dysbiosis in mouse models ofdysbiosis in mouse models of chronic gut inflammation:chronic gut...

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.