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Matam Kumar
(Vijay)
Fred Carvalho
(La Machine)
Gut microbiota, diet, chronic
inflammation and metabolic syndrome
Andrew Gewirtz, Institute for Biomedical Sciences
Georgia State University, Atlanta GA
Illustration by Micah Lidberg , Proto, MGH
Mucosal defenses maintain permit host-microbiota coexistence
Muc2 (mucin layer)
Bacteria
Actin
DNA
Antimicrobial peptides (AMP)
IgA, IgG
TLRs/NLRs
AMP
Immune cell recruitment
Dysbiosis (i.e. perturbed microbiota)
Encroaching, pro-inflammatory microbiota
Chronic inflammation
Take-home message
Impairment of mucosal anti-microbial defense
1. Innate immune deficiency
2. Chemical disturbance of microbiota
3. Mucus atrophy from low fiber diet
Colitis Metabolic Syndrome
HIV
HIV-related metabolic abnormalities
Metabolic Syndrome: A constellation of metabolic abnormalities
associating with insulin resistance including obesity,
hyperglycemia, hyperlipidemia, and hypertension. Metabolic
syndrome often progress to type 2 diabetes, hypertension,
cardiovascular disease, and/or liver disease.
Similarities between metabolic syndrome and IBD
1)Increased expression of similar pro-inflammatory genes/markers.
2)Associated with alterations in gut microbiota
3)Require microbiota – i.e. No disease in germ-free mice.
4)Feature remodeling of adipose tissue
5)Increasing incidence in last 50 years (amidst constant genetics)
Inflammatory Bowel Diseases (IBD): Collective term for chronic
inflammatory diseases of the intestine including Crohn’s disease
and ulcerative colitis.
Inflammation: Central feature of a spectrum of diseases
HIV:
✔️
✔️
✔️
?
IL-8
S. typhimurium
Flagellin
Y
NF-kB
TLR5
TJ
Commensal microbes
IBD?
Flagellin receptor TLR5 Coordinates
clearance of motile bacteria
Flagellin monomers
(About 70,000 per flagella)
IEC
Immune cell recruitment
Rapid bacterial clearance
AMP
WT T5KO0.0
0.5
1.0
1.5
2.0
2.5
40%
WT T5KO0
1
2
3
4
28%
WT T5KO0.0
0.3
0.6
0.9
1.2
1.5
27%
Co
lon
wei
gh
t [
% b
od
y m
ass
]
Cec
um
wei
gh
t [
% b
od
y m
ass
]
Sp
leen
wei
gh
t [
% b
od
y m
ass
]
T5KO mice develop spontaneous colitisT5KO
(10%)52/500
WT
500/500
Vijay-Kumar et. al. 2007. J. Clin. Invest
T5KO mice develop obesity
4 6 8 10 12 14 16 18 20
20
25
30
35
Week
Bod
y W
eigh
t (g)
4 6 8 10 12 14 16 18 20
15
20
25
Week
Bod
y W
eigh
t (g)
Males Females
WT
T5KO
WT
T5KO
WT T5KO
Fat
Pad
[g
]
Males
WT T5KO
WT T5KO
Females
0.1
0.2
0.3*
0.25
0.50
0.75
*
Vijay-Kumar et. al. 2010. Science
0 30 60 90 120
100
150
200
250
300
350
400
Time [mins]
Blo
od
Glu
co
se [
mg
/dL
]
T5KO exhibit hyperglycemia/insulin resistance
*
WT T5KO
60
85
110
135
Fasti
ng
Glu
co
se
[mg
/dL
]
Glucose Tolerance
0 30 60 90 12050
60
70
80
90
100
Time [min]Blo
od
Glu
co
se [
% s
tart
ing
]
T5KO
WT
Insulin Sensitivity
+ Ins.
*
WT T5KO
0.25
0.50
0.75
1.00
Se
rum
In
su
lin
[n
g/m
L]
0.4
0.8
1.2
1.6
Seru
m I
nsu
lin
[n
g/m
l]
Glucose: - + - +
WT T5KO
*
Insulin Production
T5KO
WT
Vijay-Kumar et. al. 2010. Science
Tracking of inflammatory marker fecal lipocalin 2 (Lcn2)
126 8 104
Fecal Lcn2
(pg/mg stool)
101
102
103
100
Age (weeks)
Colitic T5KO
Met syn T5KO
WT
Carvalho et. al. 2012. Cell Host & Microbes
Colitic T5KO
WT
Non colitic
T5KO
Colitic T5KO
T5KO mice exhibit taxonomical
alterations in the gut microbiota
Summary 1
Innate immune deficiency (T5KO)
Compensatory Activation of other components of immune system
Mismanaged gut microbiota
Severe colitis
20% Failure rate
Lack of successful breeding
80% “Success”
Plentiful breeding
(but metabolic syndrome)
Microbiota
Increased caloric consumption (obesity)
Nutrient Excess
Endoplasmic reticulum stress
Pro-inflammatory Signaling (NF-kB, MAPK, JNK)
Desensitization of insulin receptor signaling (i.e. insulin resistance)
Metabolic Syndrome
“Inflammatory explanation” for insulin resistance (Hotamisligil)
T2D CVD NASH
(& other metabolic pathways)
How would low-grade inflammation cause metabolic syndrome?
Conv WT-GF T5KO-GF0
5
10
15
20
25
Fecal L
ipo
calin
2
[ng
/g f
eces]
GF-WT GF-T5KO18
20
22
24
26
28
30
Bo
dy W
eig
ht
[g]
GF-WT GF-T5KO0.0
0.5
1.0
1.5
Fat
Pad
[% B
od
y W
eig
ht]
7-log difference in 16s RNA
Conv WT-GF T5KO-GF
100
101
102
103
104
105
106
107
108
Cecal B
acte
ria [
CF
U/m
g]
Lack of Phenotype in Germ-freeT5KO mice
Carvalho et. al. 2012. Cell Host & Microbes
0
50
100
150
200
250
WT T5KO WT T5KO WT T5KO
Weeks Post-transplant:
0 2 4
Fecal
lip
ocalin
-2 6
2
4
g/m
ou
se/2
4 h
WT T5KO
Food consumption
Conventionalizing germ-free mice restores metabolic syndrome in T5KO
600
400
200
Epididymal fat pad
(mg)
WT T5KO
100
110
120
130
140
150
160
170
0 7 14 21 27 35 42 49 56 63 70 77 84
% w
eig
ht
gain
WT
T5KO
WT T5KO
Fasting glucose
(mg/ml)
Days post-conventionalization
75
100
125
T5KO microbiota is sufficient to transfer
metabolic syndrome to WT germfree mice
WT T5KO
60
80
100
120
4.0
4.5
5.0
5.5
0 20 40 600
50
100
Days post transplant
% W
eig
ht
Gain
WT T5KO
0.50
0.75
1.00
Fo
od
In
tak
e [
g/m
ou
se
/da
y]
Se
rum
In
su
lin
[n
g/m
l]
Fa
sti
ng
Glu
co
se
[m
g/d
L]
***
0.25
0.50
0.75
1.00
Fa
t P
ad
[g]
WT T5KO
0 50 100 150 200
50
75
100
Time [min]
Blo
od
Glu
co
se
[%
sta
rtin
g v
alu
e]
WT T5KO
*
T5KO
WT
WT T5KO
WT
T5KO
Vijay-Kumar et. al. 2010. Science
Day 0PC1 (13%)
PC2 (11%)
TLR5fl/fl
TLR5DIEC
Day 42PC1 (15%)
PC2 (10%)
TLR5fl/fl
TLR5DIEC
Day 84PC1 (17%)
PC2 (14%)TLR5fl/fl
TLR5DIEC
Loss of IEC TLR5 changes microbiota composition
Fli
C (
µg
/g f
ec
es
)
6 3 5 6 3 7 7
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0 T L R 5f l / f l
T L R 5D IE C
LP
S (
µg
/g f
ec
es
)
6 3 5 6 3 7 7
0 . 0
0 . 5
1 . 0
1 . 5
2 . 0 T L R 5f l / f l
T L R 5D IE C
Fecal Flagellin Fecal LPS
Making it inherently more pro-inflammatory
Chassaing et. al. 2014. Gastro
Ba
cte
ria
/ m
g o
f co
lon
ic m
uco
sa
T L R 5f l/ f l
T L R 5D I E C
T L R 5DD C
1 0 4
1 0 5
1 0 6
Dis
tan
ce o
f ba
cte
ria
fro
m IE
C (
µm
)
T L R 5f l/ f l
T L R 5D I E C
T L R 5DD C
0
1 0
2 0
3 0
4 0
Loss of IEC TLR5 decreases microbiota-IEC distance
WT IEC-TLR5KO DC-TLR5KO
WT IEC-TLR5KO DC-TLR5KO WT IEC-TLR5KO DC-TLR5KO
Distance Adherent Bacteria
Chassaing et. al. 2014. Gastro
Microbiota encroachment is a feature of metabolic syndrome in humans
BMI = 21.1, Glucose = 114 mg/dL
HbA1C = 5.3, Diabetes mellitus = NoBMI = 34.33, Glucose =148 mg/dL
HbA1C = 7.7, Diabetes mellitus = Yes
Bacte
rial
dis
tan
ce t
o I
EC
(0-4
0m
M)
B lo o d g lu c o s e c o n c e n tra tio n (m g /d L )
Dis
tan
ce
of
ba
cte
ria
fro
m I
EC
(m
m)
1 0 0 2 0 0 3 0 0
0
1 0
2 0
3 0
4 0
B M I
Dis
tan
ce
of
ba
cte
ria
fro
m I
EC
(m
m)
2 0 3 0 4 0 5 0
0
1 0
2 0
3 0
4 0
H B A 1 C (% )
Dis
tan
ce
of
ba
cte
ria
fro
m I
EC
(m
m)
4 6 8 1 0
0
1 0
2 0
3 0
4 0
Blood Glucose50 200 20 50 4 10
BMI HBA1C
Chassaing et. al. 2017 CMGH
What might have changed the microbiota
and/or its promotion of inflammation?
Microbial environmental factors
Antibiotics
Hygiene
Loss of parasites
Presence or absence of viruses
Dietary factors
Altered macronutrient content (fat, fiber, protein carbs)
Altered micronutrient content (toxins, loss of labile nutrients)
2 Commonly used emulsifiers
Carboxymethylcellulose (CMC)Polysorbate 80 (PS80)
Increasingly used since mid-20th century in rough correlation with increasing
incidence of some chronic inflammatory diseases such as IBD and metabolic
syndrome.
Emulsifiers: Detergent-like molecules that stabilize mixtures of
immiscible liquids. Commonly used as food additives to improve
texture and homogeneity (shelf-life).
Dis
tan
ce
of
ba
cte
ria
fro
m I
EC
(m
m)
W a t e r C M C P 8 0
0
1 0
2 0
3 0
4 0
5 0
CMC and PS80 decrease bacteria-epithelial distance in WT mice
Water
CMC
PS80
Bacte
ria-e
pithelia
dis
tance, m
M
Water CMC PS80
Chassaing et. al 2015 Nature
Ba
cte
ria
/mg
of
co
lon
ic m
uc
os
a
W a t e r C M C P 8 0
1 04
1 05
Water
CMC
P80
PC1 (22%)
PC2 (13%)
WT
Emulsifiers increase adherent bacteria and change
microbiota composition
Bacte
ria/m
g c
olo
n
PC
2
PC1
Chassaing et. al 2015 Nature
Water CMC PS80
Water
CMC
PS80
T i m e ( d a y s )
FliC
(
mg
/g
fe
ce
s)
7 5 6 9 1
0
5
1 0
1 5 W a t e r
C M C
P 8 0
T i m e ( d a y s )
LP
S (µ
g/g
fe
ce
s)
7 5 6 9 1
0
1
2
3
Emulsifiers increase adherent bacteria and change
microbiota composition
WaterCMCPS80
WaterCMCPS80
Time (d) on emulsifiers
Flagellin LPS
Chassaing et. al 2015 Nature
* *
* *
#
T i m e ( d a y s )
Bo
dy
we
igh
t (
re
lativ
e v
alu
es
)
0 2 0 4 0 6 0 8 0 1 0 0
1 0 0
1 1 0
1 2 0
1 3 0
1 4 0
1 5 0
W a t e r
C M C
P 8 0
Fa
t p
ad
(m
g)
W a t e r C M C P 8 0
0
2 0 0
4 0 0
6 0 0
8 0 0
Fo
od
in
tak
e (
g p
er 2
4h
pe
r m
ou
se
)
W a t e r C M C P 8 0
4
5
6
7
15
h f
as
tin
g b
loo
d g
luc
os
e (
mg
/dL
)
W a t e r C M C P 8 0
6 0
8 0
1 0 0
1 2 0
* *#
Emulsifiers CMC and P80 induce metabolic syndrome
Body weight Fat mass Food intake Fasting glucose
T i m e ( d a y s )
Bo
dy
we
igh
t (
re
lativ
e v
alu
es
)
0 2 0 4 0 6 0 8 0 1 0 0
1 0 0
1 5 0
2 0 0
2 5 0
3 0 0 W a t e r
C M C
P 8 0
Germ-FreeF
at
pa
d (
mg
)
W a t e r C M C P 8 0
0
2 0 0
4 0 0
6 0 0
8 0 0
Fo
od
in
tak
e (
g p
er 2
4h
pe
r m
ou
se
)
W a t e r C M C P 8 0
4
5
6
7
5h
fa
sti
ng
blo
od
glu
co
se
(m
g/d
L)
W a t e r C M C P 8 0
6 0
8 0
1 0 0
1 2 0
in a microbiota-dependent manner
Body weight Fat mass Food intake Fasting glucose
Chassaing et. al 2015 Nature
Chow
“High-fat” Diet
“High-fat diet” induces microbiota encroachment
0
2
4
6
8
Dis
tan
ce
of
ba
cte
ria
to
IE
C (m
m)
Chow HFD
10
20
30
40
0
Zou et. al. Cell Host & Microbe (2018)
“High-fat” diet
Lard, Casein, Maltodextrin, Sucrose, Cellulose, Soybean Oil,
Mineral Mix, Calcium Phosphate Calcium Carbonate,
Potassium Citrate, Vitamin Mix, Choline Bitartrate, dye
Assembled from purified ingredients, 60% calories from fat:
Grain-based, relatively unrefined ingredients, 10-12% calories from fat:
Dehulled soybean meal, ground corn, dried beet pulp, fish meal, ground oats,
dehydrated alfalfa meal, cane molasses, brewers dried yeast, wheat germ,
whey, citric acid-preserved porcine animal fat, wheat middlings, porcine meat
and bone meal, salt, vitamin/mineral mix.
“Low-fat diet”: Purina 5001
18-22% fiber, both soluble (fermentable) and insoluble (not easily fermentable)
5% fiber, insoluble (not easily fermentable)
Chow
CD
D (
10%
)
CD
D (
60%
)
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
Co
lon
we
igh
t (g
/10
0g
of
BW
)
Chow
CD
D (
10%
)
CD
D (
60%
)
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
Ce
ac
um
we
igh
t (g
/10
0g
of
BW
)
Defined diets result in profound loss of gut mass
Chow HFDLow-fat
control
Chow HFDLow-fat
controlChow HFD
Low-fat
control
Colon weight
(g/100g BW)
Cecum weight
(g/100g BW)
Chassaing et. al. 2016. AJP-Gastro
Chow HFD
HFD: + Cell HFD: + Inul
Chow + Cell + Inul
HFDCho
w
50 c
ell
200
cell
200
inul
400
500
600
700
800
900
Co
lon
cry
pt
len
gth
(m
m) **
**
Chow + Cell + Inul
HFD
**
**
Chow
HFD
+50
Cel
l
HFD
+200
Cel
l
HFD
+200
Inul
100
150
200
Co
lon
weig
ht
(mg
)
“High-fat diet” (HFD) results in gut atrophy;
restored by fermentable fiber inulin
Zou et. al. Cell Host & Microbe (2018)
Cho
w
50 C
ell
200
Cel
l
200
Inul
0
5
10D
ista
nc
e o
f b
ac
teri
a t
o I
EC
(m
m)
Chow - Cell Inul
HFD
Inulin restores IEC-bacterial distance
Zou et. al. Cell Host & Microbe (2018)
Chow - Cell Inul
HFD
Chow - Cell Inul
HFD
GF miceConventional miceCho
w
50 C
ell
200 Cell
200 Ilu
n
Cho
w
50 C
ell
200 Cell
200 Ilu
n
0
100
200
300
400
Co
lon
Weig
ht
(mg
)
400
600
800
1000
Co
lon
villi len
gth
(m
m)
Effects of inulin are microbiota dependent
Chow - Cell Inul
HFD
Chow - Cell Inul
HFD
GF miceConventional mice
Zou et. al. Cell Host & Microbe (2018)
Cry
pt
len
gth
, m
M
HFD alters/depletes microbiota: Inulin restores
Chow - Cell Inul
106
107
bacteria / m
g o
f feces
HFD
Ba
cte
ria/m
g f
ece
s
106
107
Chow
HFD
+ Cellulose
+ Inulin
and changes
Zou et. al. Cell Host & Microbe (2018)
Inulin rescues some aspects of HFD-induced
metabolic syndrome
Fa
t p
ad
(m
g) **
***
**
*
Chow
HFD
+50 C
ell
HFD
+200
Cel
l
HFD
+200
Inul
Chow
HFD
+50 C
ell
HFD
+200
Cel
l
HFD
+200
Inul
Chow
HFD
+50 C
ell
HFD
+200
Cel
l
HFD
+200
Inul
500
1000
Epididymal fat
Mesenteric fat
Subcutaneous fat
Fat
pad
(m
g)
Chow
HFD
+50
Cel
l
HFD
+200
Cel
l
HFD
+200
Inul
50
100
150
200
5 h
fasti
ng
glu
co
se (
mg
/dl)
Chow 50 Cell 200 Cell 200 Inul
HFD
**
**
Cho
w
HFD
+50
Cel
l
HFD
+200
Cel
l
HFD
+200
Inul
0
20000
40000A
UC
(T
ota
l A
rea ,
a.u
.)
Chow 50 Cell 200 Cell 200 Inul
HFD
**
**
Zou et. al. Cell Host & Microbe (2018)
Inulin increases SCFA levels; blocked by hops b-acids
Zou et. al. Cell Host & Microbe (2018)
Blockade of SCFA production did not block inulin’s impact
(SCFA are overrated)
Zou et. al. Cell Host & Microbe (2018)
Inulin’s fortification of the gut requires IL-22
Zou et. al. Cell Host & Microbe (2018)
Expression of IL-22 Enterocyte
proliferation
Mucosal defense
Antimicrobial
signals (Reg3γ)ILC3
HFD enriched
with fermentable fiber
Microbiota encroachment
Colon atrophy
Adipose inflammation
Metabolic syndrome
Antimicrobial
signals
HFD lacking fermentable
fiber
Dysbiosislumen
HFD alters/depletes microbiota: Inulin restores
Chow - Cell Inul
106
107
bacteria / m
g o
f feces
HFD
Ba
cte
ria/m
g f
ece
s
106
107
Chow
HFD
+ Cellulose
+ Inulin
and changes
Zou et. al. Cell Host & Microbe (2018)
“Extreme colitis” in DSS-treated mice fed
inulin-supplemented purified diet
Chow Cell. Inul.
+ DSS
Con
Chow/DSS
Cell./DSS Inul./DSS
Con
Zou et. al. Inflamm. Bowel. Dis. (2017)
Fermentable fiber induces hepatocellular carcinoma
(HCC) in dysbiotic mice
Sing et. al. Cell, In Press
T5KO mice fed defined (10% fat) enriched with inulin
Just bring us some real food with lots of fermentable fiber
Emory/GSU
Vijay Kumar
Fred Carvalho
Benoit Chassaing
Shanthi Sitaraman
Ifor Williams
Andrew Neish
Host Defense, Osaka, JP
Shizuo Akira
Cornell/Max Planck
Ruth Ley
U. Colorado/SanDiego
Rob Knight
Acknowledgements
Supported by NIH
and CCFA
Penn State /Toledo U
Vijay Kumar
Vishal Singh
USDA
Michael Flythe
Genentech
Wenjun Ouyang
Matam Vijay Kumar
Benoit Chassaing
Jun ZouAlexis Bretin