Transcript Bacterial Infection Promotes Colon Tumorigenesis in Apcmin

Bacterial Infection Promotes
Colon Tumorigenesis in
Apcmin/+ Mice
Joseph V. Newman, Takeo Kosaka,
Barbara J. Sheppard, James G. Fox,
and David B. Schauer
Background on Diet and
Microflora
• Louis Pasteur (1822-1895)
• Adult Humans have more prokaryotic than
eukaryotic cells
• Symbiotic relationship in GI tract for more
efficient nutritional benefit
• Epidimiological studies have linked high
incidence rates for colon cancer to a western
diet (high in animal products)
Digestive Anatomy
Small vs. Large intestine:
• Different developmental
layers
• Epithelium structure
• Commensal digestive
flora
• The occurrence of
intestinal tumors in mice vs.
humans
Common Digestive Flora
• Stomach
– (Heliobacter sp.)
• Small Intestine
– Enterococci
– Lactobacilli
– (E. coli,
Psesudomonads)
• Colon
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Enterobacteria
Enterococcus faecalis
Bacteroides *
Bifidobacterium *
Clostridium
Lactobacillus *
Streptococcus
Staphylococcus
Ruminococcus
Peptostreptococcus
Peptococcus
Large Intestine
• 3 distinct regions
– Cecum
– Colon
– Rectum
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•
1011 to 1012 cells/g wet feces
>500 species
Lactobacilli
Bacteria have a few key
roles:
– Nutrient breakdown
– Preventing pathogenic
colonization
– Maintaining overall
physiological conditions
Dietary Fiber breakdown
Sloughed cells and dietary fiber
Fermentation
Glycolysis
ATP
Pyruvate
SO2
H2S
sulfidogenesis
Skin
propionate
Liver
ATP
H2
acetate
ATP
CO2 + H2
Acetyl- CoA
butyrate
ATP
methanogenesis
Gut Epithelium
CH4
Diet and its effect of gut flora
• Fermentation of SCFA  H2 + CH4
• Efficient mechanism for H2 disposal has evolved along 2 major
pathways:
– Methanogenic achea
– SRB
– Acetogenic*
• Low in colon cancers: high levels of methanogens
• western diet: higher levels of colon cancer
• Difference: Meat in the diet leads to an increase in SRB
• The composition of diet not only impacts the substrates for gut
flora, but also sets up a predictable competitive relationship
Large Intestine
• Colon mucosa has flat
epithelium with crypt
complexes
– Differentiated cells
– Proliferative stem and
precursor cells
• SCFA degredation:
proliferation  differentiation
Intestinal Epithelium
• Mucosal epithelium are bound by tight
junctions, the most luminal cell-cell junctions
• 2 major functions:
– Permeability barrier
– Protein Separation
• Tight junctions
– Occludin
– Claudin
Bacteria, inflammation, …
• Analyze KO mice to
germ-free conditions
– TCR/p53 Dbl. KO
– IL-10 deficient mice
– Apc Min mice  50%
tumor
• Hosting a bacterial
population is not without
consequence
– Maintain gene to protect
against bacterial stress:
peroxidative stress,
bacterial antigen,
inflammation
– Intact mucosal barrier
Possible models of
tumorigenesis
• Inflammation/cancer depends on aggregate
interactions
– Quorum sensing
– Alterations in flora due to diet
• Weak genetic defects and polymorphisms in
hosts might allow normal flora to induce
tumors over extended period of time
Diseases of the Colon/Large
Intestine
Crohn’s Disease
Chronic Inflammation
Inflammatory Bowel Disease (IBD)
Inflammation, Rigidity and
Thickening of Colon
Ulcerative Colitis
Chronic Inflammation
Colon Caner
Diverticulitus
Colon Develops a
pocket
Relevance of studying
Bacterial infection
Helicobater pylori
H. hepaticus
Lawsonia intracellularis
Group D Streptococcus
Increased gastric cancer
Hepatocellular carcinoma,
Liver
Intestinal epithelium
proliferation (cancer
biomarker)
Inflammation, dysplasia,
rectal carcinoma
Introduction
• C. rodentium - naturally gram (-) occurring
bacterial pathogen of lab mice
• Infection:
– Epithelial cell hyperproliferation (IBD, Chron’s,
colitis) & thickening/rigidity of colon
– Diarrhea and weight loss (suckling mice)
– Colonic hyperplasia and limited inflammation
(adult)
Transmissible Murine Colonic
Hyperplasia (TMCH)
• Colonic crypts are 2
to 3 times longer
compared to normal
mice
• Epithelium contain
twice the number of
dividing cells
• No direct evidence
linking C. rodentium
to tumorigenesis
• Increased colonic
adenoma counts in
presence of
carcinogens
AE Lesions
• Attaching and Effacing lesions during colon
infection
– Dissolution of brush border,
– cupping of adherent bacteria,
– cytoskeleton rearrangements of epithelium
• Enteropathogenic and Enterohemorrhagic E.
coli (EPEC & EHEC) infections
• Similar gene locus is required for AE
formation
 C. rodentium animal model of infection
AE Lesions (Chicken and the
egg)
• Is inflammation causing the altered
epithelium, which allows for bacterial
association?
• Does bacterial attachment cause these
lesions, which then induce
inflammation?
• AE pathogens have been shown to attach to
surface epithelial cells via type III secretion
pathway, possibly causing the release of
some inflammatory mediators
– 7 day post infection
– 21 days post infection
ApcMin/+ Mouse
• Nonsense mutation of adenomatous
polyposis coli gene
• Apc:
– Regulates cellular division frequency
– Regulates cellular attachment/movement
• Mice are pre-disposed to multiple intestinal
neoplasms (Min)
Methods
• Inoculated 4 week old
mice
–
ApcMin/+
100 L Culture
w/ 100L o/n
or
culture
– Apc+/+ w/ 100L sterile
media
• Confirmed infection 7
days post infection w/
CFU counts
• Sacrificed mice 10 days
and 5 months post
inoculation
100 L sterile media
10 days
or
5 months
the messy steps
Pathology
• The colon was removed
and examined for
hyperplasia
• Adenomas were
counted and measured
• Grossly altered tissue
was excised and
mounted for histological
analysis
Immunohistochemistry
• Representative samples
were frozen and stained
for:
 smooth muscle
actin
– F4/80 (macrophage
marker)
– COX-2
Results
• A: Mucosal epithelium,10
days post infection
• B: Intact basement
membrane and hyperplasia
• C: Dysplastic tissue with
adenoma
Colonic Adenoma
from infected
Min mouse
• D. COX-2
• E. COX-2 + F4/80
• F. COX-2 + actin
High magnification of
Adenoma
G. COX-2
H. COX-2 + F4/80
I. COX-2 +Actin
Results
ApcMin/+ mice
• Visible thickening
and rigidity of colon
(10 days post
infection)
• Mean crypt column
height 2x that of
uninfected Min mice
(significant)
Wt mice
• Visible thickening
and rigidity of colon
(10 days post
infection)
• Mean crypt column
height 2x that of
uninfected Min mice
(significant)
No significant difference
Discussion
• Infection promotes adenoma formation in Min
mice
• Could promotion be due to hyperproliferative
state induced by infection?
• COX-2 levels were not detected in colon
tissue from infection (10 day post-infection)
 Is COX-2 involved in earliest stages of
tumor promotion in Min mice?
Future Direction
• Is microbiota required for colon
tumorigenesis?
• Do A/E pathogens produce alterations of
epithelial cell cytokinetics?
• What “chemical signals” are secreted by
bacteria associated with aberrant crypts?
• Do bacterial signals influence gene activities
of colon mucosal cells?
• Heat-stable
enterotoxin
• GC-C pathway
specific to intestinal
epithelium
• Carcinoma cell
proliferation was
inhibited