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Ana Carolina PagliaroniCláudia Danella PolliPatrícia Assis
Infected Cell in Trouble: Bystander Cells Ring the Bell
Immunity 33, November 24, 2010
IL-8 (CXCL2)TNF-αGM-CSF
Phagocytes
AP-1 H3
Proinflammatory cytokines
Shigella Listeria
Curr Opin Immunol. 2008; Aug 20(4):377-82
+
Entire animal
Tissues
Pool or population of cells
+
November 2010 | Volume 6 | Issue 11 | e1001194
Listeria monocytogenes model
Adhesion and invasion
LLOphospholipases
Actin polymerization
Intercellular transmission
Listeria monocytogenes
Infection mechanism
Shigella flexneri
OspF and OspG(virulence factors)
Potente inhibitor of JNK, ERK and p38 signaling
Shigella
J Biol Chem. 2003 Sep 5 278(36) 33878-86.
Intense inflammatory
response
Hypothesis : a horizontal intercellular
communication between intestinal epithelial
cells might help to amplify the innate immune
response?
Hypothesis : a horizontal intercellular
communication between intestinal epithelial
cells might help to amplify the innate immune
response?
Immunity 33, 804–816, November 24, 2010
Ana Carolina Ana Carolina PagliaronePagliarone
Aim: to investigate whether the intercellular comunication between infected and non-infected cells contributes to innate response against Listeria
monocytogenes infection .
The activation of intestinal epithelial cells depends on the L. monocytogenes intracellular localization?
actA mutant Listeria
(LLO-deficient)
m-ICcI2 cells + siRNA
actA mutant Listeria (no cell-to-cell spread)
ELISA
4h
m-ICcI2 cells
flowcytometry
hly mutant or WT Listeria expressing GFP
The activation of intestinal epithelial cells is dependent on bacterial presence in cytosol (endosomal lysis)
Non-infected epithelial cells are activated during infection challenge?
m-ICcI2 cells flow cytometryWT Listeria –GFP(PactA-gfp)
4h
Non-infected cells are activated during infection challenge?
m-ICcI2 cells
RT-PCR
PSOD–gfp Listeria
Non-infected epithelial cells are ativated to a greater extend than infected cells during infection .
The activation of non-infected cells depends on the bacterial cell-to-cell spread?
Non-infected cells are activated even when there is no bacterial spread to neighboring cells.
m-ICcI2 cells flow cytometry or ELISA
actA PactA-gfp Listeria
Bacterial products are responsible for activating non-infected cells?
4h p.i
Bacterial products are not reponsible for activating non-infected cells.
WT Listeria
Recombinant LLO
m-ICcI2 cells
or
Cell culture medium +WT Listeria
filtrationBacteria free media
m-ICcI2 cells
4h p.i
Activation of non-infected cells occurs through gap junctions?
Gap junction inhibitors
actA PactA-gfp
4h
The non-infected cells can be activated even when gap junctions are inhibited.
actA PactA-gfp Listeria m-ICcI2 cells + gap junctions inhibitors
Flow cytometry
Secreted products of infected cells are responsible for inducting the activation of non-infected cells?
m-ICcI2 cells
Brefeldin A (BFA)
actA PSOD–gfp Listeria
30 min
OR
Brefeldin A (BFA)
actA PSOD–gfp Listeria
m-ICcI2 cells
60 min
Flow cytometry
Secreted products of infected cells are not responsible for inducting non-infected cells activation.
....but unstable and highly reactive host-derived factors cannot be excluded by the previous results!!!
Listeria infection induces reactive oxygen intermediates (ROIs) production in non-infected cells
Act A mutant Listeria
Oxygen and nitrogen radicals are involved the activation of non-infected cells?
actA PactA-gfp Listeria
DPI: NADP oxidase inhibitor
L-NAME : nitric oxide sinthase (NOS) inhibitor
Oxygen radicals synthesis are involved in the activation of non-infected cells.
flow cytometry actA PactA-gfp Listeria m-ICcI2 cells + DPI orL-NAME
4h
ROIs induce ERK activation in non-infected cells during Listeria infection?
ROIs induce Erk activation in non-infected cells .
WT Listeria
ImmunoblottingWT Listeria m-ICcI2 cells + iRNA
50min
Nox4 is responsible for the Cxcl-2 production in non-infected cells?
Immunoblotting and ELISA assay
actA PactA-gfp Listeria
m-ICcI2 cells + siRNA
4h
Nox4 induces Cxcl-2 production in non-infected cells .
professional immune cells attraction
CONCLUSION
increased host Innate response
IL-8
AP-1 H3
OspF
Hypotesis
A host cell-cell communication mechanism are circumvents the bacterial effector proteins amplifying IL-8 expression
Hypotesis
A host cell-cell communication mechanism are circumvents the bacterial effector proteins amplifying IL-8 expression
HeLa1 h
p65 nuclear translocationImmunofluorescence microscopy
S. flexneri
Inflammation Mechanism of Epithelial Cells: characterization at the single-cell level
Uninfected cells surrounding infected cells shown NF-κB activation Uninfected cells surrounding infected cells shown NF-κB activation
Shigella spread to adjacent cells by actin-based motilityShigella spread to adjacent cells by actin-based motility
Bystander NF-κB activation is due bacterial intercellular motility?
HeLa
1 hora
F-actin (FITC-phalloidin)
p65 nuclear translocationImmunofluorescence microscopy
S. flexneriWt
∆virG
NF-κB activation was not caused by intercellular motility, but reflected instead a novel host response to bacterial infection
NF-κB activation was not caused by intercellular motility, but reflected instead a novel host response to bacterial infection
HeLa90 min
p-JNK, p-ERK and p-p38Immunofluorescence microscopy
S. flexneri∆virG
JNK, ERK e p38 are also activated in bystander cells in S. flexneri infection?
p-JNK p-ERK p-p38
JNK, ERK and p38 also propagates from infected to bystander cells during S. flexneri infection
JNK, ERK and p38 also propagates from infected to bystander cells during S. flexneri infection
Bystander cells are actively producing IL-8?
HeLa6 h
ELISA IL-8S. flexneri
∆virG
Bystander cells are the main producers
of IL-8 ?
HeLa3 h
IL-8Immunofluorescence microscopy
S. flexneri
monesin
∆virG
Green: S. flexneri
Red: IL-8
Blue: Hoechst
Gray: F-actin
Bystander cells are actively producing IL-8?
Bystander cells are the main source of IL-8 during S. flexneri infectionBystander cells are the main source of IL-8 during S. flexneri infection
HeLa90 min
S. flexneri∆virG∆ospF
p-p38Immunofluorescence microscopy
Bacterial virulence factors could impair bystander cell activation?
p38 desphosphorylation by OspFp38 desphosphorylation by OspF
HeLa3 h
IL-8Immunofluorescence microscopy
S. flexneri∆virG∆ospF
OspF failed to impair the ability of the host to spread p38 activation to neighboring cells and induce IL-8 expression
OspF failed to impair the ability of the host to spread p38 activation to neighboring cells and induce IL-8 expression
Bacterial virulence factors could impair bystander cell activation?
3 h
TriDAP
IgG Alexa488
monesin
Pathogen sensing via Nod-1 is sufficient to induce bystander IL-8 expression?
IL-8Immunofluorescence microscopy
Green: TriDAP
Red: IL-8
Blue: Hoechst
Gray: F-actin
IgG
TriDAP
Nod-1-mediated recognition was necessary and sufficient to induce IL-8 expressionNod-1-mediated recognition was necessary and sufficient to induce IL-8 expressionL-Ala-D--Glu-Meso-diaminopimelic acid
What is the mechanism of cell-cell communication
between infected and bystander cells?
What is the mechanism of cell-cell communication
between infected and bystander cells?
Bystander cells activation is due to factors secreted by the infected cell?
90 min
BFAS. flexneri
∆virG
60 min
IL-8Immunofluorescence
microscopy Flow Chamber
HeLa
IL-8Immunofluorescence
microscopy
S. flexneri
∆virG 10 min
Culture Flow
Staining Imaging
Cell-cell propagation of proinflammatory signals was not mediated by secreted proteins or soluble factors
Cell-cell propagation of proinflammatory signals was not mediated by secreted proteins or soluble factors
Bystander cells activation is due to cell-cell contactwith the infected cell?
90 minImmunofluorescence
microscopy
S. flexneri∆virG
90 minImmunofluorescence
microscopy
S. flexneri∆virG
18β-GA
Subconfluent density
1
2
3
18β-glycyrrhetinic acidIL-8 expression by bystande cells was mediated by comminication through gap junctionsIL-8 expression by bystande cells was mediated by comminication through gap junctions
Bystander activation via cell-cell contact is dependent of gap-junction?
A431-Cx43
S. flexneri∆virG
A431
S. flexneri∆virG
90 minImmunofluorescence
microscopy
90 min1 2 3 4
Infected Cell
Bystander activation via cell-cell contact is dependent of gap-junction?
IL-8Immunofluorescence
microscopy
A431-Cx43
S. flexneri∆virG
A431-Cx43A431
70/30
10/90
Cx-43 IL-8
Bystander activation via cell-cell contact is dependent of gap-junction?
Immunofluorescence microscopy
The propagation of inflammation during bacterial infection of epithelial cells depends on conexin gap junctions
The propagation of inflammation during bacterial infection of epithelial cells depends on conexin gap junctions
CONCLUSION
Ca2+ IP3 cAMP
Ca2+ IP3 cAMP