Transcript No Slide Title

Dysbiosis in Mouse Models of
Chronic Gut Inflammation:
Cause or Consequence?
Matthew B. Grisham, PhD.
Department of Immunology and
Molecular Microbiology
TEXAS TECH UNIVERSITY
HEALTH SCIENCES CENTER
School of Medicine
Worldwide Incidence and Prevalence of IBD have
Increased Dramatically over the Past 50 Years
1960-79
1980-08
Moledecky et. al. Gastroenterology, 2012
CD and UC are Multifactorial Polygenic Diseases
(~163 susceptibility loci)
from Lees, C.W., Gut 2011
Jostins et. al. Nature, 2012
Environmental factors* are emerging as
major contributors to disease
pathogenesis in genetically-susceptible
individuals
● 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.
*antibiotics, hygiene, diet
Antibiotics, Hygiene and Diet Alter
Intestinal Microbiota
Stomach 0-102
Lactobacillus
Candida
Streptococcus
Helicobacter pylori
Peptostreptococcus
Duodenum 102
Lactobacillus
Streptococcus
Jejunum 102
Lactobacillus
Streptococcus
Proximal Ileum 103
Lactobacillus
Streptococcus
Colon 1011-1012
Distal Ileum
107-108
Streptococcus
Clostridium
Bacteroides
Actinomycinae
Corynebacteria
Bacteroides
Clostridium groups IV&XIV
Bifidobacterium
Enterobacteriaceae
modified from Sartor, 2008
Clinical Evidence Implicating Intestinal
Bacteria in the Pathogenesis of IBD
● 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).
Dysbiosis in IBD
from Peterson and Gordon 2008
Alterations in the Microbiota Associated
with Inflammatory Bowel Disease
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
Kostic et. al. Gastroenterology, 2014
Alterations in Microbial Function in IBD
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
Role of Intestinal Bacteria in Mouse
Models 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/HeJBir
Samp1/Yit
TLR-5-/Tbet-/- x RAG2-/- (Truc)
IL-10r2-/- x TGFβr2-/-
Mouse Models of Chronic Gut
Inflammation exhibit Dysbiosis
Healthy
Colitic
RAG-1-/-
CD45RBhigh→RAG-1-/-
Reinoso Webb, Koboziev et. al. 2014
Dysbiosis in Chronic Gut Inflammation:
Cause or Consequence?
Healthy
Colitic
RAG-1-/-
CD45RBhigh→RAG-1-/-
Reinoso Webb, Koboziev et. al. 2014
Intestinal Inflammation Promotes the Growth
of Proteobacteria (Enterobacteriales)
Control
C. rodentium infection
from Lupp et.al. 2007
Time-Dependent Dysbiosis in
IL-10-/- Mice
Wild Type
IL-10-/-
from Maharshak et. al. 2013
Intestinal Inflammation Induces
Dysbiosis in Mice
Intestinal inflammation enhances
the growth of certain facultative
anaerobes while decreasing the
growth of obligate anaerobes
Inflammation Provides a Selective Growth
Advantage for Disease-Producing Pathobionts
SO4-2
(Sulfate)
NO3(nitrate)
R3-N+-O- R2-SO
(TMAO)
(Sulfoxide)
R2-S
(Sulfide)
R3-NH
(Trimethyl Amine)
ONOO-
HOCl
H 2 O2
CHO
Mucolytic
Bacteria
Mucolytic
Bacteria
NO
O 2-
Intestinal Inflammation
Modified from Winter et. al. EMBO, 2013
Modified from Winter et. al. EMBO, 201
Products of Intestinal Inflammation:
Reactive Oxygen and Nitrogen Species
diet, bacteria
SO4-2
(Sulfate)
NO3(nitrate)
R3-N+-O- R2-SO
(TMAO)
(Sulfoxide)
R2-S
(Sulfide)
R3-NH
(Trimethylamine)
ONOOHOCl
H 2 O2
NO
CHO
O2-
Mucolytic
Bacteria
Intestinal Inflammation
Modified
Modifiedfrom
fromWinter
Winteret.
et.al.
al.EMBO,
EMBO,2013
2013
Alterations in Microbial Function in IBD
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
Proteobacteria are the only Major Group
of Bacteria that can Produce GSH
GSH
cysteine, cystine
sulfate
Glut + Gly + Cys
glucose
LOOH
H2O2
GR
Riboflavin
LOH
H2O
Pentose Phosphate
Shunt
Products of Intestinal Inflammation:
Nitric Oxide-Derived Metabolites
diet, bacteria
SO4-2
(Sulfate)
NO3(nitrate)
R3-N+-O- R2-SO
(TMAO)
(Sulfoxide)
R2-S
(Sulfide)
R3-NH
(Trimethylamine)
ONOOHOCl
H 2 O2
NO
CHO
O2-
Mucolytic
Bacteria
Intestinal Inflammation
Modified
Modifiedfrom
fromWinter
Winteret.
et.al.
al.EMBO,
EMBO,2013
2013
Anaerobic Respiration by Enterobacteriaceae:
Nitrate Reduction
Generation of Proton-Motive Force via electron Transport
H2O
Pearson Education, Inc., 2015
GSH Protects the Fumarate and Nitrate Reductase
Regulatory Protein from Oxidant-Induced
Inactivation
GSH
Ox
Active
Ox
Inactive
Products of Intestinal Inflammation:
Oxidant-Mediated formation of N- and S-Oxides
diet, bacteria
SO4-2
(Sulfate)
NO3(nitrate)
R3-N+-O- R2-SO
(TMAO)
(Sulfoxide)
R2-S
(Sulfide)
R3-NH
(Trimethylamine)
ONOOHOCl
H 2 O2
NO
CHO
O2-
Mucolytic
Bacteria
Intestinal Inflammation
Modified
Modifiedfrom
fromWinter
Winteret.
et.al.
al.EMBO,
EMBO,2013
2013
Anaerobic Respiration by Enterobacteriaceae:
TMAO and DMSO Reductases
TMAO Reductase
+ ATP
Anaerobic Respiration
TMAO
DMSO Reductase
Anaerobic Respiration
DMSO
+ ATP
Products of Intestinal Inflammation:
Mucin-Derived Sulfate
diet
SO4-2
(Sulfate)
NO3(nitrate)
R3-N+-O- R2-SO
(TMAO)
(Sulfoxide)
R2-S
(Sulfide)
R3-NH
(Trimethyl Amine)
ONOOHOCl
H 2 O2
CHO
Mucolytic
Bacteria
NO
Mucolytic
Bacteria
O2-
Intestinal Inflammation
Modified
Modifiedfrom
fromWinter
Winteret.
et.al.
al.EMBO,
EMBO,2013
2013
Alterations in Microbial Function in IBD
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
Anaerobic Respiration by δ Proteobacteria:
Sulfate Reduction
Desulfovibrio
Bilophila wadsworthia
Modified from Cypionka, Encyc. Geobiology, 2011
Products of Inflammation Feed the Expansion of
Colitogenic Pathobionts
Anaerobic
Respiration
CHO
Clostridia
Bacteriodia
Enterobacteriaceae
Modified from Winter et. al. EMBO, 2013
Sequential Generation of
Inflammation, Dysbiosis and
Disease in Susceptible Mice
modified from Craven et. al. PLOS One, 2012
Healthy
Inflammation
Dybiosis
Disease
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
5-6 Days
+
CD45RBhigh
T Cells
~ 2.0 mg/g body weight
Colitic feces
5-6 Days
Body Weight (% Original)
Colitic Fecal Transplant Accelerates
Weight Loss in the T Cell Transfer
Model of Chronic Colitis
● T cell Transfer
● RAG Feces
+ T cells
● Colitic Feces
+ T cells
Days Post T Cell Transfer
Reinoso Webb, Koboziev et. al. 2014
Colitic Fecal Transplant Induces More
Severe Colonic Inflammation
*
H is t o p a t h o lo g y s c o r e s
12
8
4
0
T cells
RAG feces
+
T Cells
Colitic feces
+
T Cells
Koboziev, Reinoso Webb et. al. 2014
Colitic Fecal Transplant Increases Myeloid Cell
Infiltration into the Inflamed Colon
Cell Number per colon (105)
15
10
Monocytes/
Macrophages
(CD11b+Ly6ChiLy6G-)
5
0
8
6
PMNs
(CD11b+Ly6CintLy6G+)
4
2
0
Koboziev, Reinoso Webb et. al. 2014
T cells
RAG feces
+ T cells
Colitic feces
+ T cells
Colitic Fecal Transplant Does Not Induce
Colitis in Wild Type or RAG-/- Mice
*
Histopathology Scores
12
6
0
Reinoso Webb, Koboziev et. al. 2014
T cells
+
Colitic Feces
WT
+
Colitic Feces
RAG-/+
Colitic Feces
Conclusions
1. 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 Webb
Iurii Koboziev
Dmitry Ostanin
Katie Furr
Rao Kottapalli
Caleb 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 reactive
oxygen and nitrogen species within the bowel lumen
(e.g. nitrate; oxidized/nitrated peptides and proteins).
● Attenuation of inflammation via transgenic overexpression 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) or
induce spontaneous colitis (GPx-1 & -2-/- mice), respectively.