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Dysbiosis in Mouse Models of
Chronic Gut Inflammation:Cause or Consequence?
Matthew B. Grisham, PhD.Department of Immunology and
Molecular Microbiology
TEXAS TECH UNIVERSITYHEALTH SCIENCES CENTERSchool of Medicine
1960-79
1980-08
Moledecky et. al. Gastroenterology, 2012
Worldwide Incidence and Prevalence of IBD have Increased Dramatically over the Past 50 Years
from Lees, C.W., Gut 2011Jostins et. al. Nature, 2012
CD and UC are Multifactorial Polygenic Diseases (~163 susceptibility loci)
● Genetically-identical twins express a relatively low concordance rates for both CD (~30-35%) and UC (~10-15%). Spehlmann et. al. IBD, 2008.
● Increased incidence and prevalence of IBD in countries that have adapted a “Modernized” lifestyle.
Environmental factors* are emerging as major contributors to disease
pathogenesis in genetically-susceptible individuals
*antibiotics, hygiene, diet
Stomach 0-102
LactobacillusCandidaStreptococcusHelicobacter pyloriPeptostreptococcus
LactobacillusStreptococcus
Duodenum 102
Distal Ileum 107-108
StreptococcusClostridiumBacteroidesActinomycinaeCorynebacteria
Jejunum 102
Proximal Ileum 103
LactobacillusStreptococcus
LactobacillusStreptococcus
Colon 1011-1012
BacteroidesClostridium groups IV&XIVBifidobacteriumEnterobacteriaceae
modified from Sartor, 2008
Antibiotics, Hygiene and Diet Alter Intestinal Microbiota
● Diversion of fecal stream prevents recurrence of Crohn’s Disease; Reinfusion of fecal contents rapidly induces disease.
● Antibiotic therapy attenuates intestinal inflammation in distal bowel disease.
● Increased numbers of bacteria are observed in intestinal tissue of patients with IBD.
● IBD-susceptibility genes are involved in bacterial killing.
● Composition of intestinal microbiota is altered in IBD (dysbiosis).
Clinical Evidence Implicating Intestinal Bacteria in the Pathogenesis of IBD
from Peterson and Gordon 2008
Dysbiosis in IBD
Decrease in alpha diversity
Decrease in Bacteroides and Firmicutes
Decreases in Clostridia, Ruminococcaceae, Lactobacillus, Faecalibacterium prausnitzii, Bifidobacterium
Increase in Proteobacteria (e.g. Enterobacteriaceae)Increases in γ-proteobacteria; E. coli (AIEC)
Increased Presence of Fusobacterium
Alterations in the Microbiota Associated with Inflammatory Bowel Disease
Kostic et. al. Gastroenterology, 2014
Increased oxidative stress protection pathways ● increased cysteine and GSH transport; ● increased riboflavin and sulfur metabolism ● increased pentose phosphate shunt pathway
Increased sulfate transport and metabolism
Increase in amino acid transport
Increase in auxotrophy
Decrease in short chain fatty acids and metabolism
Decreased in amino acid biosynthesis
from Morgan et. al., 2012; Kostic et. al. 2014
Alterations in Microbial Function in IBD
Role of Intestinal Bacteria in MouseModels of IBD
Intestinal Bacteria are Required for the Induction of Chronic Gut Inflammation in
Genetically-Susceptible Mice
CD45RBhigh T-Cell →SCID or RAG-/-
IL-10-/- IL-2-/-
TCR-α-/- or β-/-
C3H/HeJBirSamp1/Yit
TLR-5-/-
Tbet-/- x RAG2-/- (Truc)IL-10r2-/- x TGFβr2-/-
Mouse Models of Chronic GutInflammation exhibit Dysbiosis
Healthy Colitic
Reinoso Webb, Koboziev et. al. 2014
RAG-1-/- CD45RBhigh→RAG-1-/-
Dysbiosis in Chronic Gut Inflammation: Cause or Consequence?
Healthy Colitic
Reinoso Webb, Koboziev et. al. 2014
RAG-1-/- CD45RBhigh→RAG-1-/-
Intestinal Inflammation Promotes the Growth of Proteobacteria (Enterobacteriales)
from Lupp et.al. 2007
Control
C. rodentium infection
Time-Dependent Dysbiosis inIL-10-/- Mice
from Maharshak et. al. 2013
Wild Type
IL-10-/-
Intestinal inflammation enhances the growth of certain facultative anaerobes while decreasing the growth of obligate anaerobes
Intestinal Inflammation Induces Dysbiosis in Mice
R3-N+-O-
(TMAO)
R2-SO(Sulfoxide)
R2-S(Sulfide)
R3-NH(Trimethyl Amine)
O2-
H2O2
HOCl
NO3-
(nitrate)
SO4-2
(Sulfate)
Intestinal Inflammation
ONOO-
Mucolytic Bacteria NO
Modified from Winter et. al. EMBO, 2013
CHOMucolytic Bacteria
Modified from Winter et. al. EMBO, 2013
Inflammation Provides a Selective Growth Advantage for Disease-Producing Pathobionts
R3-N+-O-
(TMAO)
R2-SO(Sulfoxide)
R2-S(Sulfide)
R3-NH(Trimethylamine)
O2-
H2O2
HOCl
NO3-
(nitrate)
SO4-2
(Sulfate)
Intestinal Inflammation
ONOO-
Mucolytic Bacteria
Products of Intestinal Inflammation:Reactive Oxygen and Nitrogen Species
NO
Modified from Winter et. al. EMBO, 2013
CHO
diet, bacteria
Modified from Winter et. al. EMBO, 2013
Increased oxidative stress protection pathways ● increased cysteine and GSH transport ● increased riboflavin and sulfur metabolism ● increased pentose phosphate shunt pathway
Increased sulfate transport and metabolism
Increase in amino acid transport
Increase in auxotrophy
Decrease in short chain fatty acids and metabolism
Decreased in amino acid biosynthesis
from Morgan et. al., 2012; Kostic et. al. 2014
Alterations in Microbial Function in IBD
LOOHH2O2
LOHH2O
GRRiboflavin
GSH cysteine, cystine
Glut + Gly + Cysglucose
Proteobacteria are the only Major Group of Bacteria that can Produce GSH
Pentose Phosphate Shunt
sulfate
R3-N+-O-
(TMAO)
R2-SO(Sulfoxide)
R2-S(Sulfide)
R3-NH(Trimethylamine)
O2-
H2O2
HOCl
NO3-
(nitrate)
SO4-2
(Sulfate)
Intestinal Inflammation
ONOO-
Mucolytic Bacteria
Products of Intestinal Inflammation:Nitric Oxide-Derived Metabolites
NO
Modified from Winter et. al. EMBO, 2013
CHO
diet, bacteria
Modified from Winter et. al. EMBO, 2013
H2O
Anaerobic Respiration by Enterobacteriaceae: Nitrate Reduction
Pearson Education, Inc., 2015
Generation of Proton-Motive Force via electron Transport
GSH Protects the Fumarate and Nitrate Reductase Regulatory Protein from Oxidant-Induced
Inactivation
GSH
Ox
InactiveActive
Ox
R3-N+-O-
(TMAO)
R2-SO(Sulfoxide)
R2-S(Sulfide)
R3-NH(Trimethylamine)
O2-
H2O2
HOCl
NO3-
(nitrate)
SO4-2
(Sulfate)
Intestinal Inflammation
ONOO-
Mucolytic Bacteria
Products of Intestinal Inflammation:Oxidant-Mediated formation of N- and S-Oxides
NO
Modified from Winter et. al. EMBO, 2013
CHO
diet, bacteria
Modified from Winter et. al. EMBO, 2013
+ ATP
DMSO
+ ATPTMAO ReductaseAnaerobic Respiration
TMAO
Anaerobic Respiration by Enterobacteriaceae:TMAO and DMSO Reductases
DMSO ReductaseAnaerobic Respiration
R3-N+-O-
(TMAO)
R2-SO(Sulfoxide)
R2-S(Sulfide)
R3-NH(Trimethyl Amine)
O2-
H2O2
HOCl
NO3-
(nitrate)
SO4-2
(Sulfate)
Intestinal Inflammation
ONOO-
Mucolytic Bacteria
Products of Intestinal Inflammation:Mucin-Derived Sulfate
NO
Modified from Winter et. al. EMBO, 2013
CHOMucolytic Bacteria
Modified from Winter et. al. EMBO, 2013
diet
Increased oxidative stress protection pathways ● increased cysteine and GSH transport; ● increased riboflavin and sulfur metabolism ● increased pentose phosphate shunt pathway
Increased sulfate transport and metabolism
Increase in amino acid transport
Increase in auxotrophy
Decrease in short chain fatty acids and metabolism
Decreased in amino acid biosynthesis
from Morgan et. al., 2012; Kostic et. al. 2014
Alterations in Microbial Function in IBD
Anaerobic Respiration by δ Proteobacteria: Sulfate Reduction
Desulfovibrio
Bilophila wadsworthia
Modified from Cypionka, Encyc. Geobiology, 2011
Anaerobic Respiration
ClostridiaBacteriodia
Enterobacteriaceae
CHO
Products of Inflammation Feed the Expansion of Colitogenic Pathobionts
Modified from Winter et. al. EMBO, 2013
Healthy Inflammation Dybiosis Disease
Sequential Generation of Inflammation, Dysbiosis and Disease in Susceptible Mice
modified from Craven et. al. PLOS One, 2012
Inflammation Induces Dysbiosis
Transplant of fecal microbiota from colitic mice into healthy recipients should accelerate the onset of disease in
genetically-susceptible mice.
RAG-1-/- feces
Colitic feces
5-6 Days
+CD45RBhigh
T Cells~ 2.0 mg/g body weight
5-6 Days
Colitic Fecal Transplant AcceleratesWeight Loss in the T Cell Transfer
Model of Chronic Colitis
Days Post T Cell Transfer
Bo
dy
Wei
gh
t (%
Ori
gin
al)
● T cell Transfer
● RAG Feces+ T cells
● Colitic Feces+ T cells
Reinoso Webb, Koboziev et. al. 2014
Colitic Fecal Transplant Induces More Severe Colonic Inflammation
Koboziev, Reinoso Webb et. al. 2014
RAG feces+
T Cells
T cells Colitic feces+
T Cells
His
top
ath
olo
gy
sco
res
0
4
8
12 *
Colitic Fecal Transplant Increases Myeloid Cell Infiltration into the Inflamed Colon
15
10
5
08
6
4
2
0
Ce
ll N
um
ber
pe
r co
lon
(1
05)
Monocytes/Macrophages
(CD11b+Ly6ChiLy6G-)
PMNs(CD11b+Ly6CintLy6G+)
T cells RAG feces+ T cells
Colitic feces+ T cells
Koboziev, Reinoso Webb et. al. 2014
Colitic Fecal Transplant Does Not Induce Colitis in Wild Type or RAG-/- Mice
His
top
ath
olo
gy
Sco
res
12
6
0T cells
+Colitic Feces
Reinoso Webb, Koboziev et. al. 2014
RAG-/-
+Colitic Feces
WT+
Colitic Feces
*
Conclusions1. Intestinal inflammation induces dysbiosis via the
generation of metabolites that provide a selective growth advantage for disease-producing pathobionts (e.g. facultative anaerobes).
2. Failure to properly regulate this acute (and reversible) immune response allows for outgrowth and invasion of colitogenic microbes; This triggers the initiation and perpetuation of chronic gut inflammation.
3. Disease-producing pathobionts are not classic pathogens as they do not elicit acute or chronic inflammation in healthy wild type or lymphopenic recipients.
Acknowledgements
Cynthia Reinoso WebbIurii Koboziev
Dmitry OstaninKatie Furr
Rao KottapalliCaleb Phillips
Yava Jones-Hall
Evidence Suggesting that Intestinal Inflammation is Associated with Enhanced
Production of Reactive Oxygen and Nitrogen Species
● Detection of stable end products derived from reactiveoxygen and nitrogen species within the bowel lumen (e.g. nitrate; oxidized/nitrated peptides and proteins).
● Attenuation of inflammation via transgenic over-expression or induction of oxidant defense genes (e.g. CuZn-SOD or Mn-SOD; HO-1).
● Pharmacologic or genetic depletion of essential oxidant defenses enhances intestinal inflammation (↓GSH) orinduce spontaneous colitis (GPx-1 & -2-/- mice), respectively.