Overall
Hypothesis
In this model of inflammatory bowel disease, Helicobacter
hepaticus may form adherent biofilms in susceptible mice that disrupt the barrier
function of the mucus layer allowing closer association of colitogenic
bacteria including H. hepaticus with the intestinal mucosa and crypts.
Hypotheses
for Our Study
Aim
1: We predict that the
cecal mucus layer will be thinner and less continuous and H. hepaticus will be more intimately associated with
the mucosa in A/J mice as compared to B6 mice.
Aim
2: We predict that A/J
mice given probiotic treatment will have thicker cecal mucus and less H. hepaticus colonization when compared to mice given
vehicle alone.
A/J
+ PROBIOTICS
Effects of genetic
background and probiotic treatment on biofilm and mucosal integrity in a mouse
model of bacterial-induced inflammatory bowel disease
INTRODUCTION
EXPERIMENTAL METHODS
SELECTED REFERENCES
ACKNOWLEDGEMENTS
Rationale for Our Studies
3. Treatment with the probiotics Lactobacillus reuteri and L. paracasei decreases IBD-associated lesions in IL-10
mice.2
4. Certain mucus carbohydrates may act as
natural antibiotics against Helicobacter
pylori infection in
humans.3
5. Preliminary micorarray studies by
our laboratory show decreased expression of the relm
mucus gene in A/J mice as compared to B6 mice during infection with H. hepaticus, suggesting differences in mucus
production or content.
Helicobacter hepaticus is a gram-negative spiral rod that
colonizes the cecum of the A/JCr (A/J) and C57BL/6 (B6) mouse strains. This
bacterium causes large intestinal inflammation in A/J mice 60-90 days
following inoculation but does not cause disease in B6 mice. Thus, this infection provides a good model for studying
genetic factors in preinflammatory and inflammatory phases of
bacterial-induced mucosal inflammation.
Inflammatory bowel disease (IBD) is a collection of chronic
gastrointestinal inflammatory disorders. The two most common forms of IBD in
humans, Crohn’s disease and ulcerative colitis, have an estimated prevalence
of 175-400 cases per 100,000 persons in North America. The term IBD also
refers to idiopathic gastrointestinal syndromes in several of our domestic
companion animals including cats, dogs and horses. Although the etiology of
IBD remains unknown, most evidence suggests that it is the result of a
dysregulated mucosal immune response to microbial antigens in the intestine.
Other important factors include genetics and environment.
ONGOING STUDIES
Immunohistochemistry: Preliminary ultrastructural examination of cecal mucosa
from H. hepaticus-infected A/J and B6 mice revealed that H. hepaticus is closely associated with mucosa.
However, mucosal-associated helicobacters were more frequent in the A/J cecum.
Moreover, intestinal helicobacters were found to form adherent biofilms in
IL-10 knockout mice that ultimately developed inflammatory bowel disease.1
In the current study we are examining cecal biofilm and mucus. Biofilm is a term that describes a multispecies
community of bacteria attached to a biologic surface, and mucus refers to carbohydrate-rich secretions
produced by intestinal epithelium. The first aim
of our study is to determine if there are differences in biofilm colonization
by H. hepaticus and mucus integrity between the A/J and
B6 mouse strains that might explain their differing genetic susceptibility.
Our second aim is to investigate the effects of
treatment with the probiotics Lactobacillus
rhamnosus and Bifidobacter longum on biofilm and mucus integrity during
preinflammatory and inflammatory stages of disease.
H.
hepaticus
inoculation: Mice were
inoculated via gastric gavage with either 108 H. hepaticus or culture medium alone (“sham”).
Routine PCR was run on DNA extracted from fecal samples to confirm presence or
absence of H. hepaticus colonization.
Probiotic
administration: A
mixture of Lactobacillus
rhamnosus and Bifidobacter longum (0.5ml containing 109/ml of each bacterium)
was administered to experimental groups via gastric gavage 4 days prior to H. hepaticus or sham inoculation. Food was coated
daily with the same mixture of Lactobacillus/Bifidobacter.
Vehicle (peptone water) alone was used for control mice.
Sample
collection: A/J or B6
mice were euthanized by CO2 inhalation and necropsied at 0 and 30 days
following inoculation with H.
hepaticus. Additional mice will be necropsied at 60 and
90 days post-inoculation. Cecal tissue sections (with contents intact) were
collected in Carnoy’s fixative for histology and immunohistochemistry.
Mucus
assessment: Mucus
content and integrity was assessed by alcian blue stains prepared at pH 1.0
and 2.5 with PAS counterstains.
•At
pH 2.5, alcian blue stains carboxylated and sulfated mucopolysaccharides,
carboxylated and sulfated sialomucins and
hyaluronic acid (dark
blue).
Disease
severity: Disease
severity was assessed on hematoxalin and eosin-stained tissues. Lesions were
scored based on intensity of inflammation, longitudinal and vertical extent of
inflammation and presence and extent of mucosal hyperplasia.
Biofilm
colonization/immunohistochemistry: The location and extent of colonization by H. hepaticus was assessed via immunohistochemistry
(IHC).
conjugated to horseradish peroxidase and incubated with
diaminobenzidine substrate for visualization. All slides were pretreated
with pepsin for antigen retrieval.
Later time points: Alcian blue and H&E data from 60 and
90-day time points and IHC results generated in the coming weeks will
determine whether A/J and B6 mice show different mucosal and biofilm
characteristics as disease advances and if probiotics protect against advanced
disease by maintaining biofilm and/or mucosal integrity.
Intestinal
mucus: No differences were seen in alcian
blue-stained (pH 1 and 2.5) sections of A/J and B6 mice.
Current results suggest that
IBD-susceptible A/J mice produce less carboxylated mucosubstances than
resistant B6 mice.
The preliminary findings of differential
mucus staining of cecal contents in A/J and B6 mice generates a new
interesting question: is there a causal relationship between mucus type and
susceptibility to H.
hepaticus-induced IBD?
Future studies will include further characterization of cecal mucus from these
strains as well as assessment of differential expression of genes involved in
mucus production.
FUTURE DIRECTIONS
Katherine
A. Hodes1, Laura P. Hale, MD, PhD3, Cynthia Besch Williford, DVM,PhD1, Azlin
Mustapha, PhD2, Luxin Wang2 and Craig Franklin, DVM, PhD1
Research Animal Diagnostic Laboratory1 and Dept. of Food Science2, University of Missouri, Columbia, MO
Research Animal Diagnostic Laboratory1 and Dept. of Food Science2, University of Missouri, Columbia, MO
Dept. of
Pathology, Duke University Medical Center, Durham, NC3
HELICOBACTER & BIOFILM
B6
A/J
Therefore, we are developing an
immunohistochemistry protocol to evaluate differences in H. hepaticus colonization of the mucosal biofilm. Our
initial studies showed positive staining of the biofilm in both H. hepaticus-infected and uninfected mice, suggesting
that our antibodies were cross-reacting to other cecal flora. Adsorption of antibody to uninfected cecal
contents eliminated all reactivity. Future steps include adsorption of more
concentrated antibody over less cecal material to increase recovery of H. hepaticus-specific antibody.
1.
Correspondence with Laura P. Hale, Duke University Medical Center, Dept. of
Pathology.
2.
Pena, J.A., et.al. 2005. Probiotic Lactobacillus spp. Diminish Helicobacter
hepaticus-induced inflammatory bowel
disease in Il-10-deficient mice. Infection and Immunity 72(2):
912-920.
3. Masatomo, K., et. al. 2004. Natural
antibiotic function of a human gastric mucin against Helicobacter pylori
infection. Science 305:1003-1006.
Laura P. Hale, Duke University Medical Center
Aim 1: A/J vs. B6
Aim 2: Probiotics vs. Control
30 Day Time Point
Inflammation:
A/J mice given probiotic treatment and A/J mice given vehicle alone had
very mild inflammation at 30 days post-inoculation. No difference in lesion
severity was observed.
30 Day Time Point
RESULTS
Inflammation: A/J mice had very mild cecal
inflammation, while B6 mice had no inflammation at 30 days post-inoculation.
This difference was not statistically significant (P<0.05).
A/J, Day 30
(alcian blue, pH 2.5)
B6, Day 30
(alcian blue, pH 2.5)
Probiotic-treated A/J, Day 30
(alcian blue, pH 2.5)
Control-treated A/J, Day 30
(alcian blue, pH 2.5)
CONCLUSION
The University of Missouri Veterinary Research Scholars Program was supported by funds from Merck-Merial, Pfizer
and the MU College of Veterinary Medicine. Special thanks to Jill
Gruenkemeyer, Mathew Myles, Lydia Cook, Andrew Hillhouse, Elena Kocher, Beth
Livingston and Greg Purdy for their invaluable assistance.
“Tigger” has IBD.
Envision
method of IHC
Intestinal
mucus: Goblet epithelial mucus and luminal mucus were examined using
alcian blue stains. At pH 2.5, the luminal cecal contents of infected
A/J mice exhibited less blue staining than the contents of B6 mice. This
difference was also seen in uninfected A/J and B6 mice. These findings
suggest that A/J mice produce less acidic and carboxylated mucosubstances than
do B6 mice. No differences between A/J and B6 mice were seen in slides
stained with alcian blue at
A/J +
probiotic,
Day 30
Control,
Day 30
Probiotic-treated
A/J,
Day 30 (H & E)
Peptone-treated
A/J,
Day 30 H & E)
B6, Day 30 (H
& E)
A/J, Day 30
B6, Day 30
A/J, Day 30 (H
& E)