucosa consists of loose connective tissue, which supports blood and
lymphatic vessels, and nerve fibers. The last but not least partners are the muscularis
externa and the serosa essentially composed of, respectively, smooth muscles and
longitudinal muscles allowing intestinal contraction, basis of the peristaltic propulsion of the
bolus through the GI tract.
Reflecting specific functions dedicated to each part of the GI tract, the mucosa have
evolved differently to face low pH values in the stomach, to allows both absorption of
nutrients and delimitation of the outside versus in environment in the small intestine, and
finally to increase absorption in the large intestine. We will focus on the most complex
division, i.e. the small intestine.
2.2 Histology of the small intestine supporting the intestinal barrier
The cavity of the gut lumen is covered by a single cell monolayer underlying on loose
connective tissues, the lamina propria, which together form the basic organization of the
intestinal mucosa (Figure 2B). The mucosa is organized into villi, prominences into the gut
lumen and crypts directed into the musculous adjacent layer. Due to the villi and microvilli
present as the apical epithelium border, the intestinal mucosa presents a large surface
exceeding more than 200 times the whole skin surface. The presence of stem cells depth
into the crypts allows migration of newly generated cells from the crypts to the top of the
villi where apoptosis of senescent cells occurs allowing the total renewal of the epithelium
every 4 to 5 days2. Four different cells types derived from those stem cells compose the
intestinal epithelium: the Paneth cells, essentially present deep down into the crypts,
responsible for secretion of antimicrobial peptide such as defensins; the goblet cells, mucus
secreting cells; endocrine cells (eg: production of gastrin) and epithelial absorptive cells, the
enterocytes described in details below (Figure 3A).
The secretion of antimicrobial peptides by the Paneth cells represents the first line of
defense against the penetration of microorganisms into the underlying tissues. More than
500 different peptides have been identified.
3, 4
5-7 Among them, the cationic antimicrobial
peptides (CAMP) allow the formation of pores in the walls of various microorganisms,
17
A B
Figure 3. Cell types and cohesion basis of the epithelium of the small intestine.
A. Stems cells (orange) reside in the crypt region where proliferation takes place (yellow).
The differentiation of the epithelial cells occurs during the migration to the top of the villus. The four main
differentiated cell types (blue) found in the intestine are depicted at the bottom of the panel: the endocrine
cell, delimited by a dotted line; the enterocytes; the mucus-secreting cells (goblet cells), the endocrine cells
and the Paneth cells which, contrary to the previous cells, migrate to the bottom of the crypts. (Adapted
from Simon-Assmann et al., Cell Biol. Toxicol., 2006, 23:241-56.)4 B. The physical cohesion of the epithelial
cells is assumed by the presence of a dense network of junctions. Just below the base of the microvilli, the
plasma membranes of adjacent cells fuse at the tight junctions and the adherens junctions. Desmosomes
are located beneath the apical junctional complex and allow cell-to-cell communications. (Adapted from
Turner, Nat. Rev. Immunol., 2009, 9:799-809.)10
18
leading to a wide spectrum of activity against Gram-positive and negative bacteria, fungi,
viruses and protozoa.8
Produced by the goblet cells and epithelial cells, a dense layer of mucus composed of
mucins associated with the glycocalyx (network of carbohydrates secreted by the
epithelium) figures as physical barrier completing the chemical barrier conferred by CAMP.
This gel inhibits a possible direct contact with the huge amount of Ag permanently
challenging mucosal surfaces. Differences in size and density allow the definition of an
external layer of 100 µm harboring a looser network allowing slight bacterial anchoring and
the inner layer of 50 µm of highly dense carbohydrate network almost devoid of Ag.
These peptides represent an efficient chemical barrier against the
early stages of infection, preventing the growth of many microorganisms.
9
2.3 Intestinal epithelial cells: a physical barrier
The enterocytes, 80% of the intestinal cells found in the intestine and in the colon,
are differentiated polarized columnar epithelial cells presenting a brush border at the apical
pole called microvilli responsible for the absorption of the nutriments and the fluid
exchange. To maintain the intestinal barrier and allow at the same time the paracellular
routing of fluids, all these epithelial cells are joined together with adherens junctions,
desmosomes and tight junctions (TJ; Figure 3B).10
Found in depth at the basolateral pole, the desmosomes also known as macula
adherens fulfill the dual task to maintain cellular adhesion and to allow intercellular
communication. Desmoglein and desmocollin, members of the cadherin family of cell
adhesion molecules essentially compose the extracellular domain of the desmosoms. The
desmoplakin protein further links together transmembrane proteins to the intermediate
keratin fiber members of the cellular cytoskeleton.
Adherens junctions composed of homodimers of transmembrane proteins, the
cadherins, are responsible for the strong adhesive cohesion between cells. The cytoplasmic
tail region of the cadherin, interacts directly with β-catenin and catenin δ1. β-catenin binds
to α-catenin 1 to ensure final regulation of the actomyosine ring sustaining the global
organization of enterocytes.
19
20
The TJ also known as zonula occludens are multiple-protein complexes composed of
more than 40 proteins including proteins such as the claudin family, occludins
(transmembrane proteins) and zonula occludens proteins (ZO, peripheral membrane
proteins), ZO-1, ZO-2 and ZO-3. These latter interact with the cell cytoskeleton and in
particular with actin fibers highlighting their crucial role in maintaining cell cohesion. The
presence of this type of junction allows to discriminate the apical from the basolateral pole
of the intestinal epithelial cells (IEC). Intact TJ also limit solute flux transiting along the
paracellular pathway and are the principal determinant of the mucosal permeability.
3 THE GUT-ASSOCIATED LYMPHOID TISSUES
The underlying lamina propria is drained by vessels, lymphatic ducts and specialized
immune structures called gut associated lymphoid tissues (GALT). These GALT are well
organized structures composed of various immune cells such as B-cells, T-cells and dendritic
cells (DC; Figures 2 and 4).11
5
Inductive sites where Ag are sampled into the gut lumen are
divided into two main groups: GALT mature before birth, the mesenteric lymph nodes (MLN)
and the Peyer’s patches (PP), and the GALT with a maturation induced after birth,
represented by a continuum between the most organized isolated lymphoid follicles (ILF)
and the primary cryptopatches (CP). The effector sites are sites where the acquired
immunity is activated such as cellular responses mediated by T-cells and humoral responses
mainly by the production of secretory immunoglobulin A (SIgA) described in detail in
Introduction section .
3.1 Mesenteric lymph nodes
MLN are distributed all along the GI tract and their number varies from 100 to 150 in
the human body.12 The MLN share common structures with the highly organized lymph
nodes: encapsulated by lymphatic endothelium, MLN are organized into three parts, the
cortex, the paracortex and the medulla (Figure 4A). The cortex includes B-cells and DC.
T-cells are more diffusely distributed in surrounding paracortical areas, referred to as T-cell
zones. Some of the B-cell follicles include germinal centers, where B-cells are undergoing
intense proliferation after encountering their specific Ag. MLN are organs with a dense
21
Gut lumen Gut lumen M cell
A. MLN B. PP C. ILF D. CP
Figure 4. Inductive sites in the intestine.
A. The mesenteric lymph nodes (MLN) are encapsulated immune structures organized into B-cell zones
surrounding germinal centers and diffuse T-cell zones. This organ is highly vascularized by both lymphatic
and systemic vessels allowing diffusion of immune responses to the mucosa. (Adapted from von Andrian
and Mempel, Nat. Rev. Immunol., 2003, 3:867-78.)13 B. The follicle associated epithelium (FAE), which
covers Peyer's patches (PP), contains M cells overlying the subepithelial dome (SED) essentially composed
of DC (orange). Underlying the SED, B-cells (blue) are found concentrated in germinal center surrounded by
T-cells zones (red) in the interfollicular regions (IFR). C. Found at the end of the villus, isolated lymphoid
follicles (ILF) also harbor M cells at their surface. B-cells represent 80 % of the cells present.
Lin-cKIT+IL7-Rα+ cells, T cells and DC complete the last 20%. D. Less organized, cryptopatches (CP) are
essentially composed of (Lin)-cKIT+IL7-Rα+ cells and a few DC. (B to D adapted from Eberl, Nat. Rev.
Immunol., 2005, 5:413-20.)17
SED
IFR
FAE
22
network of lymphatic vessels coming from the peripheral GALT, converging into the
subcapsular sinus (medullary sinus) where plasma cells, macrophages and diffuse B-cells can
also be detected (medullary cord). Ag enters through the lymphatic vessels via DC or in a
soluble form. Responses can take place in interaction between afferent signals detected into
the T and B-cell zones. Besides, specialized high endothelial venules (HEV) in the paracortex
and cortical region allow the entry of immune cells such as lymphocytes from the blood and
results in activation of specialized immune responses and diffusion of these responses in the
whole organism.
3.2 Peyer’s Patches
Contrary to MLN, PP are not encapsulated structures. PP are the most organized
inductive GALT located in the mucosa and extending into the submucosa of the small
intestine, especially the ileum (Figure 4B).13 Up to 250 have been described in the human GI
tract.14 These structures are covered by an epithelium, the follicular associated epithelium
(FAE) harboring specialized cells, the microfold cells or M cells responsible for Ag sampling
from the gut lumen. Those specialized M cells display important structural changes: a poorly
organized brush-border, an absence of mucus and a unique invagination pocket at its
basolateral pole.15 Three distinct parts compose the PP: the mentioned FAE overcomes a
small dome, the subepithelial dome (SED), constituted by dense network of DC; beneath,
B-cells are found in concentrated follicular zone and germinal centers, surrounded by T-cells,
plasma cells, macrophages and DC organized in the interfollicular region (IFR) (Figure 4B).16
3.3 Isolated lymphoid follicles and cryptopatches
ILF and CP are the most recently indentified GALT. With an average number of 30 000
in humans, these structures represent a continuous consortium starting from the CP (60 μm
of diameter) to ILF (wide range of sizes) (Figure 4C and D).16 Located between the lamina
propria and the end of the crypts, CP consist of 102 to 104 lymphoid cells among which 20%
of DC and 80% of a specific phenotype called T-cell precursor, lineage negative (lin-),
expressing c-KIT and the interleukin-7 receptor α-chain (IL-7Rα) called (Lin)-c-KIT+IL7Rα+
cells. CP are induced two weeks after birth and maturation of these structures leads to the
23
Figure 5. Induction of immune responses in the intestine.
After their transport through M cells, Ag are
sampled by DC in the SED region. Maturation of
DC results in activation of T-cell either in the IFR
or in the MLN. Ag are also directly sampled
by DC extending their dendrites into the gut
lumen. Detection of these Ag results in the
secretion of a special pattern of cytokines (TGF-
β, IL-2, IL-5, IL-10, IL-6) driving lymphocyte
differentiation and homing to effector sites.
Homing of activated IgA+ B-cells allow their
differentiation into plasmocytes producing pIgA
taken in charge by epithelial cells to be secreted
in the gut lumen as SIgA. (Adapted from
Corthésy, J. Immunol., 2007, 178:27-32.)18
Figure 6. Structure and secretion of SIgA in the intestinal lumen.
pIgA is composed of two monomers linked together with a J chain (right panel). Following secretion by the
plasma cells (), pIgA is taken in charge by specific receptors called pIgR (). Bound Ab are transcytosed
through the epithelial cells (). Once at the apical pole, the ectodomain of the pIgR named secretory
component (SC) is cleaved resulting in secretion of SIgA in the intestinal lumen (). Both SC and pIgA are
highly glycosylated proteins (right panel). Additional N- and O-carbohydrates represent sites of
glycosylation specifically present on the allotype IgA1 or IgA2. For a matter of clarity, pIgA and SC have been
represented separately. Representation based on results obtained by Hughes et al. (Biochim. Biophys. Acta.,
1999, 1434:86-93)91 and Royle et al. (J. Biol. Chem., 2003, 278:20140-53.)83
24
formation of ILF.17 Essentially composed of diffusely organized B-cells (up to 70%), ILF
surmounted by M cells also harbor small populations of DC and (Lin)-c-KIT+IL-7Rα+ cells.17
3.4 Activation of immune responses at mucosal surfaces
Several mechanisms have been described to activate immune responses in the gut:
the role of sampling Ag via PP or ILF is the most described (Figure 5).18 Ag from the gut
lumen are thus phagocytosed through M cells and presented to antigen presenting cells
(APC) such as macrophages and immature DC present in the SED. Ag processing results in the
initiation of immune responses and, in particular, in the T-cell activation residing in the IFR
(CD4+ T-cells). Subsequent activation leads to the secretion of cytokines able to stimulate
B-cells of the follicular zone and their hyperproliferation in the germinal center resulting in
the induction of IgA class-switch recombination (CSR). T-cell independent activation of B-cell
has also been described: in this case, DC from the lamina propria or PP seem to be the
source of the B-cell class switching toward IgA+ B-cells.19 Secreted by DC and activated
T-cells, retinoic acid (RA), B-cell activating factor of the tumor necrosis factor (TNF) family
(BAFF), proliferation-inducing ligand (APRIL), transforming growth factor (TGF-β) and
interleukins 10 and 6 (IL-10, IL-6) represent the key cytokines and differentiation proteins
involved in this pathway of activation.19-22 Migration of activated T and B-cells to the MLN via
the lymphatic vessels amplified the response and the activation of acquired immunity. RA is
also involved in homing of IgA+ B-cells to the lamina propria, upregulating the expression of
the CC-chemokine receptor 9 (CCR-9) and the α4β7 integrin on IgA-expressing cells allowing
further migration in close contact with cells of the HEV.23, 24 IgA+ B-cells are thus actively
attracted to effector sites where immune responses are taking place. This route is followed
by activated T-cell and further enhances protective mechanisms involving both cell and
humoral responses. All together these pathways allow the differentiation of B-cells into
IgA+ B-cells and finally into plasmocytes able to secrete polymeric IgA (pIgA) once in the
lamina propria, under the influence of the surrounding cytokines (TGF-β, IL-2, IL-5, IL-10, and
IL-6). These antibodies (Ab) are recognized by specialized receptors called the polymeric
immunoglobulin receptor (pIgR) present at the basolateral pole of the IEC. Transcytosis
through IEC results in the secretion of 3 to 5 g each day of SIgA in the gut lumen where they
act as first line of defense (Figure 6).25
25
Figure 7. Spatial distribution of the commensal bacteria along the gastrointestinal tract.
Variations in the GI environment not only impact on the number of bacteria but also on their diversity:
reduced at a minimal level in the stomach, it reaches a maximum in the colon. (Adapted from Sekirov et al.,
Physiol. Rev., 2010, 90:859-904.)30
26
4 MUCOSAL CHALLENGE BY THE GUT MICROBIOTA
4.1 Phylogenetic description
The digestive tract, from the stomach to the colon, represents an ecological niche in
constant evolution. Rapidly after birth, the intestine, sterile in utero, is colonized by a
microbiota which will continuously vary during life. Whatever the mechanisms that govern
the establishment of the microflora after birth, they result in a microbial complex that
appears to be stabilized at a functional and structural level at the age of two years.26 More
than 1800 genera have been isolated which represent approximately 1014 bacteria,
exceeding by more than 10 times the body cells.27, 28 The human GI microbiota includes
members of all three domains of life (Bacteria, Archaea, and Eukarya) as well as viruses. If
the gut microflora stabilizes early in life, it is generally accepted, though rather poorly
demonstrated so far, that the composition of the flora changes with age, type of diet,
lifestyle.29
Colonization of the GI is dependent on the pH gradient.
It is therefore necessary to keep in mind that this section does not aim to define a
normal microbiota but to give an idea of the biodiversity encountered.
30 Thus, the stomach is the
less colonized due to low environmental pH which prevents from over colonization (up to
101 colony forming unit per milliter (CFU/ml) (Figure 7): Helicobacter pylori is one of the
most representative stomachal bacteria. The jejunum and the ileum have larger bacterial
charge with estimated levels of 107 CFU/ml. Those figures are only estimation because the
collection of samples in order to carry out microbial ecological studies is extremely
challenging. In contrast, the colon is the most studied compartment of the intestinal tract
with a total of 1012 microorganisms CFU/ml. Globally, the microbiota is composed of fungi,
viruses, yeasts present at low numbers (102-104 cell/ml) and overall, of strict or facultative
anaerobe bacteria, essentially Gram-positive such as Lactobacilli and Bifidobacteria
(Figure 8).
Studies based on the cultivation and metagenomic analysis of bacteria showed that
the dominant genera of the fecal microflora cultivable in adults are Bacteroides,
Eubacterium, Ruminococcus, Clostridium and Bifidobacterium, considering that the dominant
bacteria are those that represent 1% or more of total bacteria.
27, 28, 31
31 Almost 75% of the total
detected commensal belongs to the Gram-positive family. Three groups gather the bulk of
the dominant fecal bacteria (Figure 8). The Gram-positive group called
27
Figure 8. Phylogenetic tree of the indentified groups of bacteria present in human feces.
Tree obtains from 16S rRNA gene sequences representing different groups of bacteria which are most
frequently detected in human feces. (Adapted from Zoetendal et al., Mol Microbiol., 2006,
59:1639-1650.)31
28
“Eubacterium- Clostridium” also called the Firmicutes represents up to 58% of total
bacteria.31-33 It includes bacterial species belonging to the genera Eubacterium, Clostridium,
Ruminococcus, Butyruvibrio or Lactobacillus. Although less consistently detected but
averaging a few percent of total bacteria, the Gram-positive Actinobacteria such as
Bifidobacteria are found at rates ranging from 0.7 to 10%.31 Less abundant the
Gram-negative phyla of the Bacteroidetes and Proteobacteria are always present and share
dominance with the preceding groups (9-42% of total bacteria).
31, 33, 34
4.2 Recognition and immunomodulatory properties of the microbiota
Recognition by the host immune cells of the microbiota is at the center of the debate.
Sequestration in the lumen has been postulated to keep the systemic immune system
unresponsive to commensal bacteria by preventing close contacts with the subjacent host
cells.35
4.3
Nevertheless, data are accumulating demonstrating an active recognition of the
commensal bacteria leading to modulation of cellular responses at the intestinal surfaces.
These functions of commensals, maintained at steady-state level, can have profound
immunomodulatory properties either onto immune cell detection itself or IEC
(see Introduction section ).36-40 Any deregulation of the recognition of these commensals
can lead to uncontrolled inflammation involved for example in the initiation of inflammatory
bowel diseases (IBD).41 M cells present in the FAE that overlies the PP can samples Ag from
the gut lumen and deliver them by transcytosis to professional APC present in the SED such
as DC and macrophages. Limited sampling by DC in the SED region was also proposed as a
mean to educate the epithelium to the presence of the microbiota in order to assume a
peaceful relationship with these microorganisms.42 Myeloid DC distributed in the lamina
propria have also been described to extent their dendrites across IEC TJ to directly sample Ag
from the external environment.39 Activated cells then migrate to the MLN where they induce
B-cell differentiation into IgA producing cells which finally home to the lamina propria to
ensure SIgA secretion in the gut lumen.42-44 How DC and/or macrophage recognition of the
microbiota can lead to mucosal homeostasis remains an area of intense research.
Recently, commensal bacteria have also been described to govern the development of
intestinal Th17 effector T-cells and impact on the ratio Th1 and Th2 effector cells.37, 43 Last
but not least, the maturation of tertiary lymphoid tissues such as ILF in the gut lumen is
29
Figure 9. IEC responses to the microbiota at mucosal surfaces.
A. Basal recognition of the microbiota by TLR and NOD receptors leads to a minimal activation of the NF-κB
pathway, regulating local immune responses by the secretion of cytokines such as IL-10, TGF-β, TSLP, BAFF
and APRIL. B. These cytokines are responsible for the basal activation of immune responses such as the
secretion of SIgA and the activation of DC leading to modulations of T-cell population in the lamina propria.
(Adapted from Artis, Nat. Rev. Immunol., 2008, 8:411-20)44
A B
TLR2TLR4
BAFF, APRIL, IL-10, TGF-β,
TSLP
Activation
Reducedubiquitination
Reduced translocation
30
intrinsically linked with the symbiotic relationship established between the host and its
intestinal microbiota.17
4.3 Commensal-IEC crosstalk
At the interface between the gut microbes within the luminal content and the host
tissues, the intestinal epithelium must integrate pro- and anti-inflammatory signals to
regulate innate and adaptive immune responses (Figure 9A).44 IEC detection of the
continuous presence of microorganisms via specialized receptors such as Toll-like receptor
(TLR) (eg: TLR2, TLR4) or Nod-like receptors (NLR) (eg: NOD2) results in increased
proliferation of the intestinal epithelium and also enhanced cellular cohesion.45-47 Loss of TLR
or NLR signaling leads to exacerbation of intestinal inflammation when the epithelium is
affected.48-50 Contrary to detection of pathogenic components, activation of the TLR or NLR
pathways by commensal bacteria can inhibit NF-κB activation in IEC by multiple mechanisms
including the exportation of the subunit RelA of the NF-κB from the nucleus to the cytoplasm
by the peroxisome proliferator-activated receptor-γ (PPARγ) and the inhibition of the
ubiquitination of the IκBα, reducing the degradation of the cytoplasmic complex and thus
limiting the consecutive nuclear translocation of the NF-κB subunit (Figure 9A).
Commensals have also been associated with the modulation of cytokine secretion by
IEC such as BAFF, APRIL, IL-10, TGF-β and the thymic stromal lymphopoietin protein (TSLP)
involved in the class switching of B-cells into IgA-producing plasmocytes and in the reduction
of inflammatory responses (Figure 9B).
51-55
36, 47, 54, 56-58 Through these secretions, epithelial cells
also play an important role in the regulation of DC and macrophage functions by limiting
their pro-inflammatory cytokine production.54, 59, 60
Normal detection of the commensals
leads to a peaceful environment where microorganisms and IEC work in synergy to maintain
gut homeostasis. All of these mechanisms facilitate mutualism by decreasing tissue damages
and immune responses to commensal bacteria at steady-state.
31
32
4.4 Physiological roles of the microbiota
Host physiology has been described to take advantage of the presence of its diverse
intestinal microbiota. These commensal bacteria are responsible for participating in the
digestion of ingested food favoring the absorption of essential nutrients such as
carbohydrates including polysaccharides, oligosaccharides, lignin and associated plant
materials.61 These are metabolized to short chain fatty acids (SCFAs), primarily to acetate,
propionate, and butyrate.62 The latter is especially considered to be health promoting,
exerting broad anti-inflammatory activities by affecting immune cell migration, adhesion,
cytokine expression as well as affecting cellular processes such as proliferation, activation,
and apoptosis.
Beyond digestion and metabolism, the microbiota also plays a vital role in the
protection of the host from potentially pathogenic microbes. Mucosal barriers are reinforced
by the microbiota through enhanced secretion of mucus and antimicrobial peptides such as
RegIIIγ and α-defensins.
63-68
69-72
Last but not least, commensal roles also comprise immunomodulatory properties
such as the induction of SIgA secretion in the gut lumen or the development of lymphoid
tissues (see Introduction section
Another widely accepted role of the microbiota is to compete
for pathogenic bacteria, thus participating in mucosal protection.
4.2).
17
When taking into account the huge amount of commensals and the constant
presence of food Ag, these figures highlight the constant discriminating challenge
encountered by mucosal surfaces exposed to the external environment and possibly noxious
microorganisms that occasionally attack the mucosa. Thus, the mucosa must represent at
the same time an open gate for nutrient absorption and fluid transit, a peaceful niche for the
microbiota and an impenetrable wall against pathogenic infections. All these properties
explain the complexity of its organization combining physical, chemical and immune barriers.
33
34
5 FUNCTIONS OF SIGA IN THE GUT
5.1 Structural features of SIgA
SIgA produced by plasma cells in the lamina propria represents the major
immunoglobulin found at mucosal surfaces. SIgA is composed of dimeric IgA made of two
monomers (160 kDa) linked together via the joining J chain (15 kDa) and secretory
component (SC, 80 kDa), the extracellular degradation product resulting from the cleavage
at the epithelial cell surface of the precursor pIgR ensuring transcytosis of the Ab
(Figure 6).73 The pIgR only described at the basolateral pole of the IEC belongs to the type 1
transmembrane protein, is divided into three parts: the IgA binding extracellular domains
(SC), a transmembrane region and a cytoplasmic tail.74 In secretions, SC is bound covalently,
as well as non-covalently, to pIgA, and is found also as free SC.75 Two different types of IgA
are found in human secretions: IgA1 and IgA2. The second allotype is further divided into
IgA2m(1), IgA2m(2) and IgA2m(3) and constitutes the essential part of the Ab found at
intestinal mucosal surfaces whereas IgA1 is the main IgA subclass produced by the systemic
immune system.
Each monomers of IgA is composed of two disulfide bound heavy chains (α chains)
and two light chains (κ or λ chains). With respect of IgG, monomeric IgA are further divided
into one variable domain (Fab’) and three constant domains (Cα1, Cα2 and Cα3). Differences
in the structure linking the domains of the constant region sustain the existence of both IgA1
and IgA2 in human secretions: IgA1, the only one presenting an additional hinge region
between the Cα1 and Cα2 domains. α and κ chains are maintained together with additional
disulphide bounds between the Cystein (Cys) 133 residues of the Cα1 domains in IgA1, and
other residues in the allotypes IgA2m(2) and IgA2m(3). In contrast, IgA2m(1) light and heavy
chains are not linked with disulfide bounds.
76
77 The J chain, a 15 kDa-glycoprotein rich in
cysteine residues, forms two disulfide bounds, making the bridge between two monomers of
IgA via additional 18 amino acids found at the C-terminus of the Cα3 domain (tail piece).78, 79
The presence of this J chain is interlocked with the secretion of pIgA, mediating the
interaction with the pIgR. Absence of J chain results in abrogation of pIgA secretion.79, 80
Nevertheless, no covalent bridges are formed between the two partners, SC and J chain. SC
is a 70-80 kDa glycoprotein consisting in 5 different domains linked to the Fc part of the pIgA
35
Figure 10. Mechanisms of SIgA-based mucosal protection against pathogenic infections.
SIgA protection of mucosal surfaces is mediated by: A. immune exclusion inhibiting their attachment and
invasion into epithelial cells (eg, S. flexneri), B. intracellular neutralization of pathogens that have invaded
epithelial cells (eg, rotaviruses) and C. antigen excretion of antigens that have reached the lamina propria.
IgA immune complexes are then transported across the epithelial barrier to clear the starting infection
(eg, measles virus). (Adapted from Strugnell and Wijburg, Nat Rev Microbiol., 2010, 8:656-67.)97
36
by covalent and non covalent interactions involving a cysteine residue in the fifth domain
with the Cα2 domain of only one monomeric IgA.81, 82
Glycans residues have been increasingly involved in mucosal protection representing
a link between innate and adaptative immunity.
83-87 Of main importance, SIgA is a highly
glycosylated protein comprising sugar-derived residues in each part constituting the Ab
(Figure 6). With only one N-moiety on the asparagine (Asn) 48, the J chain is the less
glycosylated peptide composing SIgA.88, 89 Carbohydrate residues represent up to 25 % of the
SC molecular mass, with 7 sites of N-glycosylation identified: two on each domain I, II and V
and the ultimate one on the fourth domain.90, 91 With respect to pIgA glycosylation, both
human IgA1 and IgA2 have two conserved N-glycan sites on each heavy chain: one on the
Asn263 of the Cα2 domains and the other on the Asn459 of the tail piece. Moreover, IgA2
harbors one or two additional N-glycans present on the Cα1 domain.83 IgA1 is the only
subclass which harbors O-carbohydrates in the hinge region.83
Although IgA appears to share common features in different species, six allotypes for
the unique class of mouse IgA have been identifies in inbred mice.
92 Comparison of the
Cα domains between inbred laboratory mice with wild mice further pinpoints major
structural differences.92 Despite such differences and little information about the precise
sites of glycosylation, it is widely accepted that mouse IgA are also highly glycosylated
proteins.93 Some data revealed N-glycan residues on the surface of mouse
hybridoma-derived Ab, especially on the Cα1 and Cα3 domains.94, 95 Despite a putative site
of O-glycosylation on the hinge region of IgA from Balb/c mice, no common O-glycan residue
has been further assigned.92, 95 With 62% of homology with human SC, mouse SC is
characterized by the presence of 8 putative sites of N-glycosylation slightly differently
distributed alongside the domains: one residue on each domain I and IV, four on domain II
and two on the fifth segment.96
5.2 Protective role in pathogenesis
Beside the increasing protective role associated with carbohydrate residues present
at the surface of SIgA, the Fab-mediated specificity of the Ab has been involved in different
mucosal mechanisms of protection among which intracellular neutralization of pathogens,
antigen excretion and immune exclusion (Figure 10).97
37
Figure 11. Model of infection by the virulent bacteria S. flexneri.
Bacteria enter the mucosa through M cells, are taken in charge by underlying macrophages. Bacteria first
evade from phagocytosis, inducing the apoptosis of macrophages and monocytes. Once in the dome, the
bacteria infect the enterocytes where they disseminate from cell-to-cell. In parallel, inflammatory
molecules are produced by cells amplifying PMN cells recruitment and inflammatory processes.
(Adapted from Cossart and Sansonetti, Science, 2004, 304:242-248.)121
38
5.2.1 Neutralization, antigen excretion and immune exclusion
Intracellular neutralization occurs when apical endocytosis vesicles containing
harmful Ag such as viral proteins or bacterial toxins fuse with basolateral compartments
containing specific pIgA on their way for secretion. These Ag are thus directly neutralized by
IgA in the vesicles and secreted as complexes at the apical pole of IEC. This way of
intracellular neutralization allows arresting infection processes at early stages. It is for
example the well described case of rotavirus neutralization.98
Considering immune excretion, Ag having reached the basolateral pole of IEC are
taken in charge by specific pIgA. pIgA complexed with Ag is then recognized by the pIgR and
transcytosed through the cells to be excreted in the gut lumen. This Ab property has been
widely described in the protection against measles virus and more recently in vitro evidences
involve it in HIV excretion.
Last but not least, the protective role of SIgA has been well established in the context
of infection where the Ab acts as a first line of defense through bacterial coating, thus
preventing attachment to epithelial surfaces resulting in a process referred to as immune
exclusion.
99, 100
101 In this case, mechanisms involve neutralization of either bacterial toxin such as
Vibio cholera cholera toxin, or pathogens such as Salmonella typhimuriun or Shigella flexneri
described herein.
102-104
5.2.2 Role in Shigellosis: a mechanism with wide information gaps
Diarrheal diseases caused by bacterial, viral or parasitic pathogens are a major health
public problem. The Enterobacteriaceae S. flexneri is a Gram-negative facultative
intracellular pathogenic bacterium. Species of the genius of Shigella are among the bacterial
pathogens most frequently isolated from patients with diarrhea and are the causal agent of
1.1 million yearly fatal cases. S. flexneri, by invading epithelial cells and inducing
inflammatory responses of the colonic mucosa, causes bacillary dysentery, a bloody diarrhea
that is endemic worldwide with prevalence in young children of the developing world.
Invasive bacteria are transmitted by the fecal-oral route or via the ingestion of contaminated
food or water.105 The ingestion of as few as 100 bacteria results in severe disease.106
The pathogenesis of entry of this enteropathogen is at the centre of the debate.
39
Figure 12. Virulence plasmid mapping and architecture of the encoded proteins.
A. The genes indicated encode structural components of the Mxi-Spa T3SS, secreted translocator and
effector proteins, chaperones, and regulatory proteins. B. General architecture of the T3SS constituted by
the proteins encoded by the virulence plasmid. The needle complex starts at the cytoplasm of the
bacterium, crosses the two membranes and protrudes from the cell injecting effector proteins.
Abbreviations used: HM, host membrane, OM, outer membrane, IM, inner membrane.
(Adapted from Schroeder and Hilbi, Clin. Microbiol. Rev., 2008, 21:134-56.)117
A
B
40
None of animal model is able to faithfully mimic the pathogenesis. Nevertheless, it has been
shown that the primary site of entry of the bacterium is M cells.107 The commonly accepted
model is represented in Figure 11. Once into the PP, bacteria encounter resident
macrophages.108 The bacteria evade degradation by inducing apoptosis-like cell death
accompanied by proinflammatory signaling (eg: release of IL-1β), which results in the
recruitment of polymorphonuclear (PMN) cells that infiltrate the infected site and amplify
damages to the epithelium.109-111 PMN cells disintegrate IEC, thus exacerbating the infection
and tissue destruction by facilitating the invasion of bacteria.112, 113 Loss of integrity of the
epithelium barrier allows more bacteria to gain access to the basolateral pole of epithelial
cells. Shigella can then invade the IEC and spread from cell-to-cell with concomittent
cytokine release (IL-8, interferon gamma (INF-y)) and amplification of the inflammatory
cascade leading to massive tissue destruction.114, 115
The essential part of the molecular machinery involved in the pathogenesis is
encoded by the large plasmid of virulence.
116 This plasmid consists of 34 genes organized
into two clusters and divided into four different groups117
- the first group consists of the proteins secreted via the type III secretion system
(T3SS), the central element of the injection process allowing the translocation of
approximatively 25 proteins from the bacterial cytoplasm into the host cell. These proteins
are named Invasion plasmid antigen (Ipa) effector proteins, IpaA to IpD. IpaB to IpAD are key
virulence factors,
(Figure 12):
- the second group consists of genes expressing proteins required for the secretion of
the IpA proteins, either the membrane expression of Ipa (mxi) and surface presentation of
Ipa (spa). The mxi-spa locus encodes for the component needed for the assembly and the
function of the T3SS itself,
118-120
- the third one is composed of two transcriptional activators, VirB and MxiE,
117
- the last one represents genes encoding chaperones which are involved in the
stabilization of both the components of the T3SS and the proteins excreted through the
T3SS.117
41
Figure 13. Observation of membrane ruffling induced after the recognition of S.
flexneri by cell surface receptors.
(Adapted from Philpott et al., Philos. Trans. R.
Soc. Lond. B. Biol. Sci., 2000, 355:575-86.)125
A B
Figure 14. Intracellular movement of S. flexneri by directed actin polymerization.
A. Due to the activity of the serine protease IcsP, S. flexneri IcsA localizes to one pole of the bacterium,
where it interacts with the host cell N-WASP protein which recruits the Arp2/Arp3 complex. Elongation of
the actin tail sustains bacterial movements through the cytoplasm further facilitated by VirA, which opens a
path by degradation of the microtubule network. (Adapted from Schroeder and Hilbi, Clin. Microbiol. Rev.,
2008, 21:134-56.)117 B. Transmission micrograph of a S. flexneri-mediated protusion into a neighboring cell
demonstrating the dense accumulation of actin fibers (Adapted from Philpott et al., Philos. Trans. R. Soc.
Lond. B. Biol. Sci., 2000, 355:575-86.)125
42
Ipa effector proteins and T3SS are essential to key steps such as bacteria-cell
adhesion at the basolateral side of enterocytes, release of the bacteria from vacuole into
cytoplasm and cell-to-cell dissemination.121 The initial loci of interaction of the bacteria with
the eukaryotic cell occurs at cholesterol-rich domains called lipid rafts through receptors
such as the hyaluronan receptor CD44 or α5β1 integrins.122, 123 This first contact induces the
secretion of effector proteins via the T3SS. The entry via lipid raft implies harsh remodeling
of the cell cytoskeleton such as actin polymerization/depolymerization mediated by the IpA
and IpGB1 virulent proteins, leading to the formation of large membrane protrusion, ending
in the cellular inclusion of a large macropinocytosis pocket enclosing the bacterium
(Figure 13).124, 125 S. flexneri escape from these vacuoles in less than 15 min is again
mediated by the Mxi-Spa T3SS and the Ipa effector proteins: IpaC seems to be the key factor
mediating the lipid lysis.118 Deprived of flagellum, S. flexneri needs to hijack the host
cytoskeleton to assume its intracellular mobility. This latter observation represented in
Figure 14 is based on the ability of the intracellular spread (IscA) protein to interact with
actin fibers.126-128 IscA recruits and activated host cell factors such as neuronal
Wiskott-Aldrich syndrome protein (N-WASP) and actin-related proteins 2 and 3 (Arp2/3)
complex which further support actin elongation and bacterial propulsion though the
cytoplasm and dissemination from one cell to its neighbors (Figure 14).127 Interestingly,
intercellular bacterial protrusions are described to focus onto TJ placing them as a target for
cell dissemination in the intestinal monolayer.
In vitro models using enterocyte-derived monolayers partially or not differentiated
led to the main admitted conclusion that S. flexneri invade IEC monolayers exclusively from
the basolateral pole.
129, 130
131 Nevertheless, recently published data report the presence of an
apical infection by the bacterium, demonstrating a new potential way of entry for the
bacterium outside the PP. 100 years after its discovery, S. flexneri is still a worldwide major
health problem highlighting difficulties in understanding the pathogenesis at the molecular,
cellular and organism levels. How immune responses govern the protection against Shigella
infection in still a main subject of debate. The predominant role of SIgA has been described
in vivo using rabbit ileal loops132 and also into samples of infected patients where it plays a
major role in the protection of the intestinal environment by a mechanism called immune
exclusion.102, 133-136 Furthermore, monoclonal SIgA directed against the O-antigen
43
44
extracellular part of the lipopolysaccharides (LPS) constituting the outer membrane of
Gram-negative bacteria such as S. flexneri has been described to be the most efficient way to
limit bacterial infection as shown in the rabbit ligated ileal loop in vivo model, the best
available model of shigellosis.102, 132, 137 Experiments highlight a predominant, protective role
of the SC in the protection mediated by SIgA (SIgA is 10 fold more protective than pIgA).85
Recently published data further demonstrate a transient suppression of the T3SS when the
bacteria were incubated with LPS-specific monoclonal antibodies (Ab).138
Despite these
observations, few is known on the cellular and molecular basis pertaining to SIgA-based
protection during Shigella infection.
5.3 SIgA and mucosal homeostasis: interaction with commensals
In contrast, in order to maintain an abundant and well balanced gut flora, such a
clearance mechanism must be limited, to a level guaranteeing homeostasis. To preserve
mucosal homeostasis, a complex communication needs to be established between a narrow
triptych: the microbiota, the epithelial cells and the mucosal immune system.
Evidence is accumulating that emphasizes a complex cross-talk between the
epithelium and microbiota that triggers SIgA secretion in the gut lumen of neonates already.
In contrast, SIgA production is reduced at barely detectable level in germ-free animals,
normal values of IgA can be reached within a few weeks following intestinal recolonization
with various flora.139-142 Furthermore, the specificity of SIgA induced at mucosal surfaces has
been studied in details and allows to conclude that more than 90 % of these Ab have an
unknown specificity and are thus called “natural” SIgA.143 More recently, the induction of
strain-specific SIgA secretion following reintroduction of Enterobacter cloacae in the gut of
specific pathogen-free mice has also been highlighted.144, 145
4.2
In this particular case, the
commensals have a direct impact on the immune system maturation and activation as
mentioned in the Introduction section . Moreover, in vivo coating of commensal bacteria
by SIgA has been described in analysis of human feces.146, 147 At least 45% of intestinal
bacteria have been described to be coated by SIgA. Together with mucins, SIgA has also
been involved in the formation of intestinal biofilms.148-151 In this case, SIgA acts as an
anchoring partner allowing further adhesion in the GI tract. However, the molecular mode of
action of SIgA in regulating microbiota colonization and IEC responses remains enigmatic.
45
Figure 15. Human Fc receptor described in IgA recognition.
Each receptor harbors a cytoplasmic tail, a transmembrane domain and an extracellular domain involved in
IgA interaction. Ig-like domains are depicted as orange circles. Other receptors have been involved IgA
recognition but are not fully characterized and thus cannot be represented in this figure. (Adapted from
Monteiro and Van De Winkel, Annu. Rev. Immunol., 2003, 21:177-204.)152
pIgR
46
5.4 SIgA receptors: between pro- and anti-inflammatory responses
At mucosal surfaces, the well described pIgR acts as the major, primary receptor for
pIgA assuming IgA transport from the lamina propria to the gut lumen, actively participating
in commensal control and mucosal protection. Other receptors have been described which
can take in charge serum IgA resulting in a large spectrum of cellular responses
(Figure 15).
152
5.4.1 FcαRI (CD89)
Highly glycosylated, the FcαRI receptor is a 50-70 kDa transmembrane protein
expressed at various cell surfaces such as neutrophils, monocytes, eosinophils,
macrophages, interstitial DC and Kupffer cells (macrophages located in the liver).153
The essential population of the cells expressing the CD89 is neutrophiles present in blood
and tissues. In contrast, the intestinal mucosa harbors low levels of CD89 positive cells.154
No murine counterpart has yet been described. The structure of this receptor is closely
linked to the Fc receptor for IgG (FCγR) or for IgE (FCεRI): the FcαRI is divided into three
domains: an extracellular domain divided into two segments D1 and D2 separated by an 90°
angle, a transmembrane region and a small cytoplasmic tail. IgA interaction with the
receptor involved key amino acids present at the interface between the Cα2 and the Cα3
domains, respectively the Leucine 257 and Leucine 258 and the Proline 440 and
Phenylalanine 443 on the IgA with amino acids of the D1 domain of the receptor.155, 156
The detection of IgA complexed with Ag can initiate a broad range of biological responses
such as: Ab-dependant cell-mediated cytotoxicity, phagocytosis, superoxide generation,
release of cytokines, Ag presentation and degranulation.157-160 Interestingly, SIgA unlike IgA
is unable to mediate FcαRI phagocytosis, corroborating with its anti-inflammatory properties
at mucosal surfaces.
161
5.4.2 Fcα/μR
Found in both human and mouse cells, this receptor is so called because of its avidity
for both IgA and IgM.162, 163 It is found constitutively expressed onto B-cells and
macrophages, it is also abundant in lymph nodes, appendix, intestine and kidney.164
47
48
Fcα/μR is a type 1 transmembrane protein in which the extracellular domain is predicted to
bind to Ig-like domains presenting 43% of homology with the pIgR D1 domain.165
One can thus assume that the Fcα/μR share common IgA interaction properties with pIgR.
Nevertheless, its effective role in IgA detection remains unknown.
5.4.3 The transferrin receptor (TfR or CD71)
The CD71 is a disulfide homodimer of 180 kDa originally known as the specific
receptor for transferrin sustaining the rapid endocytosis and recycling indispensible for iron
delivery into proliferating epithelial cells. The TfR has also been described to recognize the
IgA1 allotype but not IgA2.166 Interestingly, the presence of transferrin is not able to inhibit
the IgA fixation indicating different sites of interaction involved.167 This receptor is found
expressed at the surface of mononuclear hematopoietic cells in fetal liver and bone marrow,
certain lymphocytic and myeloid cell lines.168 The TfR expression has not been yet described
on adult mononuclear or PMN cells. However, it is present on mesengial cells and epithelial
cells and further expressed in IgA nephropathy and Crohn’s affected patients.167, 168
Surexpression of CD71 in IgA nephropathy could participate in the abnormal deposition of
IgA-complexes.168 In celiac disease, increase in CD71 expression is followed by the
retrotransport of SIgA complexes from the lumen to the lamina propria triggering
inflammatory responses and mucosal injury.167
This latter case highlights a potential
receptor for SIgA retrotransport across the intestinal epithelium from the gut lumen to the
lamina propria.
5.4.4 The eosinophil SC receptor
Eosinophils support mucosal protection against parasitic infection and have potential
roles in allergic inflammation. Besides the expression of CD89 specific for the Fc part of SIgA,
a receptor for the SC has been also described.169-171 Association of the receptor with SIgA
could trigger degranulation and superoxide production leading to the creation of an
inflammatory environment. Described as a 15 kDa-protein, the receptor for SC has not been
fully characterized.
171
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50
5.4.5 Asialoglycoprotein Receptor (ASGP-R)
The ASGP-R expressed on hepathocytes has been involved in the recognition of
terminal galactose residues on serum proteins such as IgA.172, 173 Clearance of IgA from the
blood is proposed as a property for this receptor. Differential kinetic of metabolism between
the two allotypes IgA1 and IgA2 (the most efficient) may sustain the predominance of the
IgA1 allotype in the serum indicating potential role in the regulation of Ab titers in blood.
174
5.4.6 The M cell IgA receptor
Recently, immunoglobulin (Ig) transport through the M cell has been described to be
restricted to IgA indicating of the presence of a selective receptor for IgA expressed at the
cell surface.175
Nevertheless, this receptor involved in sampling luminal Ag complexed with
SIgA has not been further described at this moment.
5.4.7 Other receptors
A few other receptors have been recently identified: a receptor expressed on isolated
human natural killer cells and an unusual form of the pIgR described at the surface of the
mouse B-cell derived T560 cell line.176, 177 This latter receptor presents an unexpected avidity
for IgM compared to IgA, modifications in the pattern of glycosylation may account for this
altered selectivity.
51
AIMS OF THIS WORK
Ideally positioned at the interface separating the inside of the body and the intestinal
external environment, IEC are a crucial partner, actively participating in the maintenance of
intestinal homeostasis and the fight against infections. SIgA, considered as the major
immunoglobulin produced at the intestinal level is also implicated in mucosal protection.
Both IEC and SIgA have been suggested as partners of the intestinal homeostasis;
nevertheless, very few is known on how this Ab can modulate cellular responses.
To decipher the mode of action of commensal microorganisms and the role played by
SIgA in this relationship, polarized Caco-2 cell monolayers, mimicking the intestinal
epithelium, were incubated with bacteria alone or in complexes with non-specific SIgA.
Ab-mediated changes in IEC responsiveness to commensal bacteria were followed using
various parameters such as bacterial adhesion, NF-kB activation and selected protein
expressions leading us to conclude on the role of SIgA in maintaining mucosal homeostasis.
Coating of commensal bacteria naturally occurs in the human and mouse GI tract.
Understanding how natural, non-specific SIgA can interact with the microbiota provides a
new facet in the multiple roles of this Ab. Coating of intestinal bacteria has thus been
assessed in further details by confocal microscopy using non specific hybridoma-derived as
well as colostrum derived SIgA pinpointing the prevalent and underestimated role of glycan
residues present at its surface.
The protective role of SIgA in pathogenesis such as shigellosis has been well
established in in vivo models but very few is known about its impact at the epithelial level. A
better understanding of the mechanisms involved at the IEC level can thus provide new
perspectives in limiting the progression of this pathogenesis causing 1.1 millions fatal cases
each year. The second part is thus dedicated to the detailed study of apical infection of
Caco-2 cell monolayer using virulent bacteria alone or complexed with specific SIgA. We
have thus evaluated how SIgA could interfere with the infectious pattern of S. flexneri and
allow intestinal protection following bacterial growth and targeting cellular pillars such as
actin fibers or TJ, gatekeepers of the intercellular permeability.
Finally, we got interested in determining whether epithelial cell can also be directly
involved in a new pathway of SIgA entry. Apical incubation of Caco-2 cell monolayers with Ab
followed by a descriptive analysis were carried out giving a starting point to have a better
insight in the direct interplay involving SIgA and IEC.
52
PART I
: N-GLYCANS ON SECRETORY COMPONENT: MEDIATORS OF THE INTERACTION BETWEEN
SIGA AND GRAM-POSITIVE COMMENSALS SUSTAINING INTESTINAL HOMEOSTASIS
1 OVERVIEW OF THIS PART
The craze for gut microbiota has undergone a burst during the past decade. The roles
played by the commensal bacteria on the host physiology and more specifically the
recognition by the host immune system are increasing subjects of interest. Nevertheless, the
precise mechanisms involved in the epithelial sensing of intestinal bacteria still harbors grey
areas. In particular, the role played by SIgA in the relationship between the microbiota and
the underlying epithelium is largely unknown. Thus, in order to tackle this issue, we decided
to inoculate in vitro commensal bacteria either alone or in association with non-specific SIgA
onto polarized IEC monolayers. To analyze how epithelial cell responses can be modulated
by the presence of the bacteria alone or as a complex with SIgA, a large spectrum of
parameters was followed including bacterial adhesion, changes in protein expression and
cytokine release by IEC. These experiments allowed us to conclude on the role of the non-
specific coating of SIgA in maintaining a peaceful communication between commensals and
the intestinal epithelium mimic represented by polarized IEC.
Because abundant, low affinity, natural SIgA has been described in intestinal
secretions, we speculated that these latter can be involved in direct binding to commensals.
As substitute of natural SIgA and SC, we used non-specific recombinant mouse SIgA, pIgA
and free SC, as well as human colostral SIgA, and their deglycosylated counterpart to
examine the role of carbohydrates in binding to a selection of bacteria present in the GI tract
(Lactobacillus, Bifidobacteria, Escherichia coli and Bacteroides strains). Confocal analyses
followed by quantification led us to conclude on the crucial role played by N-glycans present
at the surface of bound and free SC in the natural coating of Gram-positive commensal
bacteria.
53
2 EXPERIMENTAL PROCEDURES, RESULTS AND DISCUSSION
Results obtained in the frame of this thematic have been published in the Journal of
Biological Chemistry in the form of two independent articles:
- Potentiation of polarized intestinal Caco-2 cell responsiveness to probiotics
complexed with secretory IgA. Mathias A. (see author contribution), Duc M., Favre L.,
Benyacoub J., Blum S., Corthésy B., J. Biol. Chem., 2010, 285:33906-13.
I have been involved in all experiments dealing with observations of bacteria (strains
provided by Nestlé Research Center) complexed with SIgA, culture and stimulation of
monolayers and analyses.
Author contribution
- Recognition of Gram-positive intestinal bacteria by hybridoma- and colostrum-
derived secretory immunoglobulin A is mediated by carbohydrates. Mathias A. (see author
contribution) and Corthésy B., J. Biol. Chem., 2011, 286:17239-47.
I have performed all experiments and analyses related to the study. I have substantially
contributed to the writing of the paper under the supervision of Blaise CORTHESY, thesis
director.
Author contribution
The role of SIgA in the relationship between the microbiota and the IEC has been
largely discussed in a review article to be published in Gut microbes:
- N-glycans on Secretory Component: mediators of the interaction between SIgA
and Gram-positive commensals sustaining intestinal homeostasis. Mathias A. (see author
contribution) and Corthésy B., Gut microbes (In press)
I have substantially contributed to the writing of the paper under the supervision of Blaise
CORTHESY, thesis director.
Author contribution
54
2.1 Potentiation of polarized intestinal Caco-2 cell responsiveness to probiotics
complexed with secretory IgA.
Mathias A., Duc M., Favre L., Benyacoub J., Blum S., Corthésy B., J. Biol. Chem., 2010,
285:33906-13.
55
Potentiation of Polarized Intestinal Caco-2 CellResponsiveness to Probiotics Complexed with Secretory IgA*
Received for publication, April 16, 2010, and in revised form, July 20, 2010 Published, JBC Papers in Press, August 20, 2010, DOI 10.1074/jbc.M110.135111
Amandine Mathias‡, Melanie Duc‡, Laurent Favre§, Jalil Benyacoub§, Stephanie Blum§, and Blaise Corthesy‡1
From the ‡R&D Laboratory of the Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois, Rue du Bugnon,1011 Lausanne, Switzerland and the §Department of Nutrition and Health, Nestec Research Center, 1000 Lausanne, Switzerland
The precise mechanisms underlying the interaction betweenintestinal bacteria and the host epithelium lead tomultiple con-sequences that remain poorly understood at themolecular level.Deciphering such events can provide valuable information as tothemode of action of commensal and probioticmicroorganismsin the gastrointestinal environment. Potential roles of suchmicroorganisms along the privileged target represented by themucosal immune system include maturation prior, during andafter weaning, and the reduction of inflammatory reactionsin pathogenic conditions. Using human intestinal epithelialCaco-2 cell grown as polarized monolayers, we found that asso-ciation of a Lactobacillus or a Bifidobacteriumwith nonspecificsecretory IgA (SIgA) enhanced probiotic adhesion by a factor of3.4-fold or more. Bacteria alone or in complex with SIgA rein-forced transepithelial electrical resistance, a phenomenon cou-pled with increased phosphorylation of tight junction proteinszonula occludens-1 and occludin. In contrast, association withSIgA resulted in both enhanced level of nuclear translocation ofNF-B and production of epithelial polymeric Ig receptor ascompared with bacteria alone. Moreover, thymic stromal lym-phopoietin productionwas increased upon exposure to bacteriaand further enhanced with SIgA-based complexes, whereas thelevel of pro-inflammatory epithelial cell mediators remainedunaffected. Interestingly, SIgA-mediated potentiation of theCaco-2 cell responsiveness to the two probiotics tested involvedFab-independent interaction with the bacteria. These findingsadd to the multiple functions of SIgA and underscore a novelrole of the antibody in interaction with intestinal bacteria.
The gastrointestinal lymphoid tissue plays an important rolein controlling transepithelial passage of bacteria across theintestinal mucosa by synthesizing more antibody (Ab)2 mole-cules than any other lymphoid tissue. This is principally initi-ated in organized lymphoid tissues referred to as Peyer’spatches, where antigen sampling by microfold (M) cells, proc-essing/presentation by dendritic cells, and subsequent T cellactivation leads ultimately to the development of immunoglob-
ulin A (IgA)-producing cells in local and distant effector muco-sal sites and glands (1–3). Antigen sampling by lamina propriadendritic cells and T cell-independent B cell maturation alsocontribute tomucosal IgAproduction (4). This class ofAb is theprincipal immunoglobulin produced in the gastrointestinaltract, and hence a key player to efficient humoral mucosalimmunity, in particular for the maintenance of the integrity ofthe epithelial barrier.In mucosal secretions, SIgA exists as a complex made of pol-
ymeric IgA (pIgA) in association with secretory component(SC), a highly glycosylated protein resulting from the cleavageof the polymeric Ig receptor (pIgR) that ensures the transport ofthe immunoglobulin across the epithelium. In the gut, boundSC contributes to both the stability of the Ab toward proteases,as well as its proper anchoring to mucus, conferring optimalbiologic activity (5, 6). In addition to its function in immuneexclusion preventing pathogen translocation across the epithe-lial barrier, abundant maternal SIgA Ab (up to 12 g/liter incolostrum and 1 g/liter in humanmilk (7)), by coating gut com-mensal bacteria in developing neonatal intestine, is thought tomodulate initial exposure to the immature immune system (8).The postnatal colonization of the gastrointestinal tract
with commensals provides bacterial stimuli that are crucialfor the functional development and homeostasis of themajorcompartments of the gastro-intestinal lymphoid tissue (9,10). Studies with various strains of mostly lactic acid probi-otics, which are normal residents of the intestinal microbiota(11, 12), represent a valuable approach to decipher the elab-orated mechanisms involved in their dynamic interactionwith intestinal epithelial cells. The relationship betweenintestinal microorganisms and SIgA led us to postulate thatcomplexes of the two might cooperate to communicate withthe interface represented by epithelial cells.To address this hypothesis, we used an in vitro system con-
sisting of polarized Caco-2 cell monolayers mimicking theintestinal epithelium, and exposed this latter apically to twoprobiotic strains Lactobacillus rhamnosus (LPR) or Bifidobac-terium lactis (BL), either alone or in association with SIgA.Important features including bacterial adhesion, NF-B activa-tion, pIgR and thymic stromal lymphopoietin production wereall potentiated upon complex formation with SIgA in a Fab-independent fashion. Tightness of the epithelial monolayer waspromoted identically by the bacteria alone or in complex withSIgA. This indicates that the combination of probiotics andSIgA has a qualitative impact on epithelial cell function, andthat this effect cannot simply be attributed to quantitative dif-ferences in bacterial binding. Our studies underscore for the
* This work was supported by Research Grant 3200-122039 from the SwissScience Research Foundation and the Nestec Research Center.
1 To whom correspondence should be addressed: R&D Laboratory of theDept. of Immunology and Allergy, University State Hospital (CHUV), Rue duBugnon 46, 1011 Lausanne, Switzerland. Tel.: 0041-21-314-07-83; Fax:0041-21-314-07-71; E-mail: [email protected].
2 The abbreviations used are: Ab, antibody; BL, B. lactis; LPR, L. rhamnosus;pIgR, polymeric Ig receptor; SC, secretory component; SIgA, secretory IgA;TER, transepithelial electrical resistance; TSLP, thymic stromal lymphopoi-etin; ZO-1, zonula occludens-1.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 44, pp. 33906 –33913, October 29, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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first time the involvement of the Ab in mechanisms associatedwith epithelium responsiveness to probiotics and maintenanceof the integrity of the intestinal barrier.
EXPERIMENTAL PROCEDURES
Caco-2 Cell Culture Conditions—Human colonic adenocar-cinoma epithelial Caco-2 cells (HTB 37, American Type TissueCollection) were grown at 37 °C in complete DMEM(C-DMEM) consisting of DMEM-Glutamax (Sigma) supple-mented with 10% FBS (Sigma), 1% non essential amino acids(Sigma), 10 mM HEPES (pH 7.0, Sigma), 0.1% transferrin(Invitrogen AG, Basel, Switzerland) and 1% streptomycin/pen-icillin (Sigma), and used between passages 23 and 37. Cells cul-tivated to 80% confluency were seeded on Transwell filters(diameter, 12 mm; pore size, 0.4 m; Corning Costar, Cam-bridge, MA) at a density of 0.8 105 cells/cm2. The culturemedium was changed every 2 days. The formation of a polar-ized Caco-2 cell monolayer at week 3 was established by mor-phology and monitoring of the transepithelial electrical resis-tance (TER; ohms cm2) using a Millicell-ERS apparatus(Millipore, Bedford, MA). TER values of well-differentiatedmonolayers were in the range of 380–450 ohms cm2 (13).Bacterial Strains—The bacterial strains L. rhamnosus
CGMCC 1.3724 and B. lactis CNCM I-3446 were obtainedfrom the China general microbiological culture collection andPasteur Institute microbiological culture collection, respec-tively. Ready to use vials of freeze-dried powders of live bacteriacoded L. rhamnosus NCC4007 (LPR) and B. lactis NCC2818(BL)were provided byNestle ResearchCenter (Lausanne, Swit-zerland). LPR was cultured from frozen stocks overnight at37 °C in Mann-Rogosa-Sharpe broth (Difco Laboratories,Detroit, MI) without agitation, and BL was grown in the sameconditions in medium complemented with 0.05% L-cysteine.The commensal strain Escherichia coli strains Nissle 1917 andthe cloning strain TG1 was grown overnight at 37 °C in Luria-Bertani broth (Difco) with agitation. Assessment of colony-forming units (CFU) per milliliter resulting from such cultureconditions was carried out by plating of successive dilutions ofthe overnight incubations, or by measurement of the opticaldensity at 600 nm (14).Proteins and Ab—Purified mouse SC was produced and pre-
pared as described in Crottet et al. (15). Hybridoma IgAC5 cellswere grown at 37 °C in Integra Biosciences Celline-350 car-tridges (Vitaris, Baar, Switzerland) in RPMI 1640 medium sup-plemented with 10% FBS, 2 mM glutamine, 2 mM sodium pyru-vate, 10mMHEPES (pH7.0), 0.1mM folic acid, and 100 units/mlpenicillin and 100 g/ml streptomycin, and purified polymericIgA was recovered from crude supernatant by sizing chroma-tography onto Sephacryl S-300 columns (GE Healthcare,Otelfingen, Switzerland (16)). SIgA Ab molecules were recon-stituted from equimolar of polymeric IgA and SC as published(5).Association of SIgA with Bacteria—Overnight bacterial cul-
tures were washed twice in phosphate-buffered saline (PBS:116.3 mM NaCl, 10.4 mM Na2HPO4, 3.2 mM KH2PO4 (pH 7.4))resuspended in PBS, and the number of bacteria was deter-mined as indicated above. 2 107 bacteria were mixed with 1g of SIgA in a final volume of 50 l of PBS and incubated at
ambient temperature for 30 min prior to use. For visualizationof the Fab-independent association between bacteria and SIgA,the proteins were labeled with indocyanin-3 (Cy3) as published(17). Following washes in PBS, mixtures were laid into 8-wellmultitest slides, fixed with 2% paraformaldehyde in PBS for 25min, washed, and mounted postcoating with Vectashield (Vec-tor Laboratories, Burlingame, CA). Observations were per-formed using a Zeiss LSM 510Meta confocal microscope (CarlZeiss, Jena, Germany). Images were taken with a 63 objectiveand processed using the microscope-related software (Zeiss).Quantification of bacterial coating by antibodies was per-formedwith ImageJ software 1.41 (NIH).Wemeasured on eachseries of pictures the area covered by bacteria (bacterial area)using the differential interference contrast (DIC) channel. Inparallel, in each area associated with bacteria, we quantify thearea containing IgA linked pixels with a fluorescence intensitysuperior to 15 units (fluorescent area) defined in our experi-mental settings as the background signal (signal range extendsfrom 0 to 256 units using transformation into 8 bit gray scale).The following ratio was used to quantify the percentage of bac-terial surface covered with fluorescent Ab molecules: 100 (fluorescent area)/(bacterial area).Exposure of Polarized Caco-2 Cell Monolayers to Bacteria
Alone or in Complexes with SIgA—The apical compartment ofCaco-2 cell monolayers was washed twice with PBS, andC-DMEMwas replacedwith the samemedium lacking FBS andantibiotics (DMEM-A). After 2 h of incubation, bacteria,SIgA, or SIgA-bacteria mixes in DMEM-A were added theco-culture was kept overnight (usually 16 h) prior to be sub-jected tomultiple analyses as described below. In control exper-iments, we found that bacterial growth rate was not affected bythe Ab.Bacterial Adhesion Assay—Transwell supports carrying
polarized Caco-2 cell monolayers were washed five times withPBS. The Caco-2 cells were then detached by a 5-min incuba-tion at 37 °C in the presence of trypsin/EDTA (Sigma) added tothe apical (0.5 ml) and the basolateral (1.5 ml) surfaces. Theadherent bacteria were dispersed by vigorous pipeting, serialdilutions (102 to 105) were applied onto triplicate agar platescast inMann-Rogosa-Sharpe or Luria-Bertani broths, and CFUwere determined after overnight incubation at 37 °C.Whole Cell Lysates and Analysis of Caco-2 Cell Proteins—At
the times indicated, Caco-2 cells grown in Transwell mem-branes were washed twice with PBS, prior to incubation for 5min in 1ml of hypotonic buffer (20 mMTris-HCl, pH 7.5, 2 mM
MgCl2). Swollen cells were lysed by up-and-down pipetting in150l of hypotonic buffer complemented with 1% (w/v) TritonX-100 (Pierce Chemical), Complete Protease InhibitorMixture(Roche Applied Science, Rotkreuz, Switzerland), 0.15 M NaCl,0.1 mM EDTA, 0.1 mM sodium orthovanadate, 0.1 mM sodiumfluoride, 2 g/ml DNase 1, and 1 mM leupeptin. The cell lysatewas transferred to a fresh siliconized 1.5-ml tube, and clearedby centrifugation at 13,000 g. An aliquot was removed forprotein determination using the bicinchoninic acid procedure(Pierce Chemical). Aliquots of the clarified lysate were stored at20 °C prior to use.For immunoprecipitation, whole Caco-2 cell extracts were
pre-cleared by incubation with protein G-Sepharose beads (GE
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Healthcare) for 2 h at 4 °C. 50 l of cleared whole Caco-2 cellextracts were incubated with 1/333 dilution of the specific Ab(anti-ZO-1 and anti-occludin from Santa Cruz Biotechnology)overnight at 4 °C. Protein G-Sepharose beads were added andincubated for an additional 1 h of incubation at 4 °C, and theimmunocomplexes were washed with TENT buffer (50 mM
Tris-HCl, pH 7.5, 5mMEDTA, pH8.0, 150mMNaCl, 1%TritonX-100). SDS-PAGE was performed according to the Laemmliprocedure using 10%polyacrylamide gels run inMini Protean IIgel apparatus (Bio-Rad). Separated proteins fromwhole Caco-2cell lysates were electrotransferred onto polyvinylenedifluoridemembranes that were saturated with 5% nonfat dry milk inPBS-Tween 20 (PBS-T). Tight junction proteins ZO-1 andoccludin were probed with 1/500 dilution of anti-ZO-1 andanti-occludin, or 1/1000 dilution of anti-phosphotyrosineand anti-phosphoserine (Transduction Laboratories, Lexing-ton, Kentucky) in 0.5% milk in PBS-T for 1 h at ambient tem-perature. After washing, the membranes were incubated withcorresponding horseradish peroxidase-linked secondary Ab(anti-mouse IgG, anti-rabbit IgG, as appropriate) for 1 h atroom temperature. Following final washes, membranes wereincubated with enhanced chemiluminescence reagents (Inter-chim, Montlucon, France) prior to exposure to photographicfilms.Polymeric Ig receptor in whole Caco-2 cell extracts was
detected by immunoblotting using rabbit antiserum against thehuman protein (1/2000 (18)). The level of pIgR was normalizedto-actin in the same sample incubatedwith 1/2000 dilution ofa specific antiserum (Alpha Diagnostic, San Antonio, TX).Quantification of human SC was carried out by ELISA asdescribed (18).Analysis of NF-B Nuclear Translocation and IB
Evaluation—Preparation of Caco-2 cell small-scale nuclearextracts and use in EMSA for the detection of NF-B was car-ried out as described in Cottet et al. (13). The radioactivityassociated with retarded oligonucleotide-NF-B complexeswas quantified on an Instant Imager reader (Packard, Palo Alto,CA).Members of the NF-B family present in the nucleus fromCaco-2 cells were identified by immunoblotting with rabbitantisera (Santa Cruz Biotechnology, Santa Cruz, CA; 1/500dilution) directed against the p50 or p65 subunits. Cytoplasmicextracts containing IB were obtained according to Cottet etal. (13), and the presence of the protein was similarly assessedbyWestern blot with a specific antiserum (Santa Cruz Biotech-nology). After binding of appropriate secondary Ab, mem-branes were processed for detection by chemiluminescence.Cytokine/Chemokine ELISA—CXCL-8 (interleukin (IL)-8),
thymic stromal lymphopoietin (TSLP), CCL-5 (regulated onactivation normal T cell expressed and secreted: RANTES),CCL-2 (monocyte chemoattractant protein (MCP)-1) possiblyreleased in the basolateral compartment of polarized Caco-2cells weremeasured by ELISAusing commercial kits for humanproteins (Biolegend, San Diego, CA), and expressed as pico-grams per ml of culture medium. Data are duplicates of 2–3independent experiments.Statistical Analysis—Statistical significance was determined
using the two-tailed nonparametric Mann-Whitney U test.Standard error means and p values were calculated using the
Prism5 application (GraphPad, SanDiego, CA), and the limit ofsignificance was set at p 0.05.
RESULTS
Adhesion of LPR Alone, BL Alone, or in Complexes with SIgAto Polarized Caco-2 Cell Monolayers—When the number ofadherent LPR and BL bacteria per 100 Caco-2 cells was plottedagainst the concentration of added bacteria, a plateau-typebinding pattern was obtained. A similar adhesion curve wasproduced with the E. coli commensal strain Nissle 1917whereas virtually no binding was observed with non-commen-salE. coliTG1 at all concentrations tested (Fig. 1A). In addition,differences in adhesion properties were detected between LPR,BL, and Nissle 1917 indicating that selective interaction withthe apical surface of Caco-2 cells mimicking the intestinal bar-rierwas indeed occurring in this simplified system.Time courseexperiments showed that there is no dependence of the finalconcentration of bacteria on adhesion.3 Because intestinal bac-teria have been shown to be coated with a mixture of specificand nonspecific, “natural” SIgA (19, 20), we sought to examine
3 B. Corthesy, unpublished results.
FIGURE 1. A, adhesion of L. rhamnosus LPR, B. lactis BL, and E. coli strains Nissle1917 and TG1 to polarized intestinal Caco-2 cell monolayers. The number ofbound bacteria per 100 Caco-2 cells is presented as a function of the concen-tration of freshly cultured bacteria added (CFU per ml). Bacterial counts weredetermined by plating of serial dilutions. Black bars, LPR; gray bars, BL; stripedbars, Nissle 1917; white bars, E. coli TG 1. B, same as in A using 2 107 bacteriaalone (white bars) or associated with SIgA (black bars) prior to incubation withCaco-2 cell monolayers. Data were obtained from four independent experi-ments performed in triplicates. Significant statistical differences are indicatedabove the lanes.
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whether the combination of LPR and BL with SIgA might haveinfluenced the adhesion properties of the bacteria. We foundthat association with SIgA enhanced the capacity to adhere topolarized Caco-2 monolayers by a factor of 3.4- for LPR, and3.9-fold for BL, respectively (Fig. 1B). Noteworthy, incubationof 2 107 bacteria in the presence of SIgA leads to more adhe-sion toCaco-2 cells thanwhen 1 108 bacteria are added alone.We concluded that SIgA potentiated the capacity of LPR andBL to adhere to polarized Caco-2 cell monolayers.Fab-independent Interaction of SIgA with LPR and BL—
Based on previous reports showing of intestinal bacteria withendogenous SIgA (19, 20), it is conceivable that the SIgA Abmolecule we used, whose specificity is directed toward S. flex-neri LPS serotype 5a, can however associate with both LPR andBL in a Fab-independent fashion, thus contributing toimproved adhesion.We examined this possibility by combiningbacteria with red Cy3-labeled SIgA. Laser-scanning confocalmicroscopy pictures show abundant red staining coveringstrings of bacteria typical of the two strains assessed, henceindicating Fab-independent association of the Ab and bacte-riumpartners (Fig. 2). Using ImageJ software (see Experimentalprocedures), quantification of fluorescent SIgA associated withbacteria indicated that the surface of all bacteria were coatedwith up to 95% (LPR) and 78% (BL) by Ab molecules. Thisdemonstrated that this was in the form of a complex with SIgAthat cellular adhesion of the bacterium was improved.Increased TER after Overnight Contact between Polarized
Caco-2 Cell Monolayers and LPR Alone, BL Alone, or in Com-plexes with SIgA—TER measures the permeability attained byepithelial cell lines grown on synthetic polycarbonate mem-branes after they have differentiated as polarized monolayersmimicking the intestinal barrier. Live commensals and probi-otics modulate TER (14, 21), and this was used in the presentstudy to investigate in vitro the possible effects of LPR or BLalone, and in complexes with SIgA, on Caco-2 cell monolayers.Functional polarity, i.e. formation of tight junctions observedby ZO-1 staining in laser scanning confocal microscope images(data not shown), was established at 350 ohms cm2 in the
model used. Exposure to either LPR or BL for 6 h ormore led toa reproducible and significant 18–25% increase in TER (0.01p 0.005), an effect that reached a plateau at 15 h (Fig. 3A).Association of SIgA with bacteria did not further modulate theTER (Fig. 3A), although binding to polarized Caco-2 monolay-ers was potentiated in the presence of the Ab (Fig. 1). The sameincrease in TERwas obtainedwhen using 5 times less or 5 timesmore bacteria (data not shown), indicating that despite of SIgA-mediated improved adhesion, the Ab had no inhibitory/activa-tory effect on the properties of the bacteria in acting on TER.Raise in TER Is Associated with IncreasedOccludin and ZO-1
Phosphorylation upon Incubation with LPR Alone, BL Alone, orin Complexes with SIgA—Increased TER implies associatedimproved tightness of the junction-connecting cells involved inmonolayer formation. Analysis of Caco-2 cell whole extracts16 h post-exposure to bacteria revealed no change in the con-tent of tight junction proteins occludin and ZO-1, despite the18–25% increase in TER previously observed at this time point.However, the degree of phosphorylation of the two proteinswas consistently increased upon incubation with LPR (Fig. 3, Band C) and BL (Fig. 3C) compared with resting polarizedCaco-2 cell monolayers. Immunodetection of occludin andZO-1 showed that very similar amounts of protein in Caco-2cell extract immunoprecipitates were loaded (Fig. 3B). Densi-tometric analysis of raw data presented in Fig. 3B indicated thatthe degree of phosphorylation increased 2-fold (occludin) andup to 3-fold (ZO-1) after exposure to the bacteria alone, andthat this value remained the same when SIgA was combinedin the assay (Fig. 3C). This correlated at themolecular level withthe contribution of probiotics in the reinforcement of the epi-thelial barrier and the absence of involvement of SIgA bound tobacteria in the raise in TER (Fig. 3A).Effect of LPR Alone, BL Alone, or in Complexes with SIgA on
NF-B Activation in Polarized Caco-2 Epithelial Cell Mono-layers—Activation of the transcription factor NF-B is a recog-nized marker of the onset of multiple signaling pathways inmany cell lines and tissues (22). In comparison with restingcells, exposure of polarized Caco-2 cells to LPR or BL led to theappearance of more shifted complexes when nuclear extractsweremixedwith a consensusNF-BDNAprobe and examinedby electrophoretic mobility shift assay (Fig. 4A). However, incomparison with the pathogen S. flexneri inducing strongnuclear translocation of NF-B, both bacteria maintained lowlevel of inducible complex formation under all conditionstested. In association with SIgA, higher levels of NF-B-probecomplexes were found in Caco-2 cell nuclear extracts as com-pared with bacteria alone, although the apparent degree ofabsolute nuclear translocation was againmuch lower than withS. flexneri-exposed Caco-2 cells. Although SIgA alone dis-played the capacity to upregulate the formation of NF-B-based complexes in EMSA, quantification of fold-inductionvalues indicated a synergic, rather than additive, effect of theAb(Fig. 4A, top of gels). We conclude that in contrast to entero-pathogenic S. flexneri, exposure to LPR and BL appears toinduce limited DNA binding by the transcription factor innuclear extracts, a fine-tune effect that can be further modu-lated upon association with SIgA.
FIGURE 2. Laser-scanning confocal microscope imaging of the Fab-inde-pendent association of SIgAC5:Cy3 with LPR and BL. Bacteria are visual-ized by differential interference contrast (DIC, Nomarski), and bound Abshows as co-localizing red fluorescent spots on the surface of bacteria. For-mation of small strings is typical of the morphology of the microorganismstested. One representative field obtained from 10 different observations afteranalysis of five different slides is shown. Bars: 5 m.
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Exposure to BL Prevents Degradation of the NF-B InhibitorIB in Polarized Caco-2 Epithelial Cell Monolayers—Toinvestigate at which level in the pathway of NF-B activationthe microbial-epithelial interaction exerts its effect, the regula-tion of the NF-B inhibitory molecule IB was examined incytoplasmic cell extracts (Fig. 4B, upper panel). Constantamounts of IB were detected by immunodetection in lysatesof Caco-2 cells incubated with BL, BL-SIgA, or SIgA. This con-trasted with the substantially reduced intensity of IB signalresulting from the analysis of Caco-2 cell lysates exposed toS. flexneri used as a positive trigger of the NF-B pathway inepithelial cells. The inverse correlation between the levels of
IB and NF-B-DNA probe com-plexes was indicative of differentdegrees of NF-B activation thatcould be finely dissected as a func-tion of the bacterium or complexestested. Occurrence of limitednuclear translocation of NF-B fol-lowing incubation with BL was fur-ther demonstrated upon directanalysis of the NF-B subunits p50and p65/relA (Fig. 4B, comparewiththe lane S. flexneri). The sum ofthese data indicated that theSIgA-BL complex (and LPR-SIgA,data not shown) adhering to the api-cal surface of polarized Caco-2 cellmonolayers was moderately activa-tory of the signaling pathway(s)leading to NF-B nuclear transloca-tion. Reinforced interaction medi-ated by SIgA increased furtherNF-B nuclear translocation, sug-gesting that “sensing” by Caco-2cells is partly controlled by the pres-ence of the Ab.LPR and BL in Complexes with
SIgA Induce pIgR Up-regulation inPolarized Caco-2 Epithelial CellMonolayers.—We then investigatedthe possible impact of the exposureof Caco-2 cells to LPR, BL, or asSIgA-based complexes, on pIgRproduction known to be involved inmucosal homeostasis (23). Cellextracts were prepared after over-night incubation and lysates wereanalyzed by immunodetection (Fig.5A). The pIgRprotein (120 kDa)waspartly converted into SC (85 kDa), afeature that reflects the rapid cleav-age of pIgR at the cell surface (24).For standardization purposes, de-tection of -actin protein was car-ried out in parallel. Densitometricanalysis relative to -actin revealedthat, in comparison with medium,
BL alone showed a slight beneficial effect on pIgR produc-tion (p 0.041), in contrast to LPR (p 0.080) (Fig. 5B).Comparison with bacteria alone showed that both LPR-SIgAand BL-SIgA immune complexes increased production ofthe pIgR/SC protein in a significant manner (p 0.0019 andp 0.0011, respectively) (Fig. 5B). SIgA alone did not haveany effect on pIgR production, whereas enteropathogenicS. flexneri used as a positive control promoted production ofthe pIgR protein by a factor of almost 4 (Fig. 5B). The abso-lute quantity of pIgR in cell lysates was measured by ELISA atdifferent time points (Fig. 5C). We found that BL in combi-nation with SIgA yielded 6.1 ng of cell-associated pIgR/mg of
FIGURE 3. A, TER of epithelial Caco-2 cell monolayers exposed to 2 107 LPR alone, 2 107 BL alone, and incomplexes with SIgA, determined at five time points. Description of symbols is given in the inset. SIgA usedalone serve as control of the stability of the Caco-2 cell monolayer TER. Compilation of data from four inde-pendent experiments performed in triplicates is shown. Codes for symbols: light blue, LPR; dark blue, LPR SIgA; pink, BL; purple, BL SIgA; black, SIgA alone. B, overnight exposure of polarized Caco-2 cell monolayers to2 107 LPR or LPR-SIgA complexes increases phosphorylation of the tight junction proteins occludin and ZO-1.Cell lysates were immunoprecipitated (IP) with specific Ab, and detected by Western blot (Wb) with anti-phosphotyrosine, anti-phosphoserine, and Ab to occludin and ZO-1. C, quantification of phosphorylatedoccludin (P-occludin) and phosphorylated ZO-1 (P-ZO-1) after exposure of Caco-2 cells to 2 107 LPR, 2 107
BL, and in complexes with SIgA. Data were obtained from three independent experiments performed in trip-licates. Comparative statistical analysis with the bar marked plain medium yielded p values 0.002 for allexperimental groups.
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total protein, while SIgA-LPR complex led to the productionof 2.9 ng of pIgR/mg of total protein. Production did notchange in a significant manner between 16 and 24 h of incu-bation. S. flexneri used as a positive control led to productionof up to 10 ng pIgR/mg of total protein. These results indi-cate that the interaction between a probiotic-SIgA complexand epithelial Caco-2 cells prompts these latter to synthesizemore pIgR involved in mucosal defense.
FIGURE 4. Effect of LPR and BL818 along the NF-B activation pathway.A, electrophoretic mobility shift assay performed with nuclear extractsfrom polarized Caco-2 cell monolayers incubated for 16 h with 2 107 LPRalone, 2 107 BL alone, or in association with SIgA, as indicated at thebottom of the lanes. Comp corresponds to a 20-fold molar excess of unla-beled NF-B oligoNT probe to identify the specific retarded complex.NF-B-probe complexes obtained when using nuclear extracts from cellsstimulated with 2 107 enteropathogic S. flexneri are shown for compar-ison. B, immunoblotting of IB in cytoplasmic extracts from Caco-2 cellsincubated as in A with BL and Ab, or S. flexneri for comparison (upperpanel). Nuclear translocation of NF-B subunits p50 and p65/relA inducedby the contact of Caco-2 cells with 2 107 BL alone and in complex withSIgA, or 2 107 S. flexneri for comparison (lower panels). Immunoblottingwas carried out on Caco-2 cell nuclear extracts. Identical amounts ofnuclear and cellular extracts based on protein concentration were usedfor each set of experiments. Panels are representative of one individualtriplicate experiment performed three times. FIGURE 5. A, Western blot analysis of lysates recovered from polarized Caco-2 cell
monolayers exposed to 2 107 LPR alone, 2 107 BL alone, and in the form ofcomplexes with SIgA, as indicated on the top of the lanes. Detection was per-formed with rabbit anti-serum against SC recognizing both SC and the precursorpIgR. Signals caused by -actin were obtained using a specific antiserum. B, den-sitometric analysis of two independent experiments was carried out with stan-dardization based on the -actin signal. The lane content is indicated below theplot. Significant statistical differences are indicated above the lanes. C, quantifica-tion of pIgR and converted SC was assessed by ELISA and is reported as a functionof the amount measured expressed in ng per mg of protein in Caco-2 cell lysates.Data were gathered from three independent experiments performed in tripli-cates. Codes for symbols: light blue, LPR; dark blue, LPR SIgA; pink, BL; purple,BL SIgA; black, SIgA alone; circle, plain medium; diamond, S. flexneri.
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LPR Alone, BL Alone, or in Complexes with SIgA PromoteTSLP Up-regulation in Polarized Caco-2 Epithelial CellMonolayers—Production of chemokines in the basolateralcompartment of polarizedCaco-2 cellmonolayers was assessedby ELISA after overnight incubation with bacteria in the pres-ence or absence of SIgA (Table 1). Upon incubation with eitherbacterium alone or in complex with SIgA, the level ofmeasuredCXCL-8 involved in recruitment of monocytes/neutrophils inepithelia was close to that obtained using plain cells, and sub-stantially below that obtained after incubationwith pro-inflam-matory S. flexneri (p 0.0002). Secretion of TSLP known toplay an important role in maintaining an intestinal noninflam-matory environment (25) favorable to the development ofsemi-mature dendritic cells (26–28) was increased after expo-sure to LPR (3-fold) and BL (4-fold) in comparisonwith controlcells (p 0.008). The release of TSLP was further enhanced(2-fold) when the bacteria were added in combination withSIgA (p 0.002), most likely a consequence of the better adhe-sion observed previously (Fig. 1). Production of bothmediatorsof neutrophil and monocyte recruitment CCL-5 (RANTES)and CCL-2 (MCP-1) by cells exposed to LPR and BL, or SIgA-based complexes thereof, were below the level of detection, incontrast to high levels obtained after incubationwith S. flexneri.Altogether, these results demonstrate a specific non-inflamma-tory type of response of Caco-2 cells in interaction with probi-otics and SIgA-based complexes.
DISCUSSION
The role of probiotic and commensal bacteria in the physiol-ogy of the gastrointestinal tract is incompletely understood,mostly because of the complex and intricate array of cellularandmolecular partners involved. In particular, themechanismsunderlying the beneficial effects of probiotics, as well as theimpact on epithelial maturation and communication with themucosal immune system are still in need of investigation. Usingpolarized Caco-2 cell monolayers, we found that incubationwith the probiotics LPR and BL led to modifications of severalfeatures including adhesion, permeability, and signaling eventsinvolved in NF-B nuclear translocation, production of pIgR,and induction of immunemediators. The further novelty of ourdata resides in the demonstration that binding of nonspecificSIgA to bacteria potentiates their effect on selected events asso-ciated with adhesion and cell signaling, reflecting that differentsensing pathways could be identified in the in vitro setting usedinhere.Preferential adhesion of LPR, BL, and Nissle 1917 probiotics
strains to Caco-2 cell monolayer in comparison with E. coli
TG1 is in agreement with previous studies using similar in vitrosystems. Our data allowed us to demonstrate that SIgAincreased adhesion toCaco-2 epithelial cells grown as polarizedmonolayers. Such an effect can find an explanation in the sur-face expression of epithelial CD71 (the transferrin receptor),which exhibits SIgAbinding properties (29), as anchoring of theAb through mucin cannot occur in Caco-2 cells unable to pro-ducemucus. Human SIgA purified from colostrum, when com-bined with bacteria, displayed the same potentiating effect onadhesion to polarizedCaCo-2 cellsmonolayers.3Unexpectedly,enhanced adhesion did not translate into further increasedTER, which ranged in between 18–25% for all conditionstested. This supports the hypothesis that SIgA-mediatedenhanced adhesion of bacteria to Caco-2 cells does not neces-sarily improve permeability, as confirmed by the stability inTER observed upon incubation of Caco-2 cells with 5 times lessor 5 timesmore bacteria. Consistent with this, phosphorylationof ZO-1 and occludin linked to epithelial tightness was thesame for bacteria and SIgA-based complexes, suggesting thatimproved adhesion to Caco-2 cells might have consequences inother pathways, pathways we identified in this work.Infection of epithelial cells with pathogenicmicrobes includ-
ing S. flexneri induces rapid degradation of IB, resulting inthe release of the NF-B complex that translocates to thenucleus where it triggers transcription of a variety of genesrequired for immune responses (30). In contrast, exposure toLPR or BL only partially induced nuclear translocation of thetranscription factor, an effect potentiated by SIgA. This indi-cates that exposure of epithelial cells to certain probioticmicro-organisms such as LPR and BL maintains a low, if not basal,degree of NF-B activation that may be instrumental for themaintenance of homeostasis (31). This is with keeping in mindthat although a central regulator in gene expression,NF-Bactsin concert with a plethora of other transcription factors to pro-mote optimal and cell-specific transcriptional control of multi-ple target genes. Consistent with this, induction of NF-B afterexposure to BL or SIgA-probiotic complexes did not lead to theproduction by Caco-2 cells of chemokines involved in therecruitment of pro-inflammatory cells. However, production ofTSLP involved in maintaining an intestinal noninflammatoryenvironment (25) was promoted byCaco-2 cells incubatedwitheither LPR or BL, and significantly further enhanced whencomplexed with SIgA.Efficient transport of IgA from the lamina propria into
mucosal secretions is mediated by pIgR produced by epithelialcells (23). Up-regulation of intestinal pIgR mRNA expression
TABLE 1Analysis of chemokines released basolaterally by polarized Caco-2 cell monolayers exposed to LPR alone, BL alone, or in complexes with SlgA, with exposure to S. flexneriused as a positive control. Values are expressed in pg/ml S.D. of chemokine measured in the basolateral compartment of Transwell filters. CXCL-8, interleukin-8; TSLP,thymic stromal lymphopoietin; CCL-5, RANTES; CCL-2, MCP-1. Data were gathered from three independent experiments performed in triplicate.
CXCL-8 TSLP CCL-5 CCL-2SlgA SlgA SlgA SlgA SlgA SlgA SlgA SlgA
Caco-2 12 4 12 3 19 3 13 6 2 2 2 2Caco-2 LPR 19 1 20 3 62 6a 144 26b 2 2 2 2Caco-2 BL 17 9 18 2 84 7a 169 28b 2 2 2 2Caco-2 S. flexneri 2000 79 N.D.c 33 11 N.D. 347 23 N.D. 478 57 N.D.
a Significant statistical differences between cells exposed to medium or bacteria alone are highlighted in bold.b Significant statistical differences between cells exposed to the bacteria alone or as SlgA-based complexes are highlighted in bold.c N.D., not determined, as the SlgA used in the assays neutralizes S. flexneri.
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has been reported in formely germ-freemice colonizedwith thecommensal Bacteroides thetaiotamicron (32). In comparisonwith LPR or BL alone, we found that exposure of Caco-2 cellmonolayers to SIgA-probiotic complexes triggered productionof the pIgR protein. Increased production of pIgR that in turnwould augment IgA translocation can be seen as an additionalstep in controlling the microbiota through a regulatory loopmechanism implying both innate and adaptive immunity.Moreover, the data presented provide a mechanistic expla-
nation to the underestimated function of natural SIgA in theregulation of the endogenous microbiota, both preweaningthrough maternal Ab, and after weaning through early, lowaffinity, Ab responses induced in the neonates (7). Indeed, byintervening in the complex interactions governing host-mi-crobe cross-talk, SIgAmay contribute to regulate epithelial cellresponses to their environment. Maternal SIgA, by coating gutcommensal bacteria in the neonatal intestine, would controlinitial exposure to the developing immune system and thussubsequentmaturation. Thismay keep the level of gut-coloniz-ing bacteria at bay until sufficient amounts of endogeneousSIgA can be produced by the neonate at the time of weaning.“Natural” existence of bacterium-SIgA complexes anytime
may contribute to maintain commensals in close associationwith the epithelium, and guarantee self-limiting control ofmicroorganisms permanently colonizing the gut (33). We pos-tulate that such a monitoring mechanism would promote opti-mal gutmicrobial colonization and ensures a dynamic and plas-tic interplay with epithelial cells. This adds to the alreadydocumented role of SIgA in limiting dissemination of microor-ganisms in the gastrointestinal lymphoid tissue (17, 34), and theimmunomodulatory properties of SIgA in the intestine (35–37). Together, this will result in the onset of modulatory path-ways crucial to maturation of both the epithelium and innateand adaptive immunity.Our results further unravel that intimate association of
microorganisms with SIgA potentiates the communicationwith epithelial monolayers to different degrees, as reflected bythe observation that not all features examined were subject tochanges, and that differences between Lactobacillus LPR andBifidobacterium BL were indeed identified. The observationthat the mucus-binding protein of another Lactobacillus spe-cies exhibits IgG and IgA binding activity might suggest a trackto explore to better understand the complexity of interactionsthat ultimately lead to gut and mucus adhesion (38). Mainte-nance of intestinal integrity and proper functioning requiresthat harmonious microbial-epithelial interactions occur, andour novel data reveal the functional importance of SIgA in par-ticipating to this complex homeostatic balance.
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Epithelial Cell Responses to Commensals Bound to SIgA
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2.2 Recognition of Gram-positive intestinal bacteria by hybridoma- and colostrum-
derived secretory immunoglobulin A is mediated by carbohydrates.
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Recognition of Gram-positive Intestinal Bacteria byHybridoma- and Colostrum-derived SecretoryImmunoglobulin A Is Mediated by Carbohydrates*
Received for publication, December 3, 2010, and in revised form, March 9, 2011 Published, JBC Papers in Press, March 21, 2011, DOI 10.1074/jbc.M110.209015
Amandine Mathias and Blaise Corthesy1
From the R&D Laboratory of the Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois, Rue du Bugnon,1011 Lausanne, Switzerland
Humans live in symbiosis with 1014 commensal bacteriaamong which >99% resides in their gastrointestinal tract. Themolecular bases pertaining to the interaction between mucosalsecretory IgA (SIgA) and bacteria residing in the intestine arenot known. Previous studies have demonstrated that commen-sals are naturally coated by SIgA in the gut lumen. Thus, under-standing how natural SIgA interacts with commensal bacteriacan provide new clues on its multiple functions at mucosal sur-faces. Using fluorescently labeled, nonspecific SIgA or secretorycomponent (SC), we visualized by confocal microscopy theinteraction with various commensal bacteria, including Lacto-bacillus, Bifidobacteria, Escherichia coli, and Bacteroidesstrains. These experiments revealed that the interactionbetween SIgA and commensal bacteria involves Fab- and Fc-independent structural motifs, featuring SC as a crucial part-ner. Removal of glycans present on free SC or bound in SIgAresulted in a drastic drop in the interaction with Gram-positivebacteria, indicating the essential role of carbohydrates in theprocess. In contrast, poor binding of Gram-positive bacteria bycontrol IgG was observed. The interaction with Gram-negativebacteria was preserved whatever the molecular form of proteinpartner used, suggesting the involvement of different bindingmotifs. Purified SIgA and SC fromeithermouse hybridoma cellsor human colostrum exhibited identical patterns of recognitionfor Gram-positive bacteria, emphasizing conserved plasticitybetween species. Thus, sugar-mediated binding of commensalsby SIgA highlights the currently underappreciated role of gly-cans in mediating the interaction between a highly diversemicrobiota and the mucosal immune system.
Humanmucosal surfaces comprising themouth, respiratory,digestive, and urogenital tracts represent 400 m2, i.e. 200times more than the global skin area. The human gastrointes-tinal tract is peacefully colonized by a large ecosystem esti-mated to belong to1800 genera, which represents1014 bac-teria, exceeding by more than 10 times the body cells (1, 2).Overall, the intestinal immune system has the dual task to pro-tect the sterile core of the organism against invasion and dis-
semination of pathogens and maintain a peaceful relationshipwith commensal microorganisms. To preserve mucosal homeo-stasis, a complex communication needs to be establishedbetween a narrow triptych: the microbiota, the epithelial cells,and the mucosal immune system.Secretory IgA (SIgA)2 produced by plasma cells in the lamina
propria represents the major immunoglobulin found at muco-sal surfaces. The protective role of SIgA has been well estab-lished in the context of infection where the antibody (Ab) actsas a first line of defense through bacterial coating, thus largelypreventing attachment to epithelial surfaces and resulting in aprocess referred to as immune exclusion (3). In contrast, tomaintain an abundant andwell balanced gutmicrobiota, such aclearance mechanism must be limited to a level guaranteeinghomeostasis. Evidence is accumulating that emphasizes a com-plex cross-talk between the epithelium and microbiota thattriggers SIgA secretion in the gut lumen of neonates already (4,5). In contrast, SIgA production is reduced at barely detectablelevel in germ-free animals, whereas normal values of IgA can bereached within a few weeks following intestinal recolonizationwith various microbiotas (6–8). Recently, data demonstratedthe induction of strain-specific SIgA secretion following rein-troduction of Enterobacter cloacae in the gut of specific patho-gen-freemice, indicating a direct impact of this microorganismon the subjacent immune cells (9). Furthermore, SIgA has beendescribed to promote biofilm formation at the gut surface,underlying a straight relationship linking mucosal Abs and thegut microorganisms (10, 11). However, the molecular mode ofaction of SIgA in regulating microbiota colonization remainsenigmatic. One can speculate that interaction between SIgAand commensals plays a role in modulating the colonization bythe microbiota in steady-state conditions. Moreover, in vivocoating of commensal bacteria by SIgA has been described inanalysis of human feces (12, 13). Because abundant intestinalsecretion of natural SIgA with unknown specificity has alsobeen described, we speculated that the latter can be involved inbinding to commensals (8, 14–16).SIgA is mostly composed of dimeric IgAmade of twomono-
mers linked together with J chain and secretory component
* This work was supported by Swiss Science Research Foundation Grant3200-122039.
1 To whom correspondence should be addressed: R&D Laboratory, Dept. ofImmunology and Allergy, University State Hospital (CHUV), Rue du Bugnon46, 1011 Lausanne, Switzerland. Tel.: 0041-21-314-07-83; Fax: 0041-21-314-07-71; E-mail: [email protected].
2 The abbreviations used are: SIgA, secretory IgA; Ab, antibody; BL, Bifidobac-terium lactis; Bt, B. thetaiotaomicron DSM 2079; Cy3, indocarbocyanine-3;D2241, E. coli strain D2241; dg suffix, deglycosylated; hSC, human SC; LPR,Lactobacillus rhamnosus; LSCM, laser scanning confocal microscopy; mSC,mouse SC; Nissle, E. coli strain Nissle 1917; pIgA, polymeric IgA; SC, secre-tory component; SCcol, colostrum-derived SC; SIgAcol, colostrum-derivedSIgA; ST11, Lactobacillus paracasei.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 19, pp. 17239 –17247, May 13, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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(SC), the extracellular degradation product resulting fromcleavage by the epithelial cells of the precursor polymeric Igreceptor ensuring transcytosis of the Ab (17). In secretions, SCis bound covalently, as well as noncovalently, to IgA, and isfound also as free SC (18). Both polymeric IgA (pIgA) and SCare heavily glycosylated (19, 20); remarkably, N- and O- carbo-hydrate residues have already been implicated in the interac-tion with bacteria, participating in the protection againstenteric pathogens (20–25) either as adhesion competitors or inanchoring SIgA in themucus to ensure optimal biological func-tion. In face of the increasing involvement of glycans in thefunction of SIgA, we sought to determine their potential role incommensal binding (25, 26). As substitute of natural SIgA andSC, we used nonspecificmouse hybridoma-derived SIgA, pIgA,and free SC, as well as their deglycosylated counterpart, toexamine the role of carbohydrates in binding to a selectionof bacteria (Lactobacillus, Bifidobacteria, Bacteroides, or Esch-erichia coli strain). The results obtained with recombinant pro-teins were validated with SIgA and SC purified from humancolostrum.
EXPERIMENTAL PROCEDURES
Microorganisms and Growth Conditions—Lactobacillusparacasei ST11 (NCC2461 provided by Nestle Research Cen-ter, Lausanne, Switzerland) and Lactobacillus rhamnosus LPR(NCC4007 provided by Nestle Research Center, Lausanne,Switzerland) were grown inMan-Rogosa-Sharpe broth at 37 °Cwithout shaking (27). Bifidobacterium lactis BL818 (BL)(NCC2818 provided by Nestle Research Center, Lausanne,Switzerland) was cultured in Man-Rogosa-Sharpe comple-mented with 0.05% L-cysteine under microaerophilic condi-tions (AnaerogenTM; Oxoid) (28). E. coliD2241 (29) and Nissle1917 (Nissle) (27) strains were grown in brain heart infusionbroth at 37 °Cwith shaking.Bacteroides thetaiotaomicronDSM2079 (Bt) was culture in anaerobic conditions as described (30).Microorganisms from frozen stocks were incubated in theirrespective growth media for 16 h, with the exception of Bt keptfor 48 h. Bacteria were washed three times in phosphate-buff-ered saline (PBS) by successive centrifugations at 2000 g for 5min. Assessment of colony-forming unit (cfu)/ml was obtainedby multiplying the optical density (O.D.) measured at 600 nmby their respective conversion factor: one O.D. corresponds to1 108 cfu/ml for Lactobacillus strains, 1.5 108 cfu/ml forE. coli strains, and 5 107 cfu/ml for Bifidobacteria. Bt wasnumbered in Neubauer chambers.Cell Lines and Protein Production—Hybridoma cells produc-
ing IgAC5 (31) or IgGC20 (32) specific for Shigella flexneri sero-type 5a LPS, IgASal4 specific for Salmonella typhimurium LPS(33), and IgAHNK20 specific for the respiratory syncytial virusfusion (F) glycoprotein (34) were cultured in RPMI 1640medium supplemented with 10% fetal calf serum, 2 mM gluta-mine, 2 mM sodium pyruvate, 10 mM HEPES (pH 7.0), 0.1 mM
folic acid, 100 units/ml penicillin, and 100 g/ml streptomycinas described (31, 32). Cell cultures were conducted in Celline-350 bioreactors (Intregra Biosciences AG). Supernatants wereharvested twice a week, filtered through 0.22-m cartridges,and separated by size-exclusion chromatography to recoverpIgA from other molecular forms and undesired ingredients
(35). Fractions containing pIgA forms were pooled, concen-trated using a Pellicon XL filter unit (100-kDa cut-off; Milli-pore) coupled to a Labscale system (Millipore), and finallystored at 4 °C. IgGC20 was purified by affinity chromatographyusing protein G-Sepharose 4 fast flow beads (GE Healthcare)according to the manufacturer’s instruction. Prior to storage at4 °C, the sample buffer was exchanged against PBS by filtrationover an Amicon Ultra 100K cartridge (Millipore). Mouse SC(mSC) was produced and purified as described (36). The puri-fied protein was stored in PBS at 4 °C until use. The bicin-choninic acid protein assay kit (Pierce) was used for proteinmeasurement. Fluorescent molecules (pIgA, IgG, mSC) wereobtained using the FluoroLink mAb indocarbocyanine-3 (Cy3)labeling kit (Amersham Biosciences) according to the proce-dure provided by themanufacturer. SIgA-Cy3were obtained bycombining pIgA-Cy3 molecules with mSC (5/1 (w/w)) in PBSaccording to the conditions defined in Crottet and Corthesy(37). The effective reassociation between pIgA and mSC pro-teins was checked by SDS-PAGE under nonreducing condi-tions using sera specific for mouse chain, J chain, or SC (38).Preparation of SIgA and SC from Colostrum—Aliquots of
individual frozen human colostral samples were centrifuged at13,000 g for 20min at 4 °C to remove lipids and cells (39). Thelower layer containing proteins was recovered and sterile-fil-tered. Delipidized colostrum was applied on a Superdex 200column (100 2.6 cm; GE Healthcare) equilibrated in PBS.Colostrum-derived SIgA (SIgAcol) and SC (SCcol) were inden-tified, respectively, in the first and the third eluting peaks andcomigrated with leftovers of unresolved IgG and lactoferrin.Pooled fractions were concentrated using Amicon Ultra 30Kcartridge to reach a final volume of 10 ml. Contaminating IgGwas removed using batch incubation with 2 ml of proteinG-Sepharose 4 fast flow beads (GE Healthcare) equilibrated inPBS; SIgA and SC were collected as unbound material. Thebuffer of the SIgA or SC solutions was exchanged for 20 mM
sodiumphosphate buffer (pH7.3) using filtration throughAmi-con Ultra 30K cartridge. To get rid of residual lactoferrin, theSIgA and SC materials were then fractionated, respectively,onto 2 ml of Q-Sepharose fast flow beads (GE Healthcare) andSP-Sepharose fast flow (GE Healthcare) equilibrated in 20 mM
sodium phosphate buffer. Stepwise elution with a range of KClconcentration (0.05–0.5M)was performed, allowing for elutionof SIgAcol between 0.05 M and 0.2 M KCl and SCcol between0.05 M and 0.1 M KCl. Pooled SIgAcol- or SCcol-containingfractions were desalted by addition of 10 volumes of PBS andconcentrated using an Amicon Ultra 30K cartridge prior tostorage at 4 °C. To ensure that Q- and SP-chromatographyproperly separated them, the resulting absence of lactoferrinwas assessed by ELISA (Lactoferrin ELISA kit; Calbiochem).The integrity of recovered SIgAcol and SCcol was checked bySDS-PAGE under nonreducing conditions. SIgAcol and SCcolwere labeled with Cy3 as described above for hybridoma-de-rived proteins.Protein Deglycosylation—Five units of N-glycosidase F (EC
3.5.1.52; Roche Applied Science) were added to either 5 g ofreassociated SIgA-Cy3 or 1 g of mSC-Cy3 and incubated at37 °C for 4 h under gentle agitation, resulting in deglycosylatedSIgAC5 (SIgAC5dg), deglycosylated SIgASal4 (SIgASal4dg),
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and deglycosylated mSC (mSCdg), respectively. Deglycosyla-tion was examined by SDS-PAGE under reducing conditions,and immunodetection was carried out using antisera againsteither chain or SC. To obtain fully deglycosylated SIgAcol(SIgAcoldg) or deglycosylated SCcol (SCcoldg), further addi-tion of 5 units of N-glycosidase F was carried out after 3 h ofincubation, and the mixture was incubated for 3 more hours.Protein Analysis—2 g of protein was used for silver staining
(35), 200 ng for chain detection, and 600 ng for other detec-tions. Samples weremixed with gel loading buffer (100mMTrisbase, 4% SDS, 0.2% bromphenol blue, and 20% glycerol), and forreducing conditions, dithiothreitol (DTT; Applichem) wasadded to a final concentration of 100 mM. Samples were heatedto 95 °C for 3 min and applied onto polyacrylamide gels ofappropriate polyacrylamide percentages as indicated in eachfigure legend. Western blot assay was performed as described(38), with proteins detected by incubation for 1 h with the fol-lowing primary and secondary Abs in PBS containing 0.05%Tween 20 and 0.5% nonfat dry milk: goat anti-mouse chain(1/3000; Sigma), goat anti-human chain (1/3000;Cappel), andrabbit anti-goat IgG conjugated to horseradish peroxidase(HRP) (1/3000; Sigma); rabbit anti-human chain (1/2000;Dako), rabbit anti-human SC (hSC; 1/3000; Dako), rabbit anti-mSC (1/3000) (36) or rabbit anti-J chain (1/1000) (40) followedbyHRP-conjugated goat anti-rabbit IgG (1/5000; Sigma); rabbitanti-human chain HRP-conjugated (1/5000; Dako). Afterfinal washing in PBS containing 0.05% Tween 20, proteins weredetected by chemiluminescence using Uptilight detection kit(Interchim) and exposed on autoradiographic films (Konica).Protein Association with Bacteria—2 107 bacteria were
mixed with 200 ng of SIgA, SIgAdg, or pIgA, or with 40 ng ofmSC and mSCdg, in a final volume of 400 l of PBS and incu-bated for 1 h at room temperature under gentle agitation. Bac-teria-protein complexes were washed three times in PBS, laidonto 8-well slides (Marienfeld), fixed in 2% paraformaldehydein PBS for 25 min, and mounted in Vectashield (Vector Labo-ratories). Complexes were observed using a Zeiss LSM 510Meta confocal microscope (Carl Zeiss) with a 63 objective(Cellular Imaging Facility, Lausanne University, Switzerland)and processed using the Zeiss LSM 510 Meta software.Quantification of Bacterial Coating by Antibodies—Quanti-
fication of bacterial coating by antibodies was performed withImageJ software 1.41 (National Institutes of Health). We mea-sured on each series of pictures the area covered by bacteria(bacterial area) using the differential interference contrastchannel. In parallel, in each area associated with bacteria, wequantify the area containing Ab- or SC-linked pixels with afluorescence intensity superior to 15 units (fluorescent area)defined in our experimental settings as the background signal(signal range extends from 0 to 256 units using transformationinto 8-bit gray scale). The following formula was used to quan-tify the percentage of bacterial surface coveredwith fluorescentmolecules: 100 (fluorescent area)/(bacterial area).Statistical Analysis—The results are given as means S.E.
Two-tailed nonparametric Mann-Whitney U test analysis wasperformed using the GraphPad 5 Prism software. Differenceswere considered as significant when p values 0.05 wereobtained.
RESULTS
SIgA Binds Bacteria in a Fab- and Fc-independent Manner—Coating of bacteria has been previously observed in feces sam-ples recovered from human andmouse (12, 41), suggesting thatthis takes place at all times in the gut lumen. We made theassumption that nonspecific SIgA serving as substitute of nat-ural SIgA can bind to commensals in a Fab-independent fash-ion.We tested this hypothesis by incubating in vitro a battery of
FIGURE 1. LSCM imaging of the association between fluorescentlylabeled proteins and LPR. Colocalization of bacteria (visualized by differ-ential interference contrast) with nonspecific proteins including SIgAC5,SIgASal4, SIgAHNK20, mSC, and IgGC20 (seen as red dots) is shown. Con-trol panels are obtained upon differential interference contrast (DIC)-me-diated visualization of LPR alone. One representative field obtained from10 different observations following analysis of 5 different slides isdepicted. Scale bars, 10 m.
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bacterial strains including Gram-positive LPR, ST11 and BL, aswell as Gram-negative E. coli strains D2241 andNissle andBac-teroides Bt with fluorescently labeled reconstituted SIgA, pIgA,SC, and IgGC20. Panels of Fig. 1 show the data obtained withLPR; similar, if not identical results were produced with thestrains ST11 and BL. Complexes with bacteria were detectedby laser scanning confocal microscopy (LSCM) after 1 h ofincubation in the presence of either SIgAC5, SIgASal4, orSIgAHNK20 (Fig. 1). Bacteria were almost entirely coated bynonspecific SIgA molecules, indicating that the formation ofcomplexes relied on Fab-independent binding mechanisms.Interestingly, incubation with SC alone led to the same obser-vation, pointing out that Fab- but also Fc-independent associ-ations sustain the coating of bacteria by SIgA.Nobacterial coat-ing could be detected using pIgA alone (data not shown).Together, this demonstrates the primordial role of SC in theprocess and indirectly indicates that constituents involved inbinding are available in both bound and free SC. Moreover,weak colocalization with IgGC20 demonstrated discriminatingproperties of SIgA and SC in the capacity to coat bacteria andfurther suggests that the variety of glycosylation patternsresults in modulable Fab- and Fc-independent binding proper-ties. Contrary towhatwas observedwith commensal bacteria ofthe Lactobacillus and Bifidobacterium genera, we found thatthe commensal Gram-negative nonpathogenic strains E. coliD2241 and Nissle and of the Bacteroides phylum were recog-nized by IgGC20 to the same extend as with either SIgA ormSC
(data not shown), indicating that different types of interactionsare involved as a function of the bacterial surface.Glycan Residues Are Involved in the Interaction between Bac-
teria and SIgA—Glycosylation is known to be involved in theinnate defense against pathogens (25). Because SIgA and SC arehighly glycosylated proteins (19, 20), we performed deglycosy-lation cleaving all types ofN-branched glycan residues bound toasparagine. Contrary tomost of othermethods,N-glycosidase Fremoves carbohydrate residues by preserving the conforma-tional structure of the protein (23). Clipping of all sevenbranched glycans carried on pIgA-bound or free SC was con-firmed by analysis on polyacrylamide denaturing gels (Fig. 2A).The shift of the apparent molecular mass from 80 kDa down to62–65 kDa is indicative of fully deglycosylated SC (24). In con-trast, carbohydratemoieties present on the chain were insen-sitive to the action of the enzyme, suggesting a nonaccessibleanchoring of the carbohydrates in pIgA (Fig. 2B). Representa-tive pictures of association between deglycosylated proteinsand LPR are depicted in Fig. 2C. Deglycosylation of either freeSC or bound to pIgA led to a drastic drop in colocalization withbacteria: only a few isolated red dots of weak fluorescenceintensity were observed by LSCM. Experiments performedwith ST11 or BL yielded to the same dramatic decline in SIgAand mSC reactivity toward bacteria (data not shown). Takentogether, these data underline the paramount role of glycanresidues and in particular those present on SC in ensuring bac-terial coating by SIgA and SC. In sharp contrast toLactobacillus
FIGURE 2. Impact of SIgA and SC deglycosylation on the association with LPR. A, Western blot analysis of mSC in reassociated SIgAC5, SIgASal4, orSIgAHNK20 and mSC was performed after 0, 2 h, or 4 h of incubation with 5 units of N-glycosidase F (see “Experimental Procedures”). Samples were separatedonto an 8% polyacrylamide gel under reducing conditions. The molecular mass (kDa) of detected protein is marked alongside the lanes. B, immunodetectionwas performed using anti- chain Ab of the same samples as in A. Proteins detected at 62 kDa represent nondeglycosylated polypeptides. C, LSCM imaging ofthe association of LPR formed with native or deglycosylated Cy3-labeled proteins is carried out as in Fig. 1. One representative field obtained from 10 differentobservations after analysis of 5 different slides is depicted. Scale bars, 10 m.
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and Bifidobacterium, interaction of the three tested Gram-neg-ative bacteria with deglycosylated or native proteins resulted insimilar high level binding, indicative of a selective role of car-bohydrates in the binding of Gram-positive bacteria only (datanot shown).Quantitative Analysis of Bacterial Coating by SIgA—LSCM
observations drove us to analyze at the quantitative level thebacterial surface covered by fluorescently labeled proteins.Data in Fig. 3 represent the quantification of the signal associ-ated with Ab- or SC-bacteria complexes using the same set ofbacteria incubated with either SIgA, SIgAdg, pIgA, mSC,mSCdg, or IgGC20. All Gram-positive bacteria mixed withSIgA or free SC resulted in almost full coating by fluorescentlylabeled proteins of LPR (95%) and high values of surface label-ing for ST11 (88.5%) and BL (73.8%) (Fig. 3,A–C). Interestingly,Lactobacillus strains LPR and ST11 displayed the most con-trasted interaction profiles, with deglycosylated moleculesleading to a drop in coating of at least 4-fold, confirming at the
quantitative level the results obtained on Figs. 1 and 2.Although resulting in a less marked 1.7-fold decrease, dataobtained for the Bifidobacterium BL (Fig. 3C) allowed us toreach the same conclusion: glycan residues carried by SC arethe keystone of the SIgA Fab- and Fc-independent coating ofbacteria. In addition, pIgA and SIgAdg exhibited a low level ofcoating in the case of LPR (3.7%), yet remaining close to 20% forST11 and varying from3% to up to 41% for BL. This later observa-tionmay reveal amodulable role for extremely heterogeneous gly-cans present on monoclonal pIgA as a function of the interactingbacterium (Fig. 3, A–C). In sharp contrast, E. coli strains D2241and Nissle, as well as Bt, were coated to at least 63% followingincubationswith all forms of proteins tested (Fig. 3,D–F), indicat-ing differentmolecular patterns involved in binding toGram-neg-ative bacteria. The very similar results obtained with deglycosy-lated and native proteins allowed us to conclude on the absence ofdirect involvement of carbohydrates present on SC in the coatingof Gram-negative bacteria by SIgA or SC.
FIGURE 3. Quantification of bacterial coating by fluorescently labeled proteins. Using ImageJ software, changes in the percentage of bacterial areacovered by nonspecific labeled proteins including SIgAC5, SIgAC5dg, pIgAC5, SIgASal4, SIgASal4dg, pIgASal4, SIgAHNK20, SIgAHNK20dg, pIgAHNK20,mSC, mSCdg, or IgGC20 were assessed in the case of LPR (A), ST11 (B), BL (C), D2241 (D), Nissle (E), and Bt (F) bacterial strains (see “ExperimentalProcedures”). Control quantifications were carried out on pictures obtained with bacteria alone. Bars represent the mean values S.E. Statisticallysignificant differences are indicated above the brackets for intragroup tests: ***, p 0.0001. Data were obtained from 5 different fields of oneexperiment repeated 5 times.
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Purification of SIgA and SC fromHumanColostrum and Bio-chemical Characterization—Colostrum and human milk areprovidential sources of SIgA containing, respectively, up to 12 gand 1 g of the Ab/liter (4, 42). We thus purified SIgA and SCfrom human colostrum to examine the interaction betweennatural SIgA and commensal bacteria. The complete purifica-tion scheme is depicted in Fig. 4A. Silver staining andWesternblot analysis revealed the coelution of SIgAwith lactoferrin andIgG (data not shown), two glycoproteins that can potentiallyinterfere with nonspecific binding properties of SIgA (43). IgGwas removed by affinity chromatography on protein G-Sepha-rose resin, whereas elimination of lactoferrin was achieved byion-exchange chromatographies. Silver staining and Westernblot analysis confirmed the integrity and purity of isolatedSIgAcol and SCcol (Fig. 4, B and C).Bacterial Coating with Natural Purified Colostrum-derived
SIgA—To confirm the validity of results obtained with mousereassociated SIgA, we combined native and deglycosylatedSIgAcol and SCcol with the same set of commensal bacteria asillustrated in Figs. 1 and 2. Treatment of SIgAcol withN-glyco-sidase F resulted in the same deglycosylation pattern of SC asfor mouse hybridoma-derived SIgA, although with some delay
(Fig. 5A), whereas sugars associated with the chain remainedundigested (Fig. 5B). The lag of sugar clipping observed is prob-ably due to slight structural differences between reassociatedSIgA and “naturally” associated SIgAcol in secretions. WhenSIgAcol was incubated with Gram-positive bacteria, we ob-served by LSCM bright dots colocalizing with LPR, whereasdeglycosylated molecules bound only marginally to bacteria(Fig. 5C and data not shown). Quantification of the bacterialpercentage covered by fluorescently labeled proteins confirmedthat the use of SIgAcol, SCcol, and control SIgAC5 resulted insimilar bacterial coating, whereas deglycosylation of colostrum-isolated molecules triggered the loss of efficient binding to Lacto-bacillus LPR (Fig. 5D). The demonstration that either humanSIgAcol or SCcol and reconstituted mouse SIgA molecules yieldsimilar binding data suggests that the heterogeneity of carbohy-drate side chains affords combinational possibilities that are pre-served between species. Selective deglycosylation of SC in bothmurine and human molecules further allows us to conclude as tothe critical roleof sugarmoieties on this polypeptide in the contextof commensal binding by the whole SIgA protein.
DISCUSSION
The intestinal microbiota have been demonstrated to beinvolved in the proper development of the intestinal immunesystem and in particular in the production of a diverse reper-toire of SIgA (4) composed of at least 90% of natural SIgA withlargely unknown specificity (8, 15, 16). The biological functionof such SIgA remains speculative; however, various studies haveunderscored the natural coating of commensal bacteria bySIgA, a process that may be involved in the homeostatic gutsensing of the microbiota (12, 41, 44).Nevertheless, the precise biochemical mechanisms underly-
ing the relation between intestinal microbiota and the immunesystem, in particular, SIgA, remain enigmatic. To tackle thisissue, various strains isolated from the human intestinal tractwere incubated with a battery of nonspecific SIgA, and thenature of the interaction was investigated qualitatively andquantitatively. The use of purifiedmurine SIgA reconstituted inthe test tube or SIgA isolated from human colostrum identifieda Fab- and Fc-independent pattern of bacterial coating. Degly-cosylation of both SC and SIgA allowed us to conclude as to thepivotal role of carbohydrates, and in particularN-branched gly-cans, in the interaction with a selection of Gram-positive bac-teria. Remarkably, SC alone was able to coat such commensalsto the same extent as thewhole SIgA.Moreover, deglycosylatedSC in the context of SIgA prevented bacterial coating by theAb,featuring surface-exposed carbohydrate moieties as crucialpartners in the process of binding.It is conceivable that the association of Gram-positive bac-
teria and SIgA is due to recognition between Ab-boundN-branched glycans and abundant cell wall components suchas peptidoglycans, lipoteichoic acid, or teichoic acid available tothe environment.Lactobacillus lactisharbors a particular, thickouter layer composed of polysaccharides acting as a protectivecapsule (45). This polysaccharide envelope conferring to thebacterium a protective barrier against phagocytosis by murinemacrophages appears to be restricted toGram-positive bacteriaand might as well serve as a particular constituent involved in
FIGURE 4. Purification and characterization of colostrum-derived SIgAand SC. A, fractionation scheme leading to the purification of SIgAcol andSCcol (for details, see “Experimental Procedures”). B, characterization of puri-fied SIgAcol performed by silver staining (lane 1) and by Western blot analysisusing antisera specific for human chain (lane 2), hSC (lane 3), chain (lane 4),and J chain (lane 5). C, characterization of purified SCcol performed by silverstaining (lane 1) and by Western blot analysis using antisera specific for hSC(lane 2). Denaturing 8% polyacrylamide gels were run under nonreducingconditions.
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recognition by SIgA or SC. In support of the importance ofbacterial surface in selective binding, the presence in theouter membrane of lipopolysaccharides in Gram-negativebacteria masking the underlying peptidoglycan layer couldexplain why both “native” and deglycosylated Ab and SCexhibit the same pattern of association. This indirectly sug-gests that other molecular patterns besides glycans on SCcontribute to genera-dependent association, opening a newavenue of research at the interface between immunology andmicrobiology.The different binding pattern between Gram-positive bacte-
ria LPR, ST11, and BL with respect to Gram-negative D2241,Nissle, and Bt revealed a fine tuning of the bacterial coating by
the Ab and SC. Perrier et al. (24) have demonstrated thatrecombinant SC prevents target HEp2 cells from being dam-aged by enteropathogenic E. coli, a property partly lost afterdeglycosylation, indicating an important contribution of carbo-hydrates in this process. These data are in sharp contrast withour results obtained with the nonpathogenic E. coli commensalstrains D2241 and Nissle for which maintenance of bindingcould be observed following SC deglycosylation. Because onlypathogenic E. coli strains including enteropathogenic E. coliexpress outer membrane intimin (46) involved in the interac-tion with glycans present on SC (24), this may explain differ-ences in SIgA binding. The sum of these data implies that theFab- and Fc-independent molecular features responsible for
FIGURE 5. Deglycosylation of colostrum-derived SIgA or SCcol and defective association with LPR observed by LSCM. A, immunodetection of the hSCwas performed after 0, 2 h, 4 h, or 6 h of incubation with 5 units of N-glycosidase F from 0 to 3 h and a further 5 units of enzyme for the last 3 h. Samples wereseparated onto a 10% polyacrylamide gel under reducing conditions. The molecular mass (kDa) of detected polypeptides is marked alongside the lanes.B, samples were separated as in A with immunodetection of the chain. The unique signal detected at 62 kDa represents nondeglycosylated chains. C, LSCMimaging of complexes formed between LPR and native or deglycosylated SIgAcol or SCcol was performed as in Fig. 1. SIgAC5 were used as a positive controlfor the interaction of bacteria with proteins. One representative field obtained from 10 different observations after analysis of 5 different slides is depicted. Scalebars, 10 m. D, quantification of LPR coating by fluorescently labeled proteins was carried out on pictures of LPR alone (control) or pictures of complexes withSIgAC5, SIgAcol, SIgAcoldg, Sccol, and SCcoldg. Bars represent the mean values S.E. Statistically significant differences are indicated above the brackets forintragroup tests: ***, p 0.0001. Data were obtained from 5 to 10 different fields of one experiment repeated 5 times.
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bacterial coating are highly plastic and flexible and result indiverse biological consequences.As SIgA carbohydrates harbor highly heterogeneous struc-
tures, these latter can contribute to combinatory-like, multi-affinity interactions with gut microorganisms (25). This wasillustrated in neonatal mice in which antibody immunity fixedto the hapten nitrophenol protected against bacterial challenge(47). Recently, the assessment of the crystal structure of amucus-binding protein present at the surface of the commensalstrain Lactobacillus reuteri identified motifs with potentialimmunoglobulin binding activity (48). The triad mucus-bind-ing protein–bacteria–SIgA may serve as an organized latticeprecluding massive commensal penetration from the intestinallumen to the underlying tissue, thus ensuring proper host-mi-crobial mutualism. Noteworthy, drug-immunosuppressed andimmunodeficient individuals have increased susceptibility tosepsis or chronic intestinal inflammation mediated by themicrobiota (49, 50), and this holds particularly true for individ-uals lacking SIgA.We found that carbohydrates present on the chain could
not be removed following exposure to N-glycosidase F. Thisobservation is consistent with themasking ofN-glycan residuesby SC in the context of the whole SIgA molecule (20, 51). It isplausible that these noncleavable carbohydrates would beresponsible for the various residual interaction observed uponincubation of Gram-positive bacteria with SIgAdg. However, asany removal of these glycan residues occurs unless harsh con-ditions (use of reducing agents, exposure to heat) are used (52),the function of residual sugarmoieties present on pIgA in bind-ing to bacteria cannot be addressed by this approach.Our study provides strong evidence thatN-branched glycans
present on SCare themain component responsible for Fab- andFc-independent specific binding to Gram-positive commensalbacteria. Due to the presence of LPS on Gram-negative bacte-ria, motifs other than carbohydrates on SIgA account for bind-ing. It is worth mentioning that introduction of commensals isassociated with the induction in the intestine of both strain-specific SIgA (9) and natural SIgAwith unknown specificity (8).Based on these observations, one can argue that SIgA present inhuman colostrum is a mixture of specific SIgA from maternalorigin and natural SIgA. Despite the potential presence of spe-cific SIgA for commensals, identical conclusions were reachedwith SIgA purified from human colostrum, compared withcoating data obtained with monoclonal Ab. Altogether, thiscontributes to explain at the biochemical level the role of colos-trum or milk SIgA with a broad maternal repertoire in shapingthe newborn microbiota before maturation of the mucosalimmune system (4). Commensal coating by colostrum-derivedSIgA would translate into controlled primary gut colonizationand the early education of the newborn immune system towardnovel antigens prone to become symbiotic partners. We pro-pose that coating of commensals by SIgA adds to the function ofmucosal SIgA inmaintaining gut homeostasis, and this processintegrates potentiation of binding to epithelial cells (28), pres-entation under noninflammatory conditions (26), and luminalsequestration or limited penetration of host tissues (53, 54).
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2.3 N-glycans on Secretory Component: mediators of the interaction between SIgA
and Gram-positive commensals sustaining intestinal homeostasis.
Mathias A. and Corthésy B., Gut microbes (In press)
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N-GLYCANS ON SECRETORY COMPONENT: MEDIATORS OF THE INTERACTION BETWEEN SIGA
AND GRAM-POSITIVE COMMENSALS SUSTAINING INTESTINAL HOMEOSTASIS Amandine Mathias, Blaise Corthésy
From the R&D Laboratory of the Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois, Rue du Bugnon, 1011 Lausanne, Switzerland
Address correspondence to: Dr. Blaise Corthésy, R&D Laboratory of the Department of Immunology and Allergy, University State Hospital (CHUV), Rue du Bugnon 46, 1011 Lausanne, Switzerland. Telephone: 0041 21 314 07 83; Telefax: 0041 21 314 07 71;
E-mail: [email protected]
Key words: Secretory IgA, Glycans, Commensal, Homeostasis, Intestinal epithelial cells, Gastro-
intestinal tract, Biofilm.
Abbreviations used: Ab, antibody; GI, gastrointestinal; IEC, intestinal epithelial cell; LPS,
lipopolysaccharide; LTA, lipoteichoic acid; pIgR, polymeric immunoglobulin receptor; PNG,
peptidoglycan; SIgA, secretory immunoglobulin A; SC, secretory component; TA, teichoic acid.
Addendum to: Mathias A, Corthésy B. Recognition of Gram-positive Intestinal Bacteria by Hybridoma-
and Colostrum-derived Secretory Immunoglobulin A Is Mediated by Carbohydrates. J. Biol. Chem.
2011; 286:17239-47. PMID: 21454510; DOI: 10.1074/jbc.M110.209015.
Human beings live in symbiosis with billions of microorganisms colonizing mucosal
surfaces. The understanding of the mechanisms underlying this fine-tuned intestinal
balance has made significant processes during the last decades. We have recently
demonstrated that the interaction of SIgA with Gram-positive bacteria is essentially based
on Fab-independent, glycan-mediated recognition. Both mouse hybridoma- and
colostrum-derived secretory IgA (SIgA) lead to the same conclusion that N-glycans present
on secretory component (SC) are a crucial partner in the process. Natural coating may
involve specific Gram-positive cell wall components, explaining at the molecular level
selective recognition. More widely, the existence of these complexes is involved in the
modulation of intestinal epithelial cell (IEC) responses in vitro and the formation of
intestinal biofilms. Thus, SIgA may act as one of the pillar for homeostatic maintenance of
the microbiota in the gut, adding another facet to its multiple roles in the mucosal
environment.
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SCAdditional N-glycans (SIgA2)
Additional O-glycans (SIgA1)
F(ab’)2 FcJ chain
pIgA
N-glycans
Figure 1. Schematic distribution of carbohydrate residues at the surface of human SIgA.
Additional N- and O-carbohydrates represent sites of glycosylation specifically present on the allotype
IgA1 or IgA2. For a matter of clarity, pIgA and SC have been represented separately. Representation
based on results obtained by Hughes et al. and Royle et al.15, 16
76
Mucosal surfaces are colonized by up to 1014, at first sight, potentially harmful
microorganisms exceeding by more than 10 times the total number of human cells.1-3 These
commensals are beneficial and also indispensable for the body wellness actively
participating in the processes of extraction of energy and digestion of otherwise unavailable
sources of nutrients such as the final degradation of carbohydrates. More interestingly, the
subjacent immune system allows this population to co-exist peacefully in the gut lumen.
Thus, it is easy to figure out how important is the maintenance of the fragile balance
between the microbiota, the intestinal epithelial cells (IEC) lining the gastrointestinal (GI)
tract and the underlying mucosal immune system, all dysfunctions leading to the
development of pathologies such as inflammatory bowel diseases (IBD).4
However, the mechanisms involved in these processes remain in need of
investigation and are an increasing subject of research. Rather than a commensalism type of
relationship, we can speak about symbiosis established between the host and his
microbiota. Commensal bacteria have been directly associated with the proper development
of gut-associated lymphoid tissues such isolated lymphoid follicle (ILF)5 or with the secretion
of normal value of secretory immunoglobulin A (SIgA)6-8, the major immunoglobulin present
at healthy intestinal mucosal surfaces. Following production by plasmocytes of the lamina
propria as polymeric IgA (pIgA) made of two monomers linked together with a joining chain
(J chain), these antibodies (Ab) are recognized by the polymeric Immunoglobulin receptors
(pIgR) present at the basolateral pole of the IEC. After transcytosis through IEC, surface
clipping of the pIgR releases the secretory component (SC) in association with transported
pIgA, resulting in the production of SIgA in the luminal secretions, where it acts as a first line
of defense against pathogens via a process known as immune exclusion.9, 10 As opposite to
gut pathogens, IgA responses against the microbiota need to be restrained to a strict, yet
optimal, minimum to allow properly controlled mucosal colonization. A fully mature mucosa
is characterized by the secretion of 3 to 5 g of SIgA actively transported through the
intestinal epithelium each day.11 This regulatory process does not take place, or at very low
levels, in the intestinal lumen of germ-free mice. Mono-associated colonization of their GI
tract is rapidly followed by the detection of normal values of SIgA and has been further
associated with the secretion of more than 90% of SIgA with unknown specificity called
“natural” SIgA.8, 12-14 Thus, mucosal secretion of SIgA is partially controlled by commensals.
Stoel et al.14 also stressed that the IgA repertoire was restricted to a minimum considering
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+ SIgAC5ST11
Cy3
DIC
Mer
ge
+ SIgAC5BL + SIgAC5D2241
Figure 2. LSCM imaging of the association between fluorescently labeled proteins and selected strains of commensal bacteria.
Colocalization of bacteria (visualized by differential interference contrast (DIC)) with nonspecific SIgAC5
(seen as red dots). Bacterial strains used include the Gram-positive strains Lactobacillus paracasei ST11
and Bifidobacterium lactis BL818 (BL) as well as the Gram-negative strain Escherichia coli D2241. Control
panels are obtained upon DIC-mediated visualization of bacteria alone. One representative field
obtained from 10 different observations following analysis of 5 different slides is depicted.
Scale bars: 10 µm.
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the enormous varieties of antigens encountered at mucosal surfaces, arguing in favor of the
presence of polyspecific, low affinity Ab in intestinal secretions. Furthermore, previous data
have demonstrated that commensal bacteria are naturally coated by SIgA in feces of humans
and mice, indicating that bacterial recognition by the host Ab is most likely necessary to
maintain a steady-state of commensal colonization. In this context, the present article
addendum will focus on the different molecular partners involved in the coating of
commensal by SIgA contributing to the fragile homeostasis between the host and its
microbiota.
N-glycans on SIgA and the interaction with the commensal cell wall: Gram-positive vs
Gram-negative bacteria
Of main importance, SIgA is a highly glycosylated protein comprising sugar-derived
residues in each polypeptide constituting the Ab (Figure 1). With only one N-moiety, the J
chain is the less glycosylated peptide composing SIgA. Carbohydrate residues represent up
to 25 % of the SC molecular mass, with 7 sites of N-glycosylation identified.15 With respect to
pIgA glycosylation, both human IgA1 and IgA2 have two conserved N-glycan sites on each
heavy chain. Moreover, IgA2 harbors one or two additional N-glycans present on the Cα1
domain. IgA1 is the only subclass which presents O-carbohydrates in the hinge region.16
Glycans residues have been increasingly involved in mucosal protection representing a link
between innate and adaptative immunity.16-20 We hypothesized that carbohydrates residues
carried by SIgA may well participate in the non-specific interaction with commensal bacteria
and thus, focused on their putative role in this process.
As a substitute of “natural” SIgA, we chose to use mouse hybridoma-derived
reconstituted SIgA with a known specificity for a pathogen (for example, IgAC5, specific for
Shigella flexneri serotype 5a LPS).21 To examine the role of glycans residues in the interaction
with the microbiota, fluorescently labeled SIgA, free SC and their deglycosylated
counterparts were then combined with a selection of bacteria isolated from human feces
(Lactobacillus, Bifidobacteria, Escherichia coli or Bacteroides strain). The subsequent
formation of complexes between bacteria and SIgA was analyzed by laser scanning confocal
microscopy (LSCM). Both Gram-positive and Gram-negative bacteria were almost fully
covered by fluorescently labeled SIgA as illustrated in Figure 2. Because identical results
were obtained with free SC, these data indicate that not only Fab-, but also Fc-, independent
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Figure 3. Model of the putative interaction profiles between SIgA, SC and components of the bacterial cell wall.
Non-deglycosylated SIgA or SC can interact directly with PNG, LTA or TA characteristic of the
Gram-positive cell wall (); this interaction is reduced at minimal level with deglycosylated proteins ().
The recently identified surface polysaccharide capsule surrounding Gram-positive bacteria is another
potential target for SIgA binding (). On the other hand, the presence of a dense outlayer of LPS in
Gram-negative cell wall prevents SIgA or SC from interacting with the subjacent PNG layer (). No effect
of deglycosylation is detected () indicative of different patterns of interaction involved in
Gram-negative coating. Abbreviations used: SIgA, deglycosylated SIgA; SCdg, deglycosylated SC.
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mechanisms are involved in bacterial coating. Association with Gram-positive commensals
revealed that it was only in the fully glycosylated form that SIgA coating could take place.
Glycan-mediated coating of Gram-positive bacteria was confirmed in experiments showing
that barely detectable interaction occurred with deglycosylated SIgA or SC. Contrary to
results obtained with Gram-positive bacteria, Gram-negative commensals associated with
either SIgA, free SC or their deglycosylated counterparts led to similar patterns of bacterial
coating: bacterial surfaces were almost fully covered by fluorescently labeled proteins. This
suggested different molecular mechanisms involved in the interaction of SIgA with Gram-
negative bacteria.
Interestingly, the pivotal role of N-glycans present at the surface of SC was then
confirmed using SIgA and SC purified from human colostrum. Although SIgA present in
human colostrum is a mixture of specific SIgA from maternal origin and natural SIgA, we
could not detect any difference in the signal intensity associated either with SIgA from
colostrum or with non specific hybridoma-derived SIgA. Thus, even if present at low rates,
potential specific SIgA in maternal colostrum does not interfere with the overall profile of
interaction with the bacteria. These latter results emphasize that both mouse monoclonal
and human polyspecific SIgA further displaying a high variety of carbohydrate side chains,
cover the bacterial surface to the same extent, thus indicating that sugar-mediated
recognition is not jeopardized by species-dependent and thus different patterns.
Distinct interaction profiles between Gram-positive and Gram-negative bacteria and
SIgA can find an explanation in the profound differences existing in the composition of the
bacterial cell wall (Figure 3, compare , with , ).). Gram-positive cell wall is
characterized by the presence of a dense external layer of peptidoglycan (PNG). In contrast,
Gram-negative cell wall is endowed with an additional lipopolysaccharides (LPS) layer which
somehow shields underlying components. It is thus conceivable that carbohydrates of SC
interact directly with the PNG envelop of Gram-positive bacteria (Figure 3, ) whereas in
the presence of LPS on the surface of Gram-negative commensals, identical coating by either
“native” or deglycosylated SIgA or SC occurs, implying that other molecular patterns govern
the direct interaction between cell wall LPS and the antibody (Figure 3, compare with ).
This argues in favor of a biochemical discrimination between Gram-positive and Gram-
negative intestinal bacteria, relying on intricate mechanisms involving glycans on both
interacting partners. Recently, the presence of a polysaccharide capsule surrounding the cell
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wall of the human commensal strain Lactococcus lactis has been described to confer
protection against phagocytosis.22 This envelop which seems to be restricted to Gram-
positive bacteria represents another putative anchoring site for SIgA and SC (Figure 3, ).
Another hypothetical target for the interaction between SIgA and bacterial cell wall is the
recently identified mucus-binding (MUB) protein present at the surface of the commensal
strain Lactobacillus reuteri.23 As the MUB protein has been described to have potential
immunoglobulin binding properties, one can thus speculate that this protein may serve as an
anchoring site for SIgA resulting in the formation of a complex network that would restrict
bacteria-SIgA complexes within the mucus layer covering the epithelium. Moreover, Gram-
positive bacteria harbor a large set of characteristic components within the PNG external
layer comprising lipoteichoic acid (LTA) or teichoic acid (TA) available to interact with
environmental ligands including SIgA (Figure 3, ). LTA and TA derived from the commensal
strain Lactobacillus plantarum have also been involved in the modulation of cell responses
through recognition by the TLR2 and NOD2 expressed on monocytes or macrophages,
inducing the production of the anti-inflammatory cytokine IL-10 and inhibiting the release of
proinflammatory TNF-α favoring intestinal homeostasis.24, 25 The interaction between SIgA
and LTA or TA may further sustain a close relationship between commensal, SIgA and
surrounding IEC or immune cells increasing the immunomodulatory properties of the Gram-
positive bacteria.
Modulation of IEC responses by SIgA-commensal complexes
Recognition of commensal bacteria by IEC has been identified to play a fundamental
role in mucosal homeostasis promoting for example cytokine release, cell expansion and
reinforcement of the barrier integrity.26-28 We have also recently demonstrated that
commensal strain coated by SIgA can potentiate the responsiveness of reconstituted IEC
monolayers in vitro.29 Intuitively, one can think that commensal bacteria associated with
SIgA would have a reduced direct impact on the epithelium through mechanisms similar to
immune exclusion. Unexpectedly, it happens that the presence of SIgA increases the
bacterial anchoring at the apical surface of IEC. At the mechanistic level, reinforcement of
the barrier integrity was characterized by an increased phosphorylation of tight junction
proteins sustaining cell-to-cell interaction. Furthermore, bacteria as a complex with SIgA
induced NF-κB nuclear translocation in a limited manner as compared with activation by the
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Figure 4. Selected roles played by SIgA-coated commensal bacteria in the intestinal homeostasis.
A. IEC pathway of activation. SIgA based-complexes or components of the cell wall can be recognized by
specialized receptors () such as the extracellular toll-like receptor 2 (TLR2) or the intracellular nucleotide-
binding oligomerization domain containing 2 (NOD2) receptor resulting in the activation of the NF-κB
pathway (). Such activation first reinforces the IEC barrier, increasing the phosphorylation of tight
junction proteins () and, secondly, leads to augmented expression of pIgR involved in mucosal defense
() and finally, promotes the secretion of the anti-inflammatory cytokine thymic stromal lymphopoietin
TSLP (). B. Role of SIgA in the formation of biofilm. Together with mucin, SIgA is also described to be
involved in the formation of intestinal biofilm, allowing the peaceful growth of commensal bacteria in close
contact with the intestinal tissue. Whether or not underlying IEC are activated upon biofilm formation has
not yet been studied.
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enteropathogen S. flexneri, suggesting a quenching of proinflammatory responses associated
with this signaling pathway. Consistent with this, reduced NF-κB activation translocated into
the absence of proinflammatory cytokines and the production of thymic stromal
lymphopoietin (TSLP), a chemokine involved in maintaining a noninflammatory environment
at mucosal surfaces (Figure 4, A). Furthermore, association of commensal with SIgA
promoted pIgR production by IEC. As pIgR is involved in transcytosis of SIgA from the
basolateral to the apical pole of IEC, one can conclude on a positive feedback of commensal
bacteria in complex with SIgA on pIgR expression, leading to more receptor available for
active SIgA transcytosis. This phenomenon could account for the sustained SIgA secretion
resulting from commensal colonization as observed decades ago.6-8 The sum of these data
argues in favor of the role of SIgA as a mediator of the fine regulation of bacterial
recognition by IEC. Thus, “sensing” of the bacteria by the epithelium is partially controlled in
presence of the Ab, leading to the reinforcement of barrier function and reduction of non-
inflammatory responses. This contributes to define the function of SIgA in maintaining
commensal bacteria at bay through a delicate balance combining appropriate neutralization
and proper sensing by the IEC. In this context, the role of maternal SIgA has thus a primordial
importance in limiting a potential inflammation induced by primary colonization of the
newborn gut. The presence of SIgA in the colostrum may serve as a first line of interaction
with the newly implanted microbiota and allows proper development of the immune
system. This noninflammatory environment is of first importance when regarding the
development of diseases such as IBD which is, among others, associated with deregulated
inflammatory responses induced by intestinal bacteria.4
SIgA as a scaffold in intestinal biofilm formation
SIgA interaction with commensals has also been described to be involved in biofilm
formation at gut surfaces. The formation of oral biofilm is widely accepted and is known as
dental plaque.30 However, the presence of biofilm formation at gut surfaces is still a matter
of debate. Ones argue that the intestinal microbiota is organized into a network in the
external layer of mucin, the first component of the intestinal mucus, without the formation
of a biofilm31; on the other hand, data are accumulating demonstrating the presence of a
matrix constituted of both bacterial and host components which seems to serve as the
cement of the colonizing microbiota.32 This debate is supported by unsatisfactory
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observations due to difficulties in recovering properly fixed tissues with undamaged mucin
layers. Data essentially based onto electron microscopy observations are now accumulating
demonstrating the presence of a “biofilm-like” covering gut surfaces in specimens of rat,
baboon, and human gut.32 Thus, the most recent emerging hypothesis favors the formation
of different types of biofilms: the “self-supported” biofilms which produce their own
extracellular matrix and the “host-supported” biofilm which are formed via a mutualistic
relationship with the host and that contains a mixture of host and microbial-derived
components.33 The latter situation is particularly well exemplified by the biofilms formed in
the mouth and along the intestine. The evaluation of the covering percentage and the
anatomical distribution are still under investigation34. In vitro models have led to the same
conclusions: the presence of both mucin35 and SIgA coating commensal bacteria36-38 support
the notion of intestinal biofilms (Figure 4, B). SIgA can act either as a scaffold linking bacteria
together or as anchoring loci between the underlying epithelium and bacteria favoring the
transition from free bacteria to a complex biofilm; the two mechanisms are not mutually
exclusive. The possible direct interaction with IEC corroborates our results demonstrating a
better adhesion of SIgA coated bacteria to IEC monolayer.29 The presence of commensal
bacteria as a biofilm may sustain the steady-state presence of bacteria but may also confer a
better resistance to pathogenic implantation, reducing potential niches to a minimal level.
Nevertheless, how underlying cells can sense the formed biofilm remains in need of further
investigation. Recent in vitro data based onto Escherichia coli-derived biofilm also highlight
the impact of SIgA in inducing an increased sensitivity to antibiotic treatment33, possibly via
changes in the organization of the extracellular matrix resulting in increased accessibility of
the antibiotic to mucus-buried bacteria. Interestingly, these new findings open new research
avenues with promising medical implications such as the use of SIgA in the treatment of
biofilm formed onto medical implants, rendering them more sensitive to antibiotic
treatments.
SIgA, as an important player regulating intestinal homeostasis
The crucial implication of SIgA in the control of commensal bacteria has become
apparent from the canonical work of the team of John Cebra.39 The availability of knock-out
mouse models for genes regulating mucosal homeostasis coupled to controlled colonization
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with intestinal commensal microbes has yielded detailed results on the cross-talk with the
host.40, 41 The identification of the role of glycans carried by SIgA in recognizing selectively
Gram-positive bacteria provides a valuable clue as to biochemical definition of the
mechanisms underlying the necessary persistence of non-pathogenic bacteria.
Fab- and Fc-independent interactions may be seen as “gentle” contributions to the
establishment of controlled microbiota, in contact to high-affinity Ab required for aggressive
disposal of pathogens. Together with its involvement in commensal sensing by IEC, its
capacity to strongly bind with mucus, its capacity to sample associated antigens to mucosal
dendritic cell in the Peyer’s patch, heavily glycosylated SIgA exerts the function of securing a
steady-state level of commensal microbes.20, 42, 43 It remains that a lot needs to be set about
to better understand how SIgA and the microbiota act in concert to educate the host
immune system to accommodate so numerous and different passengers.
ACKNOLEDGEMENTS
This work was supported by Swiss Science Research Foundation Grant 3200-122039.
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17. Wold AE, Mestecky J, Tomana M, Kobata A, Ohbayashi H, Endo T, et al. Secretory immunoglobulin A carries oligosaccharide receptors for Escherichia coli type 1 fimbrial lectin. Infect Immun 1990; 58:3073-7.
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21. Mathias A, Corthésy B. Recognition of Gram-positive Intestinal Bacteria by Hybridoma- and Colostrum-derived Secretory Immunoglobulin A Is Mediated by Carbohydrates. J Biol Chem 2011; 286:17239-47.
22. Chapot-Chartier MP, Vinogradov E, Sadovskaya I, Andre G, Mistou MY, Trieu-Cuot P, et al. Cell surface of Lactococcus lactis is covered by a protective polysaccharide pellicle. J Biol Chem 2010; 285:10464-71.
23. MacKenzie DA, Tailford LE, Hemmings AM, Juge N. Crystal structure of a mucus-binding protein repeat reveals an unexpected functional immunoglobulin binding activity. J Biol Chem 2009; 284:32444-53.
24. Kaji R, Kiyoshima-Shibata J, Nagaoka M, Nanno M, Shida K. Bacterial teichoic acids reverse predominant IL-12 production induced by certain lactobacillus strains into predominant IL-10 production via TLR2-dependent ERK activation in macrophages. J immunol 2010; 184:3505-13.
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28. Zeuthen LH, Fink LN, Frökiaer H. Epithelial cells prime the immune response to an array of gut-derived commensals towards a tolerogenic phenotype through distinct actions of thymic stromal lymphopoietin and transforming growth factor-beta. Immunology 2008; 123:197-208.
29. Mathias A, Duc M, Favre L, Benyacoub J, Blum S, Corthésy B. Potentiation of polarized intestinal Caco-2 cell responsiveness to probiotics complexed with secretory IgA. J Biol Chem 2010; 285:33906-13.
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34. Bollinger RR, Barbas AS, Bush EL, Lin SS, Parker W. Biofilms in the normal human large bowel: fact rather than fiction. Gut 2007; 56:1481-2.
35. Macfarlane S, Woodmansey EJ, Macfarlane GT. Colonization of mucin by human intestinal bacteria and establishment of biofilm communities in a two-stage continuous culture system. Appl Environ Microbiol 2005; 71:7483-92.
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38. Bollinger RR, Everett ML, Wahl SD, Lee YH, Orndorff PE, Parker W. Secretory IgA and mucin-mediated biofilm formation by environmental strains of Escherichia coli: role of type 1 pili. Mol Immunol 2006; 43:378-87.
39. Cebra JJ. Influences of microbiota on intestinal immune system development. Am J Clin Nutr 1999; 69:1046S-51S.
40. Macpherson AJ, Gatto D, Sainsbury E, Harriman GR, Hengartner H, Zinkernagel RM. A primitive T cell-independent mechanism of intestinal mucosal IgA responses to commensal bacteria. Science 2000; 288:2222-6.
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42. Macpherson AJ, Slack E. The functional interactions of commensal bacteria with intestinal secretory IgA. Curr Opin Gastroenterol 2007; 23:673-8.
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PART II:
SECRETORY IGA-MEDIATED PROTECTION OF THE INTESTINAL EPITHELIAL CELL
INTEGRITY DURING INFECTION CAUSED BY SHIGELLA FLEXNERI
1 AIM OF THIS PART
100 years after its discovery, the enteropathogen S. flexneri is still under the scope.
Bacterial entry was first described to be restricted to M cells, with subsequent propagation
in the epithelium occurring via the basolateral pole of the IEC. Furthermore, the protective
role of mucosal SIgA has been well established in pathogenic contexts such as shigellosis but
few is known about its role at the IEC level. In this project, we infected apically Caco-2 cell
monolayer with a virulent strain of S. flexneri either alone or in complexes with specific SIgA
to study the impact of the Ab on the monolayer integrity. The combination of confocal
imaging with bacterial counts and ELISA measurements aimed at better delineating the
chronological events occurring during infection by S. flexneri together with the mechanisms
underlying the protective role of SIgA at the cellular level.
2 EXPERIMENTAL PROCEDURES, RESULTS AND DISCUSSION
Results obtained in the frame of this thematic are presented as a manuscript to be
submitted entitled “Secretory IgA-mediated protection of the intestinal epithelial cell
integrity during infection caused by Shigella flexneri.” Mathias A. (see Author contribution)
and Corthésy B.
I have performed all experiments and analyses related to the study. I have substantially
contributed to the writing of the paper under the supervision of Blaise CORTHESY, thesis
director.
Author contribution
93
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SECRETORY IGA-MEDIATED PROTECTION OF THE INTESTINAL EPITHELIAL CELL
INTEGRITY DURING INFECTION CAUSED BY SHIGELLA FLEXNERI Amandine Mathias, Blaise Corthésy
From the R&D Laboratory of the Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois, Rue du Bugnon, 1011 Lausanne, Switzerland
Address correspondence to: Dr. Blaise Corthésy, R&D Laboratory of the Department of Immunology and Allergy, University State Hospital (CHUV), Rue du Bugnon 46, 1011
Lausanne, Switzerland. Telephone: 0041 21 314 07 83; Telefax: 0041 21 314 07 71;
E-mail: [email protected]
Most of invasive enterobacteria target intestinal epithelial cells (IEC) promoting
major damages to the mucosa. Shigella flexneri, by invading IEC and inducing
inflammatory responses of the colonic mucosa, causes bacillary dysentery, a bloody
diarrhea that is endemic worldwide. The mechanism of entry of this bacterium is still a
matter of debate. Although M cells overhanging Peyer’s patches are commonly considered
as the primary site of entry of the bacteria, data are accumulating demonstrating that
bacteria can also enter the lamina propria directly via IEC, underscoring IEC as another
gate of entry. In addition, the protective role of secretory IgA (SIgA) the major
immunoglobulin secreted at mucosal surfaces has been established in the context of
shigellosis but few is known about its role in maintaining cellular integrity. Here, apical
infection of polarized Caco-2 cell monolayer with a virulent strain of S. flexneri either alone
or complexed with its cognate anti-LPS SIgA allowed us to demonstrate that bacteria are
able to infect IEC through their luminal-like pole as well, inducing the complete disruption
of tight junctions before the whole depolymerization of actin and eventually cell death.
Upon neutralization and agglutination of bacteria, SIgA led to the maintenance of cellular
cohesion, limiting access of the bacteria to the TJ network, resulting in reduced NF-κB
activation, thus explaining at the cellular level the protective role of SIgA. Together with
novel insights in the chronologic events occurring during infection by Shigella, deciphering
the cellular anti-inflammatory properties of SIgA suggests a mechanism by which the
antibody circumscribes overwhelming dissemination of this pathogen.
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INTRODUCTION
Mucosal surfaces are endowed with diverse levels of protection from physiochemical,
mechanical or immunological features including secreted mucus, antimicrobial defensins,
peristaltism and cells of the immune system ubiquitously present in the lamina propria. The
intestinal lumen is also covered by a single monolayer of highly differentiated epithelial cells.
Despite their rapid turnover, the intestinal epithelial cells (IEC) lay as a critical border that
supports both the entry of essential nutrients, electrolytes and water and the segregation of
luminal microbial products from the sterile core featuring them as the first physical line of
defense. The selective permeability across intact epithelial monolayers is sustained by a
dense network of junctions organized into the apical junctional complex consisting of
desmosomes, adherens junctions and tight junctions (TJ) considered as key determinants of
the cellular cohesion. Breaching of these barriers results in profound effects on human
health and disease, as deficiencies have been associated with the development of
pathologies such as inflammatory bowel diseases (IBD) or acute diarrhea.1 It is further
generally accepted that an essential step for numerous intestinal pathogens such as Shigella
flexneri is the deleterious invasion of IEC providing a wide gate of entry in the underlying
tissue.2, 3
S. flexneri is a Gram-negative facultative intracellular pathogenic bacterium. Species
of the genius of Shigella are among the bacterial pathogens most frequently isolated from
patients with diarrhea and are the causal agent of 1.1 million yearly fatal cases.
4 S. flexneri,
by invading epithelial cells and inducing inflammatory responses of the colonic mucosa,
causes bacillary dysentery, a bloody diarrhea that is endemic worldwide with prevalence in
young children of the developing world. The mechanisms of entry of this enteropathogen
are at the centre of the debate. None of animal models is able to faithfully mimic the
pathogenesis. Nevertheless, it has been shown that the primary site of entry of the
bacterium is M cells.5 Once in the Peyer’s patches (PP), bacteria encounter resident
macrophages.6 The bacteria evade degradation by inducing apoptosis-like cell death
accompanied by proinflammatory signaling (eg: release of IL-1β), which results in the
recruitment of polymorphonuclear (PMN) cells that infiltrate the infected site and amplify
damages to the epithelium, facilitating the invasion of bacteria which gain access to the
basolateral pole of epithelial cells.7-11 Shigella can then invade the IEC and spread from
96
cell-to-cell with concomittent cytokine release (IL-8, INF-y) and amplification of the
inflammatory cascade leading to massive tissue destruction.12, 13 Despite the absence of
adherence factors or flagellum, the bacterium is able to colonize the intestinal epithelium by
remodeling epithelial cell cytoskeleton after injection of effector proteins through the
type-three secretion system (T3SS). Initially, in vitro models using enterocyte-derived
monolayers partially or not differentiated led to the main admitted conclusion that
S. flexneri invade IEC monolayers exclusively from the basolateral pole.14 However, more
recently published data report the effectiveness of an apical infection by the bacterium both
in in vitro and in in vivo models, demonstrating a novel potential way of entry for the
bacterium outside from PP.15-18 100 years after its discovery, S. flexneri is still a worldwide
major health problem highlighting difficulties in understanding the pathogenesis at the
molecular, cellular and organism levels. How immune responses govern protection against
Shigella infection in still a main subject of discussion. The predominant role of the secretion
of specific secretory IgA, the main immunoglobulins found at mucosal surface, has been
described in vivo using rabbit ileal loops19 and also into samples of infected patients where it
plays a major role in the protection of the intestinal environment by a mechanism called
immune exclusion.20-24 Recently published data further demonstrate a transient suppression
of the T3SS when the bacteria were incubated with LPS-specific monoclonal antibodies
(Ab).25
Despite these observations, little is known at the cellular and molecular bases
pertaining SIgA-based protection during Shigella infection. This prompted us to focus on the
cellular mechanisms involved during bacterial entry and proliferation inside IEC and to shed
further light on the role of SIgA in maintaining IEC integrity. To this aim, reconstituted Caco-2
cell monolayers were apically infected with virulent bacteria either alone or complexed with
specific SIgA. Combining bacterial counts to confocal observations allowed us to assign
cytoskeletal and TJ disruption as two crucial steps in bacterial dissemination, a chronologic
process delayed by the agglutination properties of SIgA.
97
EXPERIMENTAL PROCEDURES
Caco-2 cell culture and transepithelial electrical resistance measurements-
The human colonic adenocarcinoma epithelial Caco-2 cell line was obtained from American
Type Tissue Collection and was used between passages 35 and 50. The cells were grown in
complete DMEM (C-DMEM) consisting of Dulbecco's modified Eagle's medium-Glutamax
(Invitrogen) supplemented with 10% fetal calf serum (FCS, Sigma), 1% non essential amino
acids (Gibco), 10 mM HEPES (Invitrogen), 0.1% transferrin (Invitrogen), and 1%
streptomycin/penicillin (Sigma). Cells cultivated up to 80% confluency were seeded on
polyester Snapwell filters (diameter, 12 mm; pore size, 0.4 μm; Corning Costar) at a density
of 0.8 × 105 cells/cm2.26
Microorganisms and growth conditions- Bacteria used in the present study were
either the virulent strain of serotype 5a LPS S. flexneri M90T GFP (Sf) which constitutively
express green fluorescent protein (GFP) or the avirulent strain BS176 (aSf) cured of its
virulent plasmid.
The Caco-2 cell monolayer integrity was checked by measuring the
transepithelial electrical resistance (TER) using a Millicell-ERS device (Millipore). TER values
of well-differentiated monolayers were in a range of 450–550 Ω cm2.
27-29 Bacteria from frozen stock were grown in Luria-Bertani (LB) agar plate
containing 0.1‰ Congo red (Applichem) and 50 µg/ml ampicillin (Sigma-Aldrich) only for the
selection of Sf colonies for one night. One red colony was picked up and amplified in LB
liquid broth also containing 50 µg/ml ampicillin for Sf bacteria under gentle agitation for 1 h
at 37°C and then plated onto LB agar plate. After 16 h of incubation, the formed lawn was
recovered with 0.9% NaCl solution. Bacteria were washed 3 times in 0.9% NaCl solution by
successive centrifugations at 2’000 x g for 5 min. Assessment of CFU/ml was obtained by
multiplying the optical density (O.D.) measured at 600 nm by this conversion factor: one
O.D. corresponds to 5 x 108
Cell lines and protein production- Hybridoma cells producing IgAC5
CFU/ml.
30 or IgGC20 31
specific for Shigella flexneri serotype 5a lipopolysaccharide (LPS) were cultured in RPMI 1640
medium supplemented with 10% fetal calf serum, 2 mM glutamine, 2 mM sodium pyruvate,
10 mM HEPES (pH 7.0), 0.1 mM folic acid, 100 units/ml penicillin and 100 µg/ml
streptomycin as described.30, 31 Cell cultures were conducted in Celline-350 bioreactors
(Intregra Biosciences AG). Supernatants were harvested twice a week, filtered through
98
0.22-µm cartridges and separated by size exclusion chromatography to recover pIgA from
other molecular forms and undesired ingredients.32 Fractions containing pIgA forms were
pooled, concentrated using a Pellicon XL filter unit (100-kDa cut-off; Millipore) coupled to a
Labscale system (Millipore), and finally stored at 4°C. IgGC20 was purified by affinity
chromatography using protein G-Sepharose 4 fast flow beads (GE Healthcare) according to
the manufacturer’s instruction. Prior to storage at 4°C, the sample buffer was exchanged
against PBS by filtration over an Amicon Ultra 100K cartridge (Millipore). Mouse SC (mSC)
was produced and purified as described.33 The purified protein was stored in PBS at 4°C until
use. The bicinchoninic acid protein assay kit (Pierce) was used for protein measurement.
SIgA were obtained by combining pIgA molecules with mSC (5/1 : w/w) in PBS according to
the conditions defined in Crottet and Corthésy.34 The effective reassociation between pIgA
and mSC proteins was checked by SDS-PAGE under non reducing conditions using sera
specific for mouse α chain, J chain or SC.
Protein association to bacteria- Overnight bacterial cultures were washed twice in
phosphate-buffered saline (PBS) resuspended in PBS, and the number of bacteria was
determined as indicated above. 2 x 10
35
7 bacteria were mixed with 10 µg of reassociated
SIgAC5 or 2 μg of IgGC20 in a final volume of 400 µl of PBS and incubated for 1 h at room
temperature (RT) under gentle agitation. Immune complexes (IC) were washed three times
in PBS and diluted in plain DMEM (P-DMEM) complemented only with 10 mM HEPES for
bacterial growth analysis or subsequent infection of cell monolayers. IC were
immunolabeled by incubation for 1 h at RT with the following primary and secondary Ab in
PBS: biotinylated goat anti-mouse alpha chain (1/10, Cappel) or biotinylated goat anti-mouse
gamma chain (1/50, Invitrogen) and streptavidin conjugated with cyanine 5 (Cy5) (1/500,
Amersham biosciences). Labeled IC were laid onto 8-well slides (Marienfeld), fixed in
2% paraformaldehyde in PBS for 25 min at RT and mounted in Vectashield (Vector
Laboratories). IC were observed using a Leica DM2000 fluorescence microscope or a Zeiss
LSM 710 Meta confocal microscope (Carl Zeiss, Germany) with a 63X objective (Cellular
Imaging Facility, Lausanne University, Switzerland) and processed using the Zeiss ZEN 2009
light software.
99
Exposure of Caco-2 cells to bacteria- One hour before use of the Caco-2 cell
monolayers, C-DMEM was replaced by P-DMEM in both apical and basolateral
compartments. Apical medium was then replaced by either 500 µl of bacterial suspensions
(2 x 107
Bacterial counts- To measure the bacterial load having crossed the Caco-2 cell
monolayers, 2.5 ml of medium in the basolateral compartment were recovered. In parallel,
to quantify the bacterial concentration having adhered and/or infected cells, Caco-2 cells
grown in Snapwell filters exposed to bacteria were washed three times with PBS, prior to
incubation for 3 min in 500 µl lysis buffer (10 mM Tris-HCl (pH 7), 0.2 % Nonidet P40,
50 mM NaCl, 2 mM EDTA (pH 8)). Cells were lysed by up-and-down pipeting. Finally, to
measure the direct impact of the Ab on the bacterial growth, bacteria alone or associated in
IC were incubated overnight at 37°C in a cell culture incubator. In this latter case, platings
were carried out either right after the formation of IC or after the overnight incubation. All
bacterial counts were performed by serial dilutions (10
bacteria) containing either bacteria alone or IC. TER values were measured at various
time points from the beginning of the infection up to 22 h post-infection, prior to be
subjected to multiple analyses as described below
-2 to 10-10
Analysis of NF-κB nuclear translocation- Preparation of Caco-2 cell small-scale
nuclear extracts was carried out as described in Cottet et al.
) applied onto triplicate LB
agar plates further containing 50 µg/ml of ampicillin and CFU were determined after
overnight incubation at 37°C.
26 Members of the NF-κB family
present in the nucleus from Caco-2 cells were identified by immunoblotting with rabbit
antisera directed against the p50 or p65 subunits (Santa Cruz Biotechnology, Santa Cruz, CA;
1/500). Western blot assay was performed as described35
Enzyme-linked immunosorbent assay (ELISA) measurements- CXCL-8 (interleukin
(IL)-8) in the basolateral compartment of polarized Caco-2 cells was measured by ELISA using
, with proteins detected by
incubation for 1h with appropriate specific antiserum diluted in PBS containing 0.05% Tween
20 and 0.5% nonfat dry milk followed by HRP-conjugated goat anti-rabbit IgG (1/5000;
Sigma). After final washing in PBS containing 0.05% Tween 20, proteins were detected by
chemiluminescence using Uptilight detection kit (Interchim) and exposed on
autoradiographic films (Konica).
100
a commercial kit for human proteins (BD bioscience), and expressed as pictograms/ml. Data
are duplicates of three independent experiments.
Laser scanning confocal microscopy observation of Caco-2 cell monolayers- Infected
Caco-2 cell monolayers grown in Snapwell were washed twice with PBS, prior to fixation
overnight with 5 ml of 4% paraformaldehyde at 4°C. After washing with PBS, filters were
permeabilized and non-specific binding sites were blocked using PBS containing 5 % FCS and
0.2% Triton X100 at RT for 30 min. All Ab were diluted in PBS containing 5 % FCS and
0.2% Triton X100. Filters were incubated with rabbit anti-human ZO-1 (1/200, Invitrogen) for
2 h at RT, washed in PBS and the secondary Ab goat anti-rabbit IgG conjugated with
Alexa Fluor 647 (1/100, Invitrogen) was added for 90 min at RT. On some occasions,
phalloidin associated with Fluoprobes 547H (1/200, Interchim) was incubated with the
secondary Ab. After PBS washing, filters were then incubated with
4',6-diamidino-2-phenylindole (DAPI) at a concentration of 100 ng/ml in PBS (Invitrogen)
for 30 min. Filters were cut out of their holders, and mounted in Vectashield
(Vector Laboratories) for observation using a Zeiss LSM 710 Meta confocal microscope
(Carl Zeiss, Germany) equipped with either a 10X or a 40X objective (Cellular Imaging Facility
platform, Lausanne University, Switzerland) and processed using Zeiss ZEN 2009 light
software.
Quantification of the number of invasion foci and the infection area- Complete filter
observations were carried out with the 10x objective using Zeiss ZEN 2009 light software.
Numbers of invasion foci and both the global infected area and the area covered by each foci
were automatically determined using the particle analysis tool available with the ImageJ
software applied onto the channel associated with the bacteria (i.e.the green channel).
When the area affected by the bacteria reached such a level inducing partial deletion of the
monolayers, remaining cellular areas were determined upon the differential interference
contrast (DIC) channel analysis after handle delineation with the classical measurement tool.
Statistical analysis- The results are given as mean ± standard error of the mean
(SEM). Two-tailed nonparametric Mann-Whitney U-test analysis was performed using the
GraphPad 5 Prism software. Differences were considered as significant when p values <0.05
were obtained.
101
0 5 10 15 20 250
200
400
600
800
controlMM+SM+GB
TER
(Ohm
*cm
2 )
Figure 1. Effect of S. flexneri infection on Caco-2 cell monolayers: monitoring of TER changes and associated bacterial counts.
A. TER of Caco-2 cells exposed to 2 x 107 S. flexneri alone or in immune complexes with anti-LPS SIgAC5 or
IgGC20 was measured at different time points. The curves represented here resulted from one
representative experiment (n=3-10) for various conditions performed in triplicates. Three distinct phases
are observable: the early phase (), the intermediate phase (), the late phase (). Non-infected Caco-2
cell monolayers serve as control of the stability of the Caco-2 cell monolayer TER. No statistical difference
between S. flexneri alone or in immune complexes could be detected. Color code is given alongside the
curves. B. Adhesion of S. flexneri strains alone or associated with SIgA or IgG to polarized intestinal Caco-2
cell monolayers. C. Bacterial loads in the basolateral compartment of Caco-2 cell monolayers incubated with
S. flexneri strains alone or associated with SIgA or IgG. Bacterial counts were determined at different time
points [early phase (), intermediate phase (), late phase ()]. Results are expressed in absolute
number of bacteria ± SEM. Data were obtained from 3 experiments performed in triplicates. Abbreviations
used: Sf, S. flexneri; aSf, avirulent S. flexneri. Statistically significant differences calculated by comparison
with “Sf alone” is indicated above the columns: *, p<0.05; **, p<0.01; ***, p<0.001.
15 19 22
15 19 22
M
M+S
M+G
B
ControlSfSf +SIgASf +IgGaSf
Sf
Sf +SIgA
Sf +IgG
aSf
A
B
C
12
11
10
9
8
Tota
l bac
teri
a bo
und
to
Caco
-2 c
ells
( x
1010
)To
tal b
acte
ria
foun
d in
the
baso
late
ral c
ompa
rtm
ent (
x 1
010)
12
11
10
9
8
Time (h)
***
*** **
**
Sf
Sf +SIgA
Sf +IgG
aSf
102
RESULTS
TER evolution upon S. flexneri infection alone or associated in IC
As TER is associated with the difference in potential between the apical and
basolateral compartments created by the presence of polarized epithelial cells, assessment
of TER values was used to have a direct and rapid indication of the global cohesion state
between epithelial cells. Interestingly, despite an apparent tendency for a reduction in TER’s
fall in presence of SIgA, bacteria alone or in complexes with specific SIgA or IgG implied
similar fluctuations of TER values as depicted in Figure 1A. Three distinct phases could be
detected during the infection by Sf alone or in IC with SIgA or IgG: the early phase
(Figure 1A, ) where TER values gained 10%, the intermediate phase (Figure 1A, ) where
the TER progressively decreased to a basal level at 200-250 Ω x cm2 and the late phase
(Figure 1B, ) during which the TER got stabilized to a minimal level corresponding to
approximatively 50% of the initial values. Strikingly, the kinetics of entry of Sf slightly varied
upon the experiments, thus leading to the definition of two patterns: the fast infections
during which the early phase terminated after only 10 h post-infection and the late phase
was reached after 18 h post-infection (represented in Figure 1A), and the late infections
characterized by a delay of up to 3 h in the process of infection. This surprising feature
indicated that despite normalized bacterial culture conditions and bacterial counting, the
infectivity of Sf fluctuated between experiments. However, despite this variability in kinetics
of bacterial entry, the same overall conclusions developed in the next paragraphs could be
obtained. Control experiments using the avirulent strain harbored a completely different
pattern which strikingly resembled to Caco-2 cell incubation with commensal bacterial
strains36
in that a 25% increase of TER values was detected together with a complete
absence of a drastic fall up to 24 h post-infection, indicating that the virulent pattern is
involved in the process of infection at the apical surface of IEC monolayers (Figure 1A).
Adhesion profiles of S. flexneri and proliferation in the basolateral pole of Caco-2 cell
monolayers
Because decreasing TER values are intimately associated with loss of cellular
integrity, we sought to determine both the bacterial load present in contact with the
monolayers by plating of the whole cell lysates (Figure 1B) and the bacteria having reached
103
Figure 2. Confocal imaging of the Caco-2 cell monolayer integrity.
Polarized Caco-2 cell monolayers were infected either with Sf alone or in association with either
anti-LPS-specific SIgA or IgG. Bacteria constitutively express GFP and are visualized as green dots, cells are
localized via nuclear staining with DAPI (blue) while tight junctions stabilizing the Caco-2 cell monolayer are
stained in red upon detection of ZO-1. Monolayers are observed at different time points corresponding to
the early phase (, panel A) or the late phase (, panel B). Monolayers are observed at different plans by
performing Z-stack analysis and a 3D reconstruction is depicted. The pattern obtained for non-infected
control Caco-2 cell monolayers is shown in panel C. One representative field obtained from the observation
of whole Transwell filters obtained from 3 experiments performed in triplicates. Scale bars represent
50 µm. The number of infection foci (panel D) and the infected area (panel E) were determined from LSCM
pictures of whole membranes at different time points using ImageJ software [early phase (),
intermediate phase (), late phase ()]. Harsh infection leading to the formation of entire areas lacking of
cells, the quantification of the number of infection foci was thus impossible and indicated as not
measurable (nm). Data were obtained from (n=3-5) experiments performed in triplicates. Bars represent
the mean values SEM. Statistically significant differences calculated by comparison with “Sf alone” is
indicated above the columns: **, p<0.01; ***, p<0.001.
DAPI S. flexneri ZO-1 Merge
+ SI
gA+
SIgA
A:
B:
C
Con
trol
+ Ig
G
SfSf + SIgASf + IgG
2
1.5
1
0.5
0Num
ber o
f inf
ecti
on fo
ci
(x 1
000)
nm nm
***
D
10
8
6
4
2
0
Infe
cted
are
as (
mm
2 )
SfSf + SIgASf + IgG
*****
E
104
their basolateral pole by plating of the basolateral supernatants (Figure 1C). Confirming the
key role of the plasmid of virulence in the cellular invasion, control experiments performed
with aSf revealed the complete absence of bacteria in the basolateral pole underlying their
inability to affect cells as sustained by an absence of TER’s fall (Figure 1C), despite the
detection of bacteria having adhered to the surface of the monolayers (Figure 1B). During
infections by Sf alone, bacterial adhesion reached a maximum at the end of the early phase,
progressively decreasing during the intermediate phase to reach a minimum in the late
phase (Figure 1B). In parallel, the bacterial load in the basolateral supernatants seems to
have reached a steady-state level as detected by stable maximal bacterial counts (Figure 1C).
In sharp contrast, during the early phase, SIgA significantly reduced both the number of
bacteria able to adhere and infect cells (11% less abundant) and those having reached the
basolateral pole of the monolayers (33% less abundant). As time moves on, bacteria
proliferated in an exponential manner in the basolateral compartment to achieve similar
levels of bacterial loads as also observed in the whole cell lysis bacterial counts. The absence
of any effect observed on the bacterial growth in presence of IgG pinpoints a crucial and
specific role only ascribable to SIgA. All together, these results allowed us thus to further
conclude on several protective mechanisms of SIgA interfering with the process of bacterial
infection, i.e. modification of adhesion, the infectious pattern and proliferation of Sf.
Confocal imaging of Caco-2 cell monolayer integrity infected by S. flexneri alone or
associated with SIgA
The presence of invasive bacteria correlates with affected cellular cohesion. As TJ are
involved in the maintenance of epithelial integrity, we observed their aspect by LSCM
through ZO-1 detection to visually confirm the conclusions obtained with bacterial counts
and TER measurements. LSCM observations first indicate that during the early phase,
bacteria alone induced damages in the form of multiple holes in the Caco-2 cell monolayers,
whereas bacterial aggregation mediated by SIgA prevented spreading and limited damages
to a few cells, not altering dramatically the epithelial cell organization (Figure 2A). As time
moved on, bacteria progressed in the monolayers inducing cell death and thus increasing the
number and size of holes (Figure 2B, D and E). Until the late phase, outside the areas where
holes are present, the ZO-1 network is conserved in non-infected Caco-2 cell monolayers
105
Figure 3. Confocal imaging of the actin network in association with intercellular tight junctions in Caco-2 cell monolayers.
Polarized Caco-2 cell monolayers were infected either with 2 x 107 S. flexneri alone (panel A) or in
association with anti-LPS-specific SIgA (panel B). Bacteria constitutively express GFP and are visualized as
green dots, cells are localized via nuclear staining with DAPI (blue) while tight junctions or actin fibers
stabilizing the Caco-2 cell monolayer are emphasized with red labeling against respectively ZO-1 (top view)
and phalloidin (side view). Monolayers are observed at different plans by performing Z-stack analysis and
either a 3D reconstruction (lower panels) or lateral reconstructions (upper panels) are depicted for the
same monolayers. The filter position (basal side of the Caco-2 cell monolayer) is represented as a grey bar
on the merge images. Monolayers are observed at different time points either during the early phase (),
the intermediate phase or the late phase (). Sites of extensive actin remodeling are pinpointed by
white arrowheads. Non-infected control Caco-2 cell monolayers are represented in panel C.
One representative field obtained from the observation of whole filters prepared from 3 experiments
performed in triplicates. Scale bars represent 50 µm.
Act
in S
fD
API
Side
view
3D to
p vi
ew
ZO-1
Sf
DA
PI
acti
n S
fD
API
Side
view
3D to
p vi
ew
ZO-1
Sf
DA
PI
C. ControlA. Sf
B. Sf +SIgA
acti
n Ø
DA
PI
Side
view
3D to
p vi
ew
ZO-1
Ø D
API
106
(compare Figure 2 panels A with panel C). At the beginning of the late phase, Sf-induced
damages to Caco-2 cell monolayers led to the formation of large areas devoid of any cells
that can be observed macroscopically as similarly observed using IgG-mediated IC
(Figure 2B). Loss of ZO-1 signal reflects the destruction of the organized monolayer, leading
to disruption of TJ, contrary to what is observed in presence of SIgA (Figure 1B). Incubation
of Caco-2 cell monolayers with the invasive strain induced a time-dependant increase in the
area affected by the bacteria as well as the number of infection foci as depicted in Figures 2,
D and E. The quantitative analysis performed from LSCM observations allow us to further
conclude on the neutralizing and protective role of SIgA resulting in 9-times less infected
areas (Figure 2E) and preventing infected loci to merge into complete areas devoid of cells as
observed with Sf alone (Figure 2D). These latter results were in accordance with previous
conclusions emanating from bacterial counts: the presence of SIgA maintained the epithelial
monolayer integrity, most likely by neutralizing most bacteria.
Modulation of actin fibers disassembly triggered by S. flexneri in presence of SIgA
ZO-1 proteins interact with the cell cytoskeleton and in particular with actin fibers,
highlighting the function of these latter in maintaining cell cohesion. Lacking a flagellum,
S. flexneri needs consequently to hijack the host’s cytoskeleton to achieve intracellular
spreading, placing actin fibers as key targets for virulence effectors. We thus decided to
focus on LSCM observations to track changes in actin fiber integrity using
fluorescently-labeled phalloidin combined with ZO-1 detection (Figure 3). First, actin fiber
depolymerization induced as a function of intracellular dissemination of Sf was confirmed
with the complete extinction of phalloidin reactivity as depicted in Figure 3A, panels. Both
labelings further allowed us to establish a chronology in between TJ disruption and
disappearance of actin fiber: while TJ are rapidly affected during the intermediate phase
already, intact actin fibers are still detected (Figure 3A, line ). Strikingly, actin fibers
architecture drastically changes in the periphery of the hole, in support of the
rearrangement of the cytoskeleton induced by virulence effectors expressed by Sf: LSCM
observations clearly delineate a high concentration of actin detection at the borders with the
de novo induced gaps. One can thus argue that these drastic modifications aim at preventing
cellular damages during intracellular bacterial proliferation allowing a transient maintenance
107
controlSfSf + SIgASf + IgG
SfSf + SIgASf + IgG
control
150
100
50
0
Baso
late
ral I
L-8
conc
entr
atio
n (p
g/m
l)
C
A
B
Figure 4. Effect of S. flexneri alone or in complex with SIgA on nuclear translocation of NF-κB and IL-8 secretion in the basolateral compartment.
A. Nuclear translocation of NF-κB subunits p50 and p65/relA induced by the contact of Caco-2 cells with
2 x 107 bacteria alone or in complex with LPS-specific SIgAC5 and IgGC20. Immunoblotting was carried out
on nuclear extracts from polarized Caco-2 cell monolayers during the late phase (). Identical amounts of
nuclear extracts based on protein concentration were used for each set of experiments. Panels are
representative of one individual experiment (n=3 Transwell membranes per condition) performed three
times. B. Densitometric analysis of immunoblots as in A. exposed for optimal times to avoid saturation of
the photographic film. The intensity of the signals reached with Sf alone was fixed at 100%. No signal could
be detected (nd) with control extracts. C. IL-8 production in the basolateral compartment of Caco-2 cell
monolayers incubated for various times [early phase (), intermediate phase (), late phase ()].
Data were obtained from 3 experiments performed in triplicates. Bars represent the mean values SEM.
The lane content is indicated on the plot.
cont
rol
Sf Sf +
SIg
A
Sf +
IgG
p50
p65
150
100
50
0Rela
tive
abu
ndan
ce o
f nu
clea
r p50
and
p65
(arb
itra
ry u
nit)
p50 p65
nd nd
108
of a local cellular cohesion. As time moves on, the loss of both signals result in the complete
disruption of the cellular integrity and further promotes the cell death (Figure 3A, line ).
Outside infected foci, and despite a slight decrease in actin detection, Caco-2 cells
infected by Sf associated with SIgA resulted in similar observations as compared with control
filters leading us to conclude on the SIgA-mediated preservation of the monolayer,
emphasizing that both actin fibers and the TJ network are barely altered even during the late
phase (compare Figure 3 panel B with panel C). SIgA seems to block bacterial targeting of the
TJ, subsequent actin depolymerization, limiting overwhelming bacterial growth and
dissemination of Sf to the basolateral side of the monolayers.
Effect of S. flexneri alone or in complexes with SIgA on NF-κB activation and subsequent
IL-8 secretion by Caco-2 cell monolayers
Activation of the NF-κB pathways is a pro-inflammatory marker of the cellular sensing
via a various arrays of receptors in cells and tissues.37 High nuclear translocation of the NF-κB
subunits p50 and P65 occurred after incubation with Sf alone, a sign of a strong cellular
activation in response to infection (Figure 4A). Incubation with SIgA-based IC led to a
significantly decrease in nuclear translocation of either subunits p50 or p65 (Figure 4A).
Incubation of Sf with LPS-specific IgGC20 did not prevent from accumulation of p50 and p65
in the nuclei of Caco-2 cells, indicating that effective neutralization requires the SIgA isotype.
Quantitative densitometric analysis of p50 and p65 signals on blots exposed for different
times to photographic films confirms the inhibitory effect of SIgA on Sf induced NF-κB
activation (Figure 4B). The concomitant release of IL-8 involved in the recruitment of
monocytes and neutrophils in epithelia has been measured in the basolateral supernatants
after overnight incubation with bacteria in the presence or absence of SIgA or IgG.
In contrast to increased transcriptional expression described previously38
, we found no
difference at the protein level in the various conditions assayed. Thus, despite marked
differences in NF-κB nuclear activation, the protective functions of SIgA acting on the
morphology of IEC do not involve modulation of the secretion of pro-inflammatory IL-8
(Figure 4C).
109
Figure 5. Visualization of free S. flexneri or in the form of immune complexes with LPS-specific SIgA or IgG.
2 x 107 bacteria were complexed either with SIgA or IgG. Immune complexes were visualized either by
direct fluorescence microscopy (panel A) or by LSCM (panel B). The bacteria are observed by green
fluorescence associated with the constitutive expression of GFP. Coating of the bacteria by the Ab depicted
in panel B is observed by immunostaining detecting either the α chain (upper raw) or the γ chain (lower
row). Formation of immune complexes is visualized by the presence of a ring surrounding the bacteria. One
representative field obtained from 10 different observations following analysis of 3 different slides. Scale
bars represent either 30 µm (panels A) or 5 µm (panels B).
IgG
+ SIgA + IgGSf
SIgA
A
BAb Sf Merge
110
SIgA-mediated bacterial aggregation
How does SIgA promote cellular protection in comparison with IgG, and how the Ab
specifically restrains dissemination on the surface of IEC remain unclear. We thus
hypothesized that SIgA can have a direct impact on the bacterial growth. After only 1 h of
incubation with SIgA, growth of Sf was drastically reduced; viable bacteria incubated in the
presence of SIgA were 10 times less numerous as compared with Sf alone (data not shown).
After overnight culture, this difference became more marked, reaching a 30-fold decrease in
numeration performed with IC demonstrating the neutralizing activity of SIgAC5 timewise.
The mechanism underlying this difference may be either by slowing the bacterial growth or
by progressively killing the bacteria. Observations of the morphology of the IC carried out by
fluorescence microscopy surprisingly illustrated the presence of bacterial aggregation in
presence of SIgA only (Figure 5A) in contrast to free Sf observed in presence of IgG.
To confirm the formation of IC between SIgA or IgG and their cognate antigen Sf, the Ab
were immunolabeled with α chain or γ chain-specific reagents (Figure 5 B). Binding to the
bacterial surface of either SIgA or IgG was apparent from the presence of red dots
corresponding reactive IgA or IgG (Figure 5B). Moreover, almost all bacteria presenting a
signal specific for SIgA were associated in a network of bacterium indicating that the
opsonisation of the bacteria is responsible for the formation of big lattices that can explain
protection mediated by interference with the cellular surface. The same labeling steps
performed on recovered bacteria after an overnight culture indicated that IC were still
detected, although less abundantly (data not shown). These latter results revealed that the
coating by SIgA was stable even after an overnight culture and may justify the delay
observed in the process of infection.
111
Figure 6. Model of infection of the IEC monolayer by S. flexneri administered apically; putative explanation for SIgA-mediated cellular protection.
A. A few cells are first infected by the adhesion of bacteria through an unknown receptor () and/or
targeting of TJ to gain access to the basolateral pole of the cells. Bacteria rapidly proliferate () inside the
cells. S. flexneri first targets the cytosqueleton via polymerization/depolymerization of actin fibers during
the intermediate phase resulting in a complete disruption of the cytoskeletal network (). During the late
phase, the absence of both the tight junction network and the supporting actin fibers () results in the
disruption of the intestinal monolayer favoring the access to the basolateral pole resulting in exponential
bacterial growth and cell-to-cell dissemination. Detection of the bacteria leads to the rapid activation of the
NF-κB pathway () resulting in the basolateral release of the pro-inflammatory cytokine IL-8 ().
B. SIgA-mediated bacterial aggregation interferes with the cellular entry (), delays bacterial growth ()
resulting in their reduced impact on the actin fibers () and subsequently favors the maintenance of
cellular architecture (). Decreased activation of the NF-κB pathway () finally confers anti-inflammatory
properties to SIgA even associated with non-affected IL-8 release ().
112
DISCUSSION
The critical step in the pathogenesis of shigellosis is invasion of enterocytes which
takes place across M cells, followed by the propagation via basolateral infection.
This pathway does not appear to be exclusive and, alternative routes are likely to take place
outside PP: in vivo, rabbit intestinal ligated loops administered Shigella displayed a delayed
epithelial villi infection in segments devoid of PP, and thus M cells.15, 16 Overnight apical
application of virulent strain S. flexneri serotype 5a in vitro allowed us to conclude that the
consequence of the occurring infection is to damage the reconstituted monolayers
sequentially, involving molecular partners contributing to its proper organization under
normal conditions. Of note, these results are in contradiction with those of Mounier et al. 14,
who reported that the entry of S. flexneri was restricted to the basolateral pole of IEC. Major
differences in their experimental settings including time of incubation of Caco-2 cells onto
Transwell membranes may explain discrepancies between results, as fully differentiated
polarized monolayers expressed other receptors distributed along the apical and basolateral
surfaces.39 We present here the first in vitro study dissection of the sequential events
associated with S. flexneri apical invasion of the epithelium. A tentative model including the
main steps identified in this study is depicted in Figure 6: it comprises apical adhesion,
TJ disruption, intracellular proliferation leading to pro-inflammatory responses that reaches
a state of no return, and eventually cell death when complete depolymerization of actin
filaments is achieved. Whether or not apical recognition of the bacteria is mediated by a
specific receptor remains open at this stage, as all identified cell adhesion receptors for
Shigella (α5β1 integrin and CD44) are located at the basolateral pole of the enterocytes.40
In this respect, Mounier at al. 14 pinpointed a colocalization between the distribution of a cell
surface receptor, the transferrin receptor (CD71), and sites of bacterial entry. Although
overproduction of CD71 occurs at the apical pole in a pathologic context such as active celiac
disease, it is found to be confined to the basolateral pole of normal biopsies.41 Early infection
and detection of CD71 at the apical surface of Caco-2 cell monolayers did not allow us to
further conclude on its involvement in bacterial recognition (data not shown). Despite the
current lack of a defined apical receptor, bacteria were clearly found to infect initially a
limited number of cells, leading to the privileged targeting of the TJ network. This feature
allows more bacteria to gain access to the basolateral side of the IEC monolayer where they
can freely proliferate exponentially latter on. Interestingly, a recent article published by
113
Sakaguchi et al. 18 is in support of our results: another strain of S. flexneri was able to infect
epithelial cells from the apical pole, and after targeting TJ, to spread along the monolayer.
Described for other pathogen as enterohemorrhagic Escherichia coli (EHEC) or Salmonella
strains3, TJ rearrangement seems to be also a central target of entry of S. flexneri serotype
5a. Bacterial internalization allows the presentation of components of the cell wall such as
LPS recognized by the NOD1 receptor, sufficient for the initiation of inflammatory responses
characterized by the nuclear translocation of the NF-κB and the resulting secretion of IL-8
known to increase recruitment of PMN and further amplification of tissue damages.40, 42
Actin fibers are also known to play a fundamental role in S. flexneri epithelial invasion.
Modifications in the distribution of actin filaments as demonstrated by their abnormal
concentration at the border of induced holes appear to sustain a temporary reinforcement
of the local adherence of IEC; such a preservation allows bacteria to keep proliferating
intracellularly. The rapid turnover and exfoliation of the mucosal epithelium is an intrinsic
innate defense against harmful intruders, thus interfering with this program represents a
new infectious stratagem to circumvent host’s mechanisms of protection.43 In accordance
with this hypothesis, S. flexneri has recently been described to interfere with the epithelial
renewal through the OspE virulence effector, which function is to reinforce adherence of IEC
to their basal membrane, eventually increasing the number of focal adhesions and finally
reducing cell motility.44
A S. flexneri LPS-specific SIgA has been recently involved in the transient suppression
of the T3SS, the partial reduction of the potential of the bacterial membrane and a decrease
in the intracellular generation of ATP.
Despite their fundamental role in cell-to-cell dissemination,
overwhelming proliferation of S. flexneri induces major and irreversible damages to the cell
architecture as observed by the complete disorganization of the network of actin fibers. The
resulting destruction of the epithelial integrity is a well-accepted feature of the bacillary
dysentery faithfully mimicked in our in vitro model leading to strong inflammation as
discussed below.
25 Nevertheless, how SIgA neutralization of S. flexneri
impacts at the interface between the bacteria and the target IEC harbors wide information
gaps. Besides novel insights in the sequence of infectious events, SIgA-mediated cellular
protection during S. flexneri invasion has been assessed. Contrary to what was observed
using specific IgG, the neutralizing properties of SIgA allow to delay the infection via
limitation of bacterial growth and proliferation inside the intestinal cells, maintenance of the
114
TJ network, interference with the activation of the pro-inflammatory NF-κB cascade, and
finally to slow down depolymerization of actin fibers, pillars of the cell architecture.
Surprisingly, despite the demonstrated anti-inflammatory properties of SIgA via restrained
NF-κB activation, no changes in IL-8 secretion could be detected. Although transcriptional
activation of IL-8 has been reported after S. flexneri-mediated infection of IEC, the picture
can be totally different at the protein level. In this respect, the secretion of IL-8 has been
recently described to be reduced as a function of the OspF virulence effector protein
injected by S. flexneri preventing access of NF-κB to its chromatin target sites.45, 46
Facing the emergence of Shigella strains resistant to multiple antibiotics and the
increasing number of infected persons in some areas of the world, it is easy to figure the
fundamental importance of a better appreciation of the mechanisms involved to develop
safe and long-term treatments such as vaccination already tested in clinical trials.
S. flexneri
thus hijacks normal cellular responses, limiting IL-8 secretion which appears not to be
modulable in presence of the Ab and consequently reduces the recruitment of surrounding
immune cells, further allowing the intracellular proliferation to persist. Interestingly, we
further observed that after an overnight culture in vitro, 50% of the total bacteria are no
longer coated by SIgA, suggesting that bacterial division still occurs after SIgA coating, or
alternatively, that complete blocking does not take place, thus leading to delayed infection.
The presence of SIgA thus partially abrogates, but not fully blocks, bacterial adhesion and/or
bacterial growth mechanisms, most likely based onto the agglutining properties of this type
of Ab.
47-50
As a consequence, deciphering at the cellular and molecular levels the patterns involved in
SIgA-mucosal protection represent valuable targets to develop effective strategies to fight
against S. flexneri mucosal invasion.
115
FOOTNOTES
This work was supported by research grant number 3200-122039 from the Swiss
Science Research Foundation.
The abbreviations used are: Ab, antibody; aSf, avirulent strain of S. flexneri BS176;
GFP, green fluorescent protein; IC, immune complexe; IEC, intestinal epithelial cell; IL-8,
interleukin 8; LSCM, laser scanning confocal microscopy; M cell, microfold cell; PMN,
polymorphonuclear; Sf, S. flexneri strain M90T GFP; SIgA, secretory immunoglobulin A; T3SS,
type three secretion system; TER, transelectrical epithelial resistance; TJ, tight junction; ZO-
1, zonula occludens protein 1.
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120
PART III:
RETROTRANSPORT OF SIGA THROUGH INTESTINAL EPITHELIAL CELL, A NEW GATE OF
RE-ENTRY FOR THE ANTIBODY
1 AIM OF THIS PART
pIgR mediated-IgA transport from the basolateral to the apical pole ensures the
secretion of these Ab into the lumen of the GI tract. The function of this secretory route in
mucosal protection has been discussed in details in Introduction section 5. In the intestinal
environment, retrotransport of SIgA has been described to occur through M cells in in vivo
model178 or across IEC once complexed with an Ag (gliadin) in the context of the celiac
disease, a peculiarity involving the transferrin receptor CD71 also capable to bind SIgA.167
Nevertheless, retrotransport of SIgA from the apical to the basolateral pole of the healthy
IEC has not been described at this point. Deciphering such a way of internalization of the Ab
alone or complexed with an Ag could open new perspectives in the role played by SIgA at the
interface between the intestinal epithelium and the external environment in maintaining gut
homeostasis. In the next section, we thus aimed at investigating the retrotransport of SIgA
directly across the epithelial cells: in vitro reconstituted, Caco-2 cell monolayers were
incubated in presence of fluorescently-labeled SIgA and the fate of these latter further
analyzed by laser scanning confocal microscopy (LSCM).
121
2 EXPERIMENTAL PROCEDURES
2.1 Caco-2 cell culture and measurements of transepithelial electrical resistance
The human colonic adenocarcinoma epithelial Caco-2, the human hepatocellular
carcinoma HepG2 and the human embryonic kidney HEK 293 cell lines were obtained from
American Type Tissue Collection and were used between passages 35 and 50. The cells were
grown in complete DMEM (C-DMEM) consisting of Dulbecco's modified Eagle's
medium-Glutamax (Invitrogen) supplemented with 10% fetal calf serum (FCS, Sigma),
1% non essential amino acids (Gibco), 10 mM HEPES (Invitrogen), 0.1% transferrin
(Invitrogen), and 1% streptomycin/penicillin (Sigma). Caco-2 cells cultivated up to 80%
confluency were seeded on polyester Snapwell filters (diameter, 12 mm; pore size, 0.4 μm;
Corning Costar) at a density of 0.8 × 105 cells/cm2.179
The Caco-2 cell monolayer integrity was
checked by measuring the transepithelial electrical resistance (TER) using a Millicell-ERS
device (Millipore). TER values of well-differentiated (presence of microvilli) and polarized
(presence of the apical junctional ring defining the apical from the basolateral pole)
monolayers were in a range of 450–550 Ω cm2.
2.2 Cell lines and protein production
Hybridoma cells producing IgAC5 specific for S. flexneri serotype 5a LPS was cultured
in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, 2 mM sodium
pyruvate, 10 mM HEPES (pH 7.0), 0.1 mM folic acid, 100 units/ml penicillin and 100 µg/ml
streptomycin as described.137, 180 Cell cultures were conducted in Celline-350 bioreactors
(Intregra Biosciences AG). Supernatants were harvested twice a week, filtered through
0.22-µm cartridges and separated by size exclusion chromatography to recover pIgA from
other molecular forms and undesired ingredients.181 Fractions containing pIgA forms were
pooled, concentrated using a Pellicon XL filter unit (100-kDa cut-off; Millipore) coupled to a
Labscale system (Millipore), and finally stored at 4°C. Prior to storage at 4°C, the sample
buffer was exchanged against PBS by filtration over an Amicon Ultra 100K cartridge
(Millipore). Mouse SC (mSC) was produced and purified as described.182 The purified protein
was stored in PBS at 4°C until use. The bicinchoninic acid protein assay kit (Pierce) was used
for protein measurement. Fluorescent pIgA were obtained using the FluoroLink mAb
122
Indocarbocyanine-3 (Cy3) labeling kit (Amersham Biosciences) according to the procedure
provided by the manufacturer. SIgA:Cy3 were obtained by combining pIgA:Cy3 molecules
with mSC (5/1 : w/w) in PBS according to the conditions defined in Crottet and Corthésy.183
The effective reassociation between pIgA and mSC proteins was checked by SDS-PAGE under
non reducing conditions using sera specific for mouse α chain, J chain or SC.184
2.3 Flow cytometry
Undifferentiated Caco-2 cells, HepG2 and HEK 293 cells cultivated up to
80% confluency were washed twice with PBS. 2 x 105 cells were fixed using PBS containing
1% paraformaldehyde (PFA) and 2% glucose at 4°C for 20 min. All Ab were diluted in PBS
containing 5 % FCS alone or with 0.2% saponin. After washing with cold PBS containing
5% FCS, cells were incubated for 20 min at 4°C with 100 μl of the primary Ab (mouse
anti-human CD71 diluted 1/50; clone A24168, a gift from Renato Monteiro’s lab, Necker
Hospital, Paris), washed in cold PBS containing 5% FCS and finally labeled with 100 μl
containing goat anti-mouse IgG conjugated with fluorescein isothiocyanate (
FITC, 1/100,
Dako) for 20 min at 4°C. After washing, cells were resuspended in PBS containing 5% FCS and
cellular associated fluorescence intensity was evaluated by flow cytometry (FACScan flow
cytometer, Becton-Dickinson).
2.4 Exposure of Caco-2 cell monolayers to pIgA and SIgA
One hour before use, C-DMEM was replaced by plain DMEM (P-DMEM) containing
only 10 mM HEPES. Apical medium was then replaced by 500 µl containing 2 μg of either
SIgA or pIgA. When used, dextran-FITC (70 kDa, anionic, lysine-fixable, Invitrogen) and
transferrin-FITC (Invitrogen) were added simultaneously to SIgA respectively at 1 mg/ml and
50 μg/ml. TER values were measured at various time points from the beginning of the
infection to 18 h post-infection.
123
2.5 Laser scanning confocal microscopy observation of Caco-2 cell monolayers
Caco-2 cell monolayers exposed to SIgA or pIgA were washed twice with PBS, prior to
fixation overnight with 5 ml of 4% PFA at 4°C. After washing with PBS, filters were
permeabilized and non-specific binding sites were blocked using PBS containing 5 % FCS and
0.2% saponin at RT for 30 min. All Ab were diluted in PBS containing 5 % FCS and
0.2% saponin. Filters were incubated for 2 h at RT with primary Ab listed in Table 1, washed
in PBS and then incubated for 90 min at RT with the corresponding secondary Ab listed in
Table 2. After PBS washing, filters were then incubated with 4',6-diamidino-2-phenylindole
(DAPI) at a concentration of 100 ng/ml in PBS (Invitrogen) for 30 min. Filters were cut out of
their holders, and mounted in Vectashield (Vector Laboratories) for observation using a Zeiss
LSM 710 Meta confocal microscope (Carl Zeiss, Germany) equipped with a 40X objective
(Cellular Imaging Facility platform, Lausanne University, Switzerland) and processed using
Zeiss ZEN 2009 light software.
Primary antibodies
Table 1: Primary antibodies, sources and working dilutions used for the labeling of Caco-2 cell monolayers
Description Source Working dilution
goat anti-human early endosome antigen 1 (EEA-1) Santa Cruz 1/50
mouse anti-human CD71 Clone A24, gift from
Monteiro R. 1/50
mouse anti-human lysosomal-associated membrane protein 1 (LAMP-1)
BD Pharmingen 1/10
rabbit anti-human pIgR Labmade 1/50
rabbit anti-human ZO-1 Invitrogen 1/200
Secondary antibodies
Table 2: Secondary antibodies, sources and working dilutions used for the labeling of Caco-2 cell monolayers
Description Source Working dilution
rabbit anti-goat IgG conjugated with Alexa Fluor 647 Invitrogen 1/100
goat anti-mouse IgG conjugated with FITC Dako 1/100
goat anti-rabbit IgG conjugated with Alexa Fluor 647 Invitrogen 1/100
124
2.6 Enzyme-linked immunosorbent assay (ELISA)
Basolateral compartment were recovered after apical incubation with various
stimulators and conserved at -20°C before ELISA measurements. 96-well plates
(Nunc-Immuno Plate Maxisorp surface, Nalge Nunc International) were coated overnight at
4°C with goat anti-mouse alpha chain (1/1000, Sigma) diluted in 100 µl of carbonate buffer
(pH 9.6). Plates were washed in PBS containing 0.05% Tween 20 (Biorad, PBS-T) before
blocking in 250 µl of PBS containing 1% bovine serum albumin (BSA, Sigma) for 2 h at room
temperature (RT). After washing in PBS-T, 100 µl of standards and samples were incubated
for 2 h at RT. Standards used were obtained with dilution of pIgAC5 with a known
concentration. All Ab and reagents were diluted in PBS containing 0.1% BSA. After washing,
plates were then incubated with biotinylated goat anti-mouse alpha chain (1/2000, Cappel)
for 2 h at RT. Final incubation with extravidin coupled with horseradish peroxidase (HRP)
(1/5000, Sigma) were performed for 1 h at room temperature and revealed with a 0.1 M
citrate sodium solution (pH 5) containing 1 mg/ml of O-phenylenediamine (OPD, Sigma) and
0.01% H2O2
. The reaction was stopped by the addition of 2 M sulfuric acid and the
absorbance was measured at 490 nm with 630 nm as reference.
2.7 Statistical analysis
The results are given as means ± the standard error of the mean (SEM). Two-tailed
nonparametric Mann-Whitney U test analysis was performed using the GraphPad 5 Prism
software. Differences were considered as significant when p values <0.05 were obtained
125
Figure 1. Confocal imaging of SIgA retrotransport across IEC reconstituted monolayers.
Caco-2 cell monolayers were apically incubated either with P-DMEM alone (panel A) or with 2 µg of
SIgAC5:Cy3 (panel B) for 18 h. Cells are localized via nuclear staining with DAPI while tight junctions
stabilizing the Caco-2 cell monolayer are emphasized with green labeling against ZO-1. Monolayers are
observed at different levels by performing Z-stack analysis. Representative pictures are depicted for
selected confocal plans: in the microvilli, at the apical junctional ring and at the level of the nucleus, as
schematically represented on the right. The presence of red dots detected only in the upper stage of the
monolayers incubated with fluorescently labeled SIgA confirms the retrotransport of these Ab from the
lumen-like pole to the basolateral pole. One representative field obtained from the observation of whole
filters prepared from 3 experiments performed in triplicates. Scale bars represent 20 µm.
DAPI ZO-1Cy3 MergeP-
DM
EMSI
gAC5
Cellular level
A
B
126
3 RESULTS
3.1 SIgA retrotransport across Caco-2 cell monolayers.
SIgA retrotransport across IEC has been described previously allowing the transport
of Ag into the lamina propria in the context of the celiac disease.167
In contrast,
retrotransport of SIgA alone has not yet been studied in the healthy epithelium. As a first
step toward this open issue, incubation of fluorescently labeled SIgA in the apical pole of
Caco-2 cell monolayer was thus performed. LSCM analysis revealed the strong
retrotransport of fluorescent molecules detected as intracellular red vesicles completely
absent in the control panels (Figure 1). Labeling of ZO-1 proteins, a partner in the TJ defining
the apical from the basolateral pole, allowed us to localize the vesicles inside the cells:
strikingly, SIgA positive intracellular compartments were restricted to the upper part of the
columnar cells, over the nuclei. In conclusion, LSCM observations of SIgA allowed us to first
demonstrate that SIgA uptake by epithelial cells can occur in our model of reconstituted IEC
monolayers. Whether or not this uptake resulted in basolateral exocytosis was assessed by
performing ELISA measurements of the Ab in the basolateral supernatants recovered after
initial incubation in the apical compartment. Concentration of SIgA in the basolateral pole
could be detected and reached mean values of 42 ng/ml. The series of preceding LSCM
observations combined with ELISA measurements (data not shown) clearly demonstrate the
uptake and retrotransport of SIgA across polarized IEC monolayers.
127
128
Figure 2. Multiple portals of entry for solutes into mammalian cells.
Macropinocytosis vesicles (or macropinosomes) dependent on the Rac/Rho family of GTPases mature to
early endosomes (EEA-1 positive vesicles).190 Clathrin-coated vesicles (CCVs) also mature to early
endosomes in a Rab5- dependent manner. In IEC, early endosomes can either recycle to the cell surface or
to the basolateral pole of IEC (controlled by Rab4 and/or Rab11) or progress to the degradation pathway of
the late endosome/lysosome (controlled by Rab7, LAMP-1 positive vesicles). Other possible endocytic
pathways are non-coated pits (non-CCV), or caveolae (dependent on caveolin). The Rab family which
represent a large spectrum of small GTPases allows the regulation and sorting of endocytic vesicles. Both the
secretory and the endocytic pathways are influenced by the Golgi apparatus . (Adapted from Sieczkarski and
Whittaker, J. Gen. Virol., 2002, 83:1535-45.)185
EEA-1
LAMP-1
RECEPTOR-MEDIATEDENDOCYTOSIS
or
129
Figure 3. Colocalization of IgAC5 with early endosomes but not with lysosomes in Caco-2 cell monolayers.
Caco-2 cell monolayers were apically incubated either with P-DMEM alone (first raw) or with 2 µg of
SIgAC5:Cy3 (second raw) or pIgAC5:Cy3 (third raw) for 18 h. Cells are localized via nuclear staining with
DAPI while intracellular compartments are labeled either for LAMP-1 (blue) or EEA-1 (green).
3D reconstitutions of Z-stack acquisitions are depicted: A. LAMP-1, EEA-1 and Ab detection; B. DAPI channel
alone; C. Side view combining SIgA and nuclei detection. The filter position (basal side of the Caco-2 cell
monolayer) is represented as a grey bar. The intracellular vesicles containing the (S)IgA are restricted to the
apical side of the monolayers. The presence of yellow dots demonstrates the presence of Ab in early
endosomes (EEA-1 positive vesicles). Nevertheless, no colocalization between SIgA and LAMP-1 associated
signals can be detected at this time point, indicating that Ab retrotransport does not result in lysosomal
addressing. One representative field obtained from the observation of whole filters prepared from
3 experiments performed in triplicates. Scale bars represent 20 µm.
DAPI
SIgA
C5pI
gAC5
Lamp-1 EEA-1 DAPIA. B. C.
P-D
MEM
130
3.2 SIgA retrotransport involves early endosome vesicles.
Among various mechanisms of endocytosis depicted in Figure 2185, IEC have been
described to use both the receptor-mediated entry indispensible for the internalization of
nutrients such as ferritin, and macropinocytosis responsible for bacterial toxin entry for
example.186, 187
To better characterize the pathway implicated in IgA retrotransport,
monolayers exposed to reconstituted SIgA and pIgA were first labeled for intracellular
compartments classically involved in endocytosis, i.e. early endosomes detected as EEA-1
positive vesicles and late endosomes/lysosomes characterized by LAMP-1 marker.
LSCM observations are depicted in Figure 3. Control panels obtained with Caco-2 cell
monolayers incubated in presence of P-DMEM alone demonstrated the dichotomy between
early and late endosomes: very few vesicles harbor both markers (Figure 3A). In presence of
SIgA or pIgA, a clear colocalization of IgA with EEA-1-positive intracellular compartments
seen as yellow dots suggests that early endosomes are involved in the retrotransport of IgA
across the monolayers (Figure 3B and C). In sharp contrast, no colocalization between IgA
and LAMP-1 positive vesicles could be detected, stressing that the internalization of IgA
molecules does not engage the lysosomal pathway, possibly protecting engulfed molecules
from potential intracellular degradation (Figure 3B and C). Interestingly SIgA and pIgA harbor
similar patterns of internalization suggesting that SC contribution to Ab uptake is reduced to
a minimum or is not necessary. Furthermore, the detection of red vesicles implies that other
intracellular compartments such as early macropinosomes not involved in classical
receptor-mediated endocytosis also participate in IgA cellular entry. These later results lead
us to the main conclusion that entry of SIgA results from receptor-mediated retrotransport
and potentially another pathway of endocytosis such as macropinocytosis; this issue was
assessed in the next section.
3.3 Pathways of internalization induced during SIgA retrotransport.
To confirm the involvement of receptor-mediated endocytosis in SIgA retrotransport,
Caco-2 cell monolayers were incubated with fluorescently-labeled transferrin recognized by
its specific receptor CD71, which serves as a marker of this route of entry.188 On the other
hand, in order to examine whether macropinocytosis was also involved, Caco-2 cell
131
Figure 4. SIgA retrotransport involves both macropinocytosis and receptor-mediated endocytosis.
Caco-2 cell monolayers were apically incubated either with P-DMEM alone (panels A-C) or with
2 µg of SIgAC5:Cy3 (panels D-F) for 4 h in combination with either P-DMEM (panels A and D), dextran
(green, panels B and E) or transferrin (green, panels C and F). Cells are localized via nuclear staining with
DAPI while tight junctions stabilizing the Caco-2 cell monolayer are emphasized with blue labeling against
ZO-1. Monolayers are observed at different confocal plans by performing Z-stack analysis. Representative
pictures are depicted for a selected cellular stage corresponding to the apical junctional ring. Yellow dots
detected in presence of SIgA and either the dextran or the transferrin indicate that both macropinocytosis
and receptor-mediated endocytosis are involved in the retrotransport of the Ab through the IEC
monolayers. One representative field obtained from the observation of whole filters prepared from
2 experiments performed in triplicates. Scale bar represent 20 µm.
DAPI ZO-1
P-D
MEM
SIgA
C5
P-DMEM dextran transferrin
A B C
D E F
132
Figure 5. Macropinocytosis and receptor-mediated endocytosis lead to SIgA retrotransport via early endosomes.
Caco-2 cell monolayers were apically incubated either with P-DMEM alone (panels A-C) or with 2 µg of
SIgAC5:Cy3 (panels D-I) for 4 h in combination with either P-DMEM (panels A, D and G), dextran (green,
panels B, E and H) or transferrin (green, panels C, F and I). Panels G to I represent higher magnification of
indicated regions on the panels D to F. Cells are localized via nuclear staining with DAPI while early
endosomes are detected with an Ab directed against EEA-1 marker. Monolayers are observed at different
levels by performing Z-stack analysis. Representative pictures are depicted for a selected confocal plan
corresponding to the apical junctional ring. Uptake of both dextran and transferrin led to the appearance of
EEA-1 positive intracellular compartments (panels B and C, arrow headed). Colocalization of SIgA with EEA-1
positive vesicles is confirmed by the detection of purple dots in panels A, D and G (arrow headed).
Finally, vesicles positives for SIgA, EEA-1 and dextran or transferrin seen as white dots indicate that both
pathways of SIgA retrotransport involve early endosomes at incubation time (Panels E, H and F, I).
One representative field obtained from the observation of whole filters prepared from 2 experiments
performed in triplicates. Scale bars represent either 20 µm (panels A-F) or 10 µm (panels G-I).
10 µm
A B C
D E F
DAPI EEA-1P-
DM
EMSI
gAC5
P-DMEM dextran transferrinSI
gAC5
20 µm
G H I
133
Figure 6. Basolateral concentrations of IgA upon Caco-2 cell apical incubation with SIgA alone or associated with dextran or transferrin.
SIgA concentration were measured by ELISA in basolateral supernatants recovered after apical incubation
with SIgA alone (white bar) or associated with either 1 mg/ml of dextran (grey bar) or 50 µg/ml of
transferrin (black bar) for 4 h. The presence of dextran reduced SIgA retrotransport in a non-significant
manner contrary to transferrin for which blockade was almost complete, indicating a competition between
transferrin and SIgA transport. Bars represent the mean values ± SEM. Statistically significant difference
between incubation with SIgA alone is indicated above the column. Data were obtained after analysis of
one experiment performed in triplicates.
0
10
20
30
40
50
*SIgAdextran+SIgAtransferrin+SIgA
IgA
conc
entra
tion
(ng/
ml)
p=0.024
134
monolayers were similarly incubated with fluorescently labeled-dextran, which transport
should be only fluid phase as dextran does not bind to the vesicular surface.
Double-positive vesicles for SIgA and either dextran or transferrin underscored that
both pathways of retrotransport are responsible for the Ab internalization (Figure 4).
Surprisingly, labeling depicted in Figure 5 further indicates that a few dextran-containing
intracellular compartments colocalized with EEA-1-positive compartments alone (turquoise
dots, Figure 5 B, E and H), or with fluorescently labeled Ab seen as white dots
(Figure 5 E and H). We also observed similar patterns of colocalization on filters co-incubated
with SIgA and transferrin, confirming that receptor-mediated endocytosis is also involved.
Retrotransport of SIgA across IEC monolayers induces the formation of early endosomes
and/or EEA-1-positive macropinosomes but not late endosomes, protecting it from potential
degradation. Concentrations of retro-transcytosed Ab in the basolateral compartment were
also assessed by ELISA (Figure 6). Noteworthy, the presence of transferrin inhibited the
basolateral release of Ab, suggesting a role for transferrin receptor in Ab recognition.
In sharp contrast, incubation with dextran resulted only in a limited non-significant drop in
SIgA retrotransport. These drastic differences may suggest that the essential part of the
delivery of the Ab to the basolateral compartment would be due to receptor-mediated
entry. It remains to be examined whether equimolar concentration of transferrin will lead to
similar observations. Nevertheless, confocal observations did not allow us to ascribe a
competitive role of transferrin in the cellular uptake of Ab as similar levels of red vesicles
were observed for all Transwell membranes. Taken together, these results led to two main
conclusions: macropinocytosis and receptor-mediated endocytosis, via early endosomes, are
responsible of SIgA uptake and transport across IEC monolayers and the transferrin receptor
seems to be involved in the latter pathway. Last but not least, inhibition of Ab exocytosis at
the basolateral pole does not imply a reduction of its apical uptake, suggesting that the
presence of transferrin with competitive functions affects the fate of SIgA along the
internalization pathways.
189
3.4 Receptor(s) involved in the retrotransport of IgA.
Our previous results suggest that the transferrin receptor (CD71) may be involved in
Ab retrotransport underlying the necessity to better characterize the receptor involved in
135
Figure 7. Expression of the transferrin receptor in the Caco-2 cell line.
A. Flow cytometric analysis of the surface (non-permeabilized) and intracellular (permeabilized) expression
of the CD71 (analyzed with the A24 clone) in the colonic Caco-2, the hepatic HepG2 and the renal HEK 293
cell lines. Open curves represent cells incubated with the secondary Ab alone. Shaded curves represent
counts of CD71-positive cells. B. and C. Quantification of the mean of fluorescence intensity (MFI) of the
cytometric analysis either onto non-permeabilized (B) or permeabilized (C) cells. As expected the hepatic
cell line HepG2 expresses both surface and intracellular CD71. This pattern of expression further marked in
the colonic Caco-2 cell line contrary to HEK293 cells harboring a highly reduced expression at their surface.
Bars represent the mean values. D. 3D reconstitution of a Z-stack acquisition is depicted: on the left, side
views of the CD71 detection combined with nuclei detection; on the right, the top view. The filter position
(basal side of the Caco-2 cell monolayer) is represented as a grey bar on the side views. The confocal
imaging of Caco-2 cell monolayers labeled for the CD71 figures out the effective expression of CD71 onto
differentiated cells. One representative field obtained from the observation of whole filters prepared from
2 experiments performed in triplicates. Scale bars represent 20 µm.
DAPI DAPI CD71MergeCD71
Non
-per
mea
biliz
edPe
rmea
biliz
ed
Caco-2 HEK 293HepG2 A
B C
D Side view Top view
MFI
Caco2 HepG2 HEK 2930
500
1000
1500
2000cellssecondary AbCD71
MFI
Caco2 HepG2 HEK 2930
2000
4000
6000
8000cellssecondary AbCD71
136
Figure 8. IgA containing vesicles are not detected colocalizing with the transferrin receptor.
Caco-2 cell monolayers were apically incubated with either P-DMEM alone (first raw), SIgAC5:Cy3 (second
raw) or pIgAC5:Cy3 (third raw) for 18 h. Cells are localized via nuclear staining with DAPI while the
transferrin receptors (CD71) is labeled in green. 3D reconstitutions of Z-stack acquisitions are depicted:
A. CD71 and Ab detection; B. DAPI channel alone; C. Side view combining the Ab and the nuclei detection.
The filter position (basal side of the Caco-2 cell monolayer) is represented as a grey bar. The intracellular
vesicles containing the Ab are restricted to the apical side of the monolayers. The absence of colocalization
(no yellow dots) do not allow us to further conclude on IgA recognition by the CD71. One representative
field obtained from the observation of whole filters prepared from 3 experiments performed in triplicates.
Scale bars represent 20 µm.
P-D
MEM
SIgA
C5pI
gAC5
DAPICD71 DAPIA. B. C.
137
Figure 9. IgA retrotransport is not mediated by pIgR.
Caco-2 cell monolayers were apically incubated with 2 µg of either with P-DMEM alone (first raw),
SIgAC5:Cy3 (second raw) or pIgAC5:Cy3 (third raw) for 18 h. Cells are localized via nuclear staining with
DAPI while the polymeric immunoglobulin receptors (pIgR) are labeled in green. 3D reconstitutions of
Z-stack acquisitions are depicted: A. CD71 and Ab detection; B. DAPI channel alone; C. Side view combining
the Ab, pIgR and the nuclei detection. The filter position (basal side of the Caco-2 cell monolayer) is
represented as a grey bar. The absence of colocalization (no yellow dots) indicates that the Ab
retrotransport does not result from IgA recognition by the pIgR. One representative field obtained from the
observation of whole filters prepared from 3 experiments performed in triplicates. Scale bars represent
20 µm.
P-D
MEM
SIgA
C5pI
gAC5
DAPIpIgR DAPI pIgRA. B. C.
138
the recognition of SIgA at the Caco-2 cell surface. Detection of the CD71 at the epithelial
surface has been essentially performed with biopsies obtained from individuals suffering
from celiac disease.167 Expression of CD71 was thus assessed in various epithelial-like cell
lines: the HEK293 cell line derived from kidney cells expected to express low levels of the
receptor, the HepG2 supposed to have high level of expression and the cell line Caco-2.188, 190
The other receptor present at the IEC surface is the pIgR.
Interestingly, the Caco-2 cells harbors the highest expression of both extracellular and
intracellular CD71 as compared with HepG2 cells, displaying an intermediate expression with
more than 2.5 less receptor detected intracellularly and extracellularly (Figure 7 A, B and C).
Finally, although expressed intracellularly (4 times less that Caco-2 cells), CD71 was, as
expected, barely detected at the surface of the HEK293 cell line. While flow cytometry
confirmed the expression of CD71 at the cell surface of non-differentiated cells,
LSCM complete these observations by demonstrating the expression of CD71 in polarized
monolayers (Figure 7D). Contrasting with ELISA results (Figure 6), LSCM observations of
monolayers incubated with pIgA or SIgA do not lead to the detection of IgA containing
vesicles positive for CD71 (Figure 8). This does not allow us to further conclude on the role of
CD71 in SIgA retrotransport, although it remains an open possibility that the most
appropriate time-point was not selected.
191 Although the presence of
SC in SIgA molecules should prevent IgA binding to pIgR, we decided to analyze whether or
not this receptor may allow retrotransport of pIgA. Interestingly, labeling of pIgR revealed
that this receptor was not only present at the basolateral pole but also at the upper
intracellular area of the monolayers (Figure 9, first line). Whether or not this receptor was
present at the cellular surface will be sorted out using membrane markers such as syntaxin 3
and 4.192
As expected, vesicles containing SIgA do not colocalize with pIgR
(Figure 9, second line) similarly to what is observed for pIgA (Figure 9, third line).
As already visualized in Figure 1, SIgA-positive vesicles are localized at the apical junctional
ring contrasting with a deeper intracellular presence of pIgR, indicating that various
intracellular compartments are involved in IgA retrotransport and pIgR intracellular
transport (Figure 9 C).
139
Figure 10. Hypothetical pathways of SIgA retrotransport across IEC monolayers.
SIgA retrotransport across IEC seems to be sustained by two distinct pathways: macropinocytosis
(highlighted by dextran uptake) and receptor mediated-endocytosis. Vesicles coming from both
pathways can fuse together resulting in the same early-endosome vesicles and a unique final secretion of
SIgA at the basolateral pole of enterocytes. This hypothetical fusion may also exclude
macropinocytosis-mediated entry of the Ab: dextran may enter independently from SIgA
(receptor-mediated entry exclusively). Colocalization between SIgA and dextran would thus result from the
late fusion within early endosome vesicles. Receptor-mediated endocytosis, demonstrated by
colocalization between SIgA and transferrin, involves CD71. This coincubation between SIgA and a large
excess of transferrin does not interfere with Ab uptake but results in the inhibition of SIgA release at the
basolateral pole of the monolayers. Differential redistribution of the Ab seems to be dependent on the
cellular sensing of the external environment and in particular, the transferrin concentration.
140
4 DISCUSSION
Our preliminary results demonstrate in vitro the presence of a pronounced
retrotransport of SIgA across polarized IEC monolayers. Both macropinocytosis and
receptor-mediated endocytosis (also known as clathrin-mediated endocytosis) seem to be
involved in the process. Interestingly but not conclusively, LSCM observations allow us to
describe the pathways involved in this entry leading to the transport of the Ab in early
endosomes but not along the late endosome/lysosome pathway (Figures 3 and 10, ).
Strikingly, entry by macropinocytosis of either dextran alone or combined with SIgA
leads to the formation of EEA-1-positive macropinosomes, commonly not associated with
the classical macropinocytosis pathway (Figure 5). Maturation of macropinosomes is still a
matter of debate: while well-defined in the context of professional APC such as
macrophages or DC, its contribution in the uptake of extracellular solutes as for example
nutrients and Ag at the surface of epithelial cells is much less delineated.193, 194
EEA-1-positive macropinosomes were recently described to be present in the human
epithelial carcinoma cell line A431.195 Thus, it is conceivable that the expression of the EEA-1
marker is not only involved in endocytosis, but also in other endocytic pathways.
In this scenario, resulting macropinocytosis vesicles would fuse with early endosomes and
allow both pathways to merge and converge toward basolateral secretion of Ab
(Figure 10, ). This latter hypothesis could also jeopardize the effective role of
macropinocytosis, in that SIgA could be taken in charge only via receptor-mediated
endocytosis in parallel with dextran-containing vesicles coming from macropinocytosis; the
colocalization in early endosomes would thus result from the fusion of both types of vesicles
(Figure 10, ). Besides the association of EEA-1 marker with macropinosomes, the study on
the A431 cell line also demonstrates that only a minority of vesicles containing both
transferrin and dextran can be observed, suggesting that, even if detectable, the fused
endosomes and macropinosomes represent only a small proportion of endocytic vesicles.195
Confocal imaging revealed a role of receptor-mediated endocytosis in SIgA uptake
based on transferrin colocalization with fluorescently-labeled SIgA (Figures 4 and 5).
Thus, to more precisely address the relative contribution of either macropinocytosis or
receptor-mediated endocytosis, specific inhibitors (eg: wortmannin for macropinocytosis,
chlorpromazine for receptor-mediated endocytosis) used either alone or in combination,
would bring valuable information with respect to SIgA retrotransport.
141
Similar detection of IgA-positive intracellular vesicles were obtained in presence of SIgA
alone, indicating that transferrin do not interfere with recognition and therefore SIgA
uptake. Measurement of SIgA retrotransported in the basolateral compartment led to
different conclusions regarding the final destination of the Ab-containing vesicles: the
release of SIgA at the basolateral pole was abolished. Thus, one can speculate that IEC
sensing of the lumenal environment such as high amounts of transferrin results in
modification of the intracellular addressing of the Ab (Figure 10, ).
Despite a clear detection of the receptor with the monoclonal Ab A24, confocal
imaging did not allow us to conclude to the effective SIgA-CD71 interplay as no colocalization
could be detected (Figure 8). This result suggests that either the interaction between the
receptor and the Ab is transient and could only be detected at earlier time points of
incubation, or the monoclonal Ab used does not allow proper observation by confocal
microscopy. The use of a commercial polyclonal Ab against CD71 could help to sort out this
issue. Furthermore, experiments were performed for either 4 h or 18 h of incubation,
indicating that the process detected results from a constitutive and steadily mechanism of
uptake. Nevertheless, the very early stages of Ab uptake have already been triggered,
underlying the crucial relevance of performing earlier time points to conclude on the
involvement of CD71.
In addition to the biological questions remaining open, more technical approaches
can also be suggested as for example whether high expression of CD71 in our reconstituted
IEC monolayer model could be directly related to the presence of transferrin in the culture
medium. Analysis of CD71 expression in cells deprived of transferrin would help us to
conclude on the mechanisms leading to expression of CD71 at the IEC surface. Furthermore,
results obtained in the present work were conducted with reconstituted mouse monoclonal
SIgA, providing precious information regarding the mechanisms involved in the Ab
recognition and, further putting forth a cross-reactivity in between species. To conclude on
hypothetical interspecies-conserved mechanisms, similar experiments should be carried out
using SIgA purified from human colostrum for example.
It becomes obvious that the preliminary descriptive results presented herein raise
more questions than we can answer. Thus, performing the below-proposed experiments
would further help in the understanding of a likely new specialized, not yet fully
characterized, pathway of internalization for mucosal Ab. In addition, whether or not this
142
phenomenon occurs in in vivo models has not yet been evaluated and will need to be tackled
in future experiments.
Notwithstanding, the contribution of SIgA recognition by IEC can be discussed in
further details. Because CD71 is known to preferentially bind IgA1 to IgA2 and moreover
pIgA1 as compared with SIgA1, such a described selectivity would imply that this receptor
could have a limited role in SIgA retrotransport in the intestinal context. Nevertheless, the
recognition and retrotransport of SIgA complexed with Ag across biopsies of patients
suffering from celiac disease has been published.167
In conclusion, we report on an in vitro model where polarized Caco-2 cell
monolayers-mediated SIgA retrotransport could be used as a basis for cellular and molecular
analyses of the role of IEC in the development of inflammatory diseases such as the celiac
disease, or in the education of underlying immune cells. Furthermore, understanding at the
cellular level how SIgA can be retrotransported from the lumen-like pole of enterocytes
represents another underestimated role of IEC in sensing the external environment and in
the modulation of mucosal homeostasis.
While CD71 expression has been
restricted to the basolateral pole of normal epithelial cells, abnormal apical expression has
been associated with the retrotransport of SIgA complexed with gliadin peptides. The
protection of these Ag by the Ab from potential intracellular degradation explains at the
molecular level the presence of Ag in the lamina propria inducing pathological inflammatory
processes. Our preliminary results argue for the effective retrotransport of SIgA associated
with a high expression of transferrin receptor that may further represent a new gate of entry
of potential harmful Ag, disrupting a fragile equilibrium. From a drastically opposite point of
view, this retrotransport would also be crucial in passive acquisition of Ab, present in high
amounts in maternal milk, into the newborn intestine exhibiting a not fully functional
mucosal immune system. Similarly, in newborns, retrotransported SIgA in the lamina propria
would thus actively participate in the clearance of potentially harmful Ag having escaped the
first line of intestinal defense. In vivo experiments will thus help to reach a better perception
of how SIgA retrotransport across the epithelium may occur physiologically.
143
Figure 1. SIgA and IEC: sensors of the intestinal content mediating mucosal homeostasis and
protection.
SIgA mediated IEC sensing of the gut microbiota may serve as a mean to educate and regulate the
underlying immune system leading to mucosal homeostasis. The protection of the epithelial monolayer
against infection results from diverse mechanisms involving the presence of specific SIgA. Co-incubation
with invasive S. flexneri, commensal bacteria and/or SIgA would further bring other clues on the combined
roles of the microbiota together with immune responses in fighting against invasion by intruders. Active
retrotransport of SIgA across IEC would also be involved in the modulation of immune responses in
particular in the newborn intestine. The mechanisms pertaining to the induction, regulation and
maintenance of the underlying immune responses remain in need of investigation.
144
CONCLUDING REMARKS / OUTLOOKS
Previous works obtained in the laboratory drove to assign M cells as a main portal of
sensing of the lumenal content allowing sampling of SIgA alone or in complexes with Ag by
immune cells of the lamina propria.178, 196
In the first part of this work, we provided new evidence on the multiple roles of SIgA
in maintaining intestinal mucosal homeostasis by modulating IEC responsiveness in presence
of commensal or probiotic bacteria. One can thus speculate that IEC sensing of the gut
microbiota serves as a mean to educate the underlying immune cells such as DC and
macrophages in order to maintain adequate immune regulation. Whether the production of
IEC-derived mediators modulated in presence of SIgA influences the development and the
regulation of the immune responses would be evaluated for example by co-culture
experiments between Caco-2 monolayers and DC purified from fresh peripheral blood
(Figure 1 ). Furthermore, the constant presence of bacteria in the gut lumen has been
described to play a direct role in IEC responsiveness (see Introduction section
Here, polarized Caco-2 monolayers allowed us to
decipher at the cellular level the pathways of activation leading to either homeostasis with
commensal bacteria or protection against pathogens. Despite their relative static position
between the outside environment and the inner compartment, it becomes clear that IEC is
an underestimated partner, which probably actively intervenes on the modulation of innate
and adaptative immunity.
4.3).
Nevertheless, how SIgA can modulate these responses in vivo remains to be elucidated. This
latter may be addressed upon injection of commensal bacteria either alone or in complexes
with non-specific SIgA in mouse ileal ligated loops in area deprived of PP. Subsequent
analyses would then focus on cell subtypes potentially involved in this not yet described
natural Ag sampling and their maturation.
We further demonstrate for the first time the involvement of carbohydrate residues
at the surface of SIgA in mediating the natural interaction with commensal bacteria.
Nevertheless, the precise motifs, either on the bacterial cell wall or on the sugar residues,
involved in this interaction remain to be identified. Competitive experiments using excess of
selected carbohydrates or, specific deglycosylation directed against glycan motifs such as
145
β-galactose, fructose or mannose should be performed to define key sugar motifs.
On the other hand, competitive experiments using excess of purified bacterial cell wall
components such as peptidoglycan or lipoteichoic acid or, specific bacterial mutagenesis
directed against cell wall components would further help in triggering the crucial bacterial
counterparts. Exploring such aspects would bring further information on the role of SIgA in
maintaining mucosal homeostasis, and reinforce the contribution of N-glycans at mucosal
surface.
A notion recently emerges that opposes symbiont-associated molecular patterns
(SAMP) to pathogen-associated molecular patterns (PAMP) revealing that how cells can
distinguish commensals from pathogens is still a “crowd-puller” with grays areas.197
In a second part, the protective role of SIgA at the cellular level during S. flexneri
infection has been described in vitro involving Ab agglutinating properties. SIgA thus
interferes with bacterial growth, the cellular recognition of invasive bacteria and the
disruption of the TJ and the cytoskeleton. Interestingly, we have also demonstrated that the
presence of commensal bacteria promoted the expression of TJ proteins, thus increasing
cellular cohesion. One can thus imagine that commensal bacteria could reinforce SIgA
mediated anti-inflammatory properties by promoting cell-cell interactions and by competing
with recognition sites at the IEC surfaces during infection by S. flexneri (Figure 1 ).
First evidences are emerging demonstrating a potential role of probiotics in limiting Shigella
invasion but the precise mechanisms underlying these processes remain unclear.
In accordance with this notion, IEC low responsiveness facing commensals or probiotics
contrasts with the aggressive events occurring during the inflammatory and destructive
processes caused by S. flexneri. This latter dichotomy further emphasizes the crucial role of
performing in vitro experiments to better define the cellular components resulting in a
differential recognition of bacterial patterns leading either to homeostasis or to
inflammation.
198, 199
Thus performing in vitro experiments using polarized IEC monolayers infected with invasive
S. flexneri, commensal bacteria and/or SIgA would also bring valuable insights in the process
involved and would help in the overall understanding of the multiple layers in mucosal
protection. Interestingly, despite in vitro and in vivo studies aiming at developing a detailed
picture of how S. flexneri causes disease, 100 years from their discovery have not been
146
enough to develop safe treatments to cure this endemic pathogenesis as the extensive use
of antibiotic has led to the emergence of resistant strains.200 Together with this latter
observation and the cost of antibiotic treatments, the World Health Organization has
preconised the development of safe and effective treatments such as vaccines against
S. flexneri.105
In the last part of this work, we described IEC as a novel partner in the direct SIgA
uptake from the lumen-like pole, associated with two main pathways of entry i.e. the
macropinocytosis and the receptor-mediated endocytosis. Future in vitro experiments
detailed in the Part III section
Such a novel vaccine or treatment requires a precise dissection of the
mechanisms involved at the cellular level leading in particular to the maintenance of
acquired mucosal immune responses which remain poorly understood to date.
Thus, deciphering in vitro the role of SIgA together with selected commensal strains in
mediating the education of the underlying immune system via the direct sensing by the IEC
may represent a first step toward the development of effective treatments.
4 will allow us to better characterize this new gate of re-entry
of the Ab. Our preliminary data did not permit us to conclude on the identity of the
receptor(s) expressed at the apical pole of IEC involved in SIgA recognition; further
characterization of this potential receptor(s) would allow us to better understand at the
cellular level the mechanisms underlying the complex dialogue established between IEC and
SIgA during homeostasis with the microbiota and the protection against pathogens.
The Ab retrotransport was also proposed among others as a way to educate the immune
system especially in the newborn intestine (Figure 1 ). In vivo experiments using injection
of SIgA in mouse ileal ligated loops in areas deprived of PP will also help us to tackle whether
this phenomenon could naturally occurs in the normal intestine.
In conclusion, this work shed light on how both SIgA and IEC work in synergy to allow
both the maintenance of a fragile intestinal equilibrium with the microbiota and the
protection against the entry of intruders. The dissection of this fine tuned balance finally
opens new avenue of researches in understanding how non-immune cells can mediate the
education of the underlying immune cells in the intestinal environment.
147
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