Helicobacter-induced inflammatory bowel disease in
IL-10- and T cell-deficient mice
Andrew
Burich1,5,
Robert
Hershberg3,
Kim
Waggie4,
Weiping
Zeng1,
Thea
Brabb1,2,
Gina
Westrich5,
Joanne L.
Viney5, and
Lillian
Maggio-Price1,5
Departments of 1 Comparative Medicine and 2 Molecular
Biotechnology, University of Washington, Seattle 98195; 3 Corixa
Corporation, Seattle 98104; 4 Zymogenetics Incorporated, Seattle
98102; and 5 Department of Molecular Immunology, Immunex
Corporation, Seattle, Washington 98101
 |
ABSTRACT |
Inflammatory bowel disease (IBD) is
thought to result from a dysregulated mucosal immune response to
luminal microbial antigens, with T lymphocytes mediating the colonic
pathology. Infection with Helicobacter spp has been reported
to cause IBD in immunodeficient mice, some of which lack T lymphocytes.
To further understand the role of T cells and microbial antigens in
triggering IBD, we infected interleukin (IL)-10
/
,
recombinase-activating gene (Rag)1
/
,
T-cell receptor (TCR)-
/
, TCR-
/
,
and wild-type mice with Helicobacter hepaticus or
Helicobacter bilis and compared the histopathological IBD
phenotype. IL-10
/
mice developed severe diffuse IBD
with either H. bilis or H. hepaticus, whereas
Rag1
/
, TCR-
/
,
TCR-
/
, and wild-type mice showed different
susceptibilities to Helicobacter spp infection.
Proinflammatory cytokine mRNA expression was increased in the colons of
Helicobacter-infected IL-10
/
and
TCR-
/
mice with IBD. These results confirm and
extend the role of Helicobacter as a useful tool for
investigating microbial-induced IBD and show the importance, but not
strict dependence, of T cells in the development of bacterial-induced IBD.
Helicobacter bilis; Helicobacter hepaticus; colitis; proinflammatory cytokines
 |
INTRODUCTION |
THE PRECISE ETIOLOGY
of the human idiopathic inflammatory bowel disorders ulcerative colitis
and Crohn's disease is unknown. Proposed factors include infection
with bacterial pathogens, disruption of the intestinal mucosal barrier,
dysregulated immune responses to luminal antigens, and genetic
susceptibility (2, 35), but their exact roles have not
been defined. Within the past decade, numerous mouse models have
emerged as tools to investigate mechanisms of initiating and
perpetuating inflammatory bowel disease (IBD). Spontaneous intestinal
inflammation has been observed in genetically altered mice with various
immunologic defects. These include interleukin (IL)-2
/
,
IL-10
/
, major histocompatibility complex (MHC)
class II
/
, T-cell receptor (TCR) mutants, IL-7
transgenics, and G
i-2
/
mice (18,
25, 32, 33, 44). Although the underlying genetic defect that
confers susceptibility to IBD varies considerably in these mouse
models, a common and central feature is that the development of IBD is
dependent on the presence of intestinal luminal flora. For example,
IL-2
/
mice maintained in a conventional facility
develop IBD, but when rederived into specific pathogen-free (SPF) or
germ-free conditions, intestinal lesions are delayed or prevented,
respectively (33). Similarly, IL-10
/
mice
spontaneously develop IBD when housed under conventional conditions
(18), but there is no evidence of colitis when animals are
maintained under germ-free conditions (37). In the
CD4+ CD45RBhi adoptive transfer model, mice
with reduced intestinal flora show less severe wasting disease
(1), whereas mice treated with bacitracin and streptomycin
are rescued from weight loss (27), again stressing the
role that luminal bacteria play in initiating IBD. The adoptive
transfer model has also shown that the colitis is mediated by a
CD45RBhi subset of CD4+ T cells, (20, 27,
28, 30), highlighting an important role for T cells in the
disease process. Few studies have analyzed, in a controlled manner, the
relative contribution of bacterial flora and T cell dysregulation in
the development of IBD (4).
Infection with murine Helicobacter species has been
implicated as a potential cause of "spontaneous" gastrointestinal
inflammation mimicking IBD in various mouse strains raised in
conventional facilities. Indeed, natural and experimental infections of
certain strains of laboratory rodents with particular species of
Helicobacter have been shown to cause IBD. For example,
immunodeficient mice with natural infections of H. hepaticus
exhibit a proliferative typhlocolitis and proctitis (21, 42,
43), and experimental inoculation of severe combined
immunodeficiency mice with H. bilis has been reported to
result in typhlitis and colitis (14, 39). Recently
identified Helicobacter species have also been associated with IBD in IL-10
/
(11) and severe
combined immunodeficiency mice (15). Although these data
by no means suggest that Helicobacter is the only organism capable of initiating murine IBD, they do underscore the importance of
this widely endemic bacteria in inducing gastrointestinal inflammation. Therefore, Helicobacter spp may be an excellent model
organism with which to investigate the complex role of luminal bacteria in initiating experimental IBD.
Although the above-cited reports have solidified a link between
experimental IBD and Helicobacter infection, important
questions remain. Given the observation that various
Helicobacter species can cause disease, what is the relative
efficiency of the different species to induce IBD? Is there identical
or disparate pathology? In addition, given the importance of T
lymphocytes in experimental IBD, how can one interpret the occurrence
of Helicobacter-induced IBD in mice that lack T and B cells
(41)? Therefore, the aims of this study were to determine
1) whether H. hepaticus or H. bilis
was more potent in producing bowel pathology, 2) whether the
IBD induced by Helicobacter spp differs phenotypically in mice with a dysregulated cytokine network compared with mice deficient in specific immune cell subsets, and 3) whether
proinflammatory cytokines are regulated during the disease process. To
this end, a histopathological scoring system allowed us to quantitate
the severity and regional extent of disease, and MHC class II
expression and cytokine production were analyzed in colonic tissue.
Here we report that IL-10
/
mice develop severe IBD
after H. bilis or H. hepaticus infection. Furthermore, the IBD that develops is associated with increased MHC
class II expression in the intestinal epithelium. None of the C57BL/10J
or C57BL/6J immunocompetent mice infected with H. hepaticus
or H. bilis showed any evidence of IBD. We also report that
mice with absent [recombinase-activating gene (Rag)1
/
mice] or dramatically altered T cell populations [T cell receptor (TCR)-
/
mice] show mild intestinal inflammation
after Helicobacter infection. We analyzed cytokine mRNA
expression associated with disease and show that T helper (Th)1-type
cytokines are upregulated in the colons of
Helicobacter-infected mouse strains with IBD, regardless of
whether the model has been previously associated with a Th1 or Th2
cytokine profile.
 |
MATERIALS AND METHODS |
Animals.
Three- to seven-week-old female C57BL/10J, C57BL/6J,
IL-10
/
(C57BL/10J), IL-10
/
(C57BL/6J),
Rag1
/
(C57BL/6J), TCR-
/
(C57BL/6J),
and TCR-
/
(C57BL/6J) mice were obtained from Jackson
Laboratories (Bar Harbor, ME). All mice were certified free of
Helicobacter spp by the vendor and retested in our animal
facilities. Cohorts of mice were housed at the University of Washington
(UW) or at Immunex Corporation (IMNX). Animals were housed
in a SPF room in polycarbonate microisolator cages containing
Bed-O'cob (The Andersons, Maumee, OH) and a nestlet. Mice were fed
irradiated Picolab rodent diet 20 (PMI Nutrition International,
Brentwood, MO) and autoclaved, acidified water. All supplies entering
the animal rooms were autoclaved, and the room was maintained at
70-74°F, 45-55% humidity, with 28 air changes per hour and
a 12:12-h light-dark cycle. To prevent cross contamination, uninfected
and infected mice were housed in separate cubicles or separate rooms.
At UW, mice were housed in separate cubicles, depending on infection
status, within the same room, and cages were changed in
"uninfected" or "infected" laminar flow changing stations. At
IMNX, uninfected and infected animals were housed in separate rooms,
each containing its own laminar flow changing station. Sentinel mice at
the UW were tested quarterly for endo- and ectoparasites, mouse
hepatitis virus, mouse parvovirus, and rotavirus and annually for
Mycoplasma pulmonis, pneumonia virus of mice, reovirus-3,
Sendai virus, and Theiler's murine encephalomyelitis virus. Also,
colon samples were screened quarterly for Citrobacter
rodentium, non-lactose-fermenting Escherichia coli,
Salmonella spp, Klebsiella spp, and
Clostridium spp (Phoenix Laboratories, Seattle, WA). At
IMNX, random sentinel mice from all rooms were tested weekly for mouse
hepatitis virus and monthly for 14 mouse viruses (Charles River
Laboratories, Wilmington, MA). Biannually, sentinel mice from each room
were sent to Charles River Laboratories, necropsied, and screened for
several important murine viral, bacteriological, and parasitic
pathogens and for Helicobacter spp. The UW Institutional
Animal Care and Use Committee and the IMNX Animal Use and Care
Committee approved all animal procedures.
Experimental design.
Before inoculation, mice were determined to be negative for
Helicobacter spp by fecal PCR (see Clinical and
histopathological findings associated with Helicobacter spp infection
in IL-10
/
mice). In six separate
experiments, 5-15 mice were given H. hepaticus or
H. bilis, and 3-15 mice were given broth alone.
Initially, fecal Helicobacter culture and species-specific
fecal PCR were done on pooled cage samples taken every 2 wk until the
end of the study. Later experiments used fecal PCR as the sole means to
determine Helicobacter infection status. Between 3 and 36 wk postinfection (PI), uninfected and infected mice were euthanized with
CO2 and necropsied. Tissues were taken for histopathology, immunohistochemistry, or cytokine analysis or combinations of the
three. Mice were weighed weekly and monitored for weight loss, dehydration, and diarrhea. Additionally, fecal samples from both infected and uninfected mice were periodically tested by PCR for cross
contamination with another Helicobacter spp in infected mice
and to confirm the absence of Helicobacter infection in
uninfected animals.
Inoculation.
Mice were inoculated by oral gavage with ~2 × 107 H. hepaticus or H. bilis colony-forming
units (CFU) in 0.2 ml of Brucella broth or with
Brucella broth alone three times within a 9-day period. H. hepaticus was obtained from the American
Type Culture Collection (ATCC 51448), and H. bilis was
kindly provided by Dr. Lela Riley (University of Missouri, Columbia,
MO). Briefly, organisms were streaked onto Brucella blood
agar plates (Dept. of Microbiology, Media Laboratory, UW, Seattle, WA),
grown in microaerobic conditions (90% N2, 5%
H2, and 5% CO2) in vented jars (Oxoid), and
kept at 37°C. Bacteria were harvested and inoculated into
flasks containing 150 ml of Brucella broth supplemented with
5% FCS (Sigma Chemical, St. Louis, MO). The flasks were placed on a
continuous shaker and incubated for 24-48 h at 37°C in
microaerobic conditions. The organisms were centrifuged at 10,000 rpm
at 4°C for 20 min. The resultant pellet was examined by Gram's stain
and phase microscopy for purity, morphology, and motility (catalase,
urease, and oxidase positive). The pellet was resuspended in
Brucella broth, and optical density was adjusted to 1.0, as
measured at 600 nm, for an estimated 108 CFU/ml (2 × 107 CFU/0.2 ml).
Helicobacter PCR and fecal culture.
Feces were analyzed for Helicobacter spp, H. hepaticus, and H. bilis, as described previously
(12, 31, 38), with slight modifications. For
generic Helicobacter PCR, 5 µl of unquantitated fecal DNA
were used as a template, and for H. hepaticus and H. bilis PCR, 10 µl of unquantitated fecal DNA were used as a
template for all samples. Primers, their sequences, and the PCR
conditions used are summarized in Table
1. To correlate fecal culture results and
PCR status, fecal pellets were vortexed in PBS and incubated at room
temperature for 30 min. Fecal slurries were then filtered through an
0.8-µm filter, and the filtrate was streaked onto Brucella blood agar plates containing trimethoprim, vancomycin, and polymyxin and incubated as described in Inoculation. Cultures were
maintained for 2-3 wk before determination of negative growth was
made.
Pathology.
The colon was fixed in 10% buffered formalin with a "Swiss roll"
technique (26). The liver, cecum, rectum, and mesenteric lymph nodes were also fixed in formalin. Fixed tissues were routinely processed, embedded in paraffin, sectioned at 5 µm, and stained with
hematoxylin and eosin. Tissue sections were coded to blind the
pathologist (K. Waggie) to the strain and infection status of the
animal. The cecum, proximal colon, middle colon, distal colon, and
rectum from each mouse were scored on severity of mucosal epithelial
changes, degree of inflammation, and extent of pathology (Table
2). The segment score was derived by
summing the severity scores [segment score = mucosal score + inflammation score + extent of segment affected in any manner
(extent 1) + extent of segment affected at level 3 or 4 (extent 2); maximum segment score was 15]. The total score
for each mouse was derived by summing the scores from the individual
segments (maximum total score was 75). Subsequently, the mean segment
and total scores were derived for each treatment group.
MHC class II immunohistochemistry.
MHC class II expression was analyzed in H. bilis- and
H. hepaticus-infected and uninfected IL-10
/
,
Rag1
/
, and C57BL/10J mice. Briefly, 3- to 4-mm sections
of proximal colon and distal colon were snap-frozen in optimal cutting
temperature compound, and detection of MHC II expression was
performed on 8-µm frozen sections. Sections were fixed with acetone
and then incubated with a 1:200 dilution of biotinylated mouse
anti-mouse I-Ab monoclonal antibody (PharMingen, San Diego, CA).
Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide
for 20 min followed by detection of signal with the
biotin-streptavidin-peroxidase VECTASTAIN ABC Kit (Vector Laboratories,
Burlingame, CA). The chromagen used was 3,3'-diaminobenzidine.
Intensity of MHC II staining was based on the following scoring system:
0 = no expression, 1 = minimal expression, 2 = moderate
expression, and 3 = maximal expression. As a negative control,
colonic tissue from MHC class II
/
mice was stained for
class II expression. All slides were scored independently by two
investigators blinded to the strain and infection status of the animal.
Colonic RNA extraction and cytokine analysis by RT-PCR and RNase
protection assay.
Qualitative analysis of cytokine mRNA expression in colons from
H. bilis-infected or uninfected IL-10
/
,
Rag1
/
, C57BL/6J, TCR-
/
, and
TCR-
/
mice was performed between 6 and 17.5 wk PI.
Briefly, colonic fecal contents were emptied, and two to four colons
were pooled and homogenized in guanidinium isothiocyanate buffer [4.5
M guanidinium isothiocyanate, 50 mM sodium citrate, and 0.5% (wt/vol)
sodium sarcosyl] containing 2% 2-mercaptoethanol (Life
Technologies, Rockville, MD). RNA was isolated over a cesium chloride
cushion and subjected to phenol chloroform extraction as previously
described (34). cDNA was prepared from 5 µg of isolated
RNA with SuperScript II reverse transcriptase (Life Technologies,
Rockville, MD) in the presence of 0.3 µg of random primers, 5× RT
buffer, 0.1 M dithiothreitol, 10 mM deoxynucleotide triphosphate, and
water. Interferon-
and IL-4 messages were analyzed by RT-PCR with
the mouse TH1/TH2 Switch Cytokine kit (BioSource
International, Camarillo, CA). The following profile was used for
amplification: 96°C for 1 min followed by 2 cycles of 96°C for 1 min and 59°C for 4 min, then 30 cycles of 94°C for 1 min and 59°C
for 2.5 min, and a final cycle of 70°C for 10 min. As a negative
control, cDNA was replaced with water, and the sample was subjected to
the same PCR conditions. The PCR product and 1 µl of loading buffer
were loaded on a 1.0-mm, 15-well, 10% TBE precast gel (Novex, San
Diego, CA) and run at 126 V. The gel was stained with ethidium bromide
and photographed. For RNase protection assay analysis, H. bilis-induced IL-1
, IL-1
, and IL-1 receptor antagonist
(IL-1RA) levels were measured at selected time points in
IL-10
/
, C57BL/6J, and Rag1
/
mice with
the RiboQuant Multiprobe RNase Protection Assay kit mCK-2b
(PharMingen). The assay was run according to the manufacturer's instructions, and the values obtained for each sample were normalized to glyceraldehyde-3-phosphate dehydrogenase.
Statistical analyses.
Differences between mean total pathology scores in
IL-10
/
mice infected with either H. bilis or
H. hepaticus were evaluated by two-way analysis of variance
(Sigma Stat 2.0; Jandel, San Rafael, CA). Statistical significance was
set at a P value <0.05.
 |
RESULTS |
Colonization with H. hepaticus or H. bilis.
Mice inoculated with H. hepaticus or H. bilis
became fecal PCR positive 2-4 wk postinoculation and remained
persistently positive for the duration of these experiments (12-36
wk PI). Consistent with other studies, PCR was a more sensitive and
specific means to detect Helicobacter spp infection compared
with culture (data not shown) (22). Therefore, PCR was the
sole method used to confirm infection. Uninfected animals that received
broth alone remained Helicobacter fecal PCR negative
throughout these studies, as determined with Helicobacter
genus primers. Periodic fecal PCR for the alternate
Helicobacter spp with species-specific primers revealed that
at no time were the mice cross contaminated.
Clinical and histopathological findings associated with
Helicobacter spp infection in IL-10
/
mice.
An initial study (study 1) was performed to determine
whether different species of Helicobacter induced similar or
dissimilar forms of IBD after infection with either H. bilis
or H. hepaticus in a well-characterized IBD model, the
IL-10
/
mouse. Diarrhea was evident in the
Helicobacter spp-infected IL-10
/
mice, but
there were notable differences between the clinical course of H. bilis and H. hepaticus-infected animals. The earliest time to development of diarrhea differed depending on whether IL-10
/
mice were infected with H. hepaticus
(1.5 wk) or H. bilis (3 wk). Despite the more rapid
induction of diarrhea with H. hepaticus, weight gain in
H. bilis-infected IL-10
/
mice was less than
that seen in uninfected controls (Fig.
1A), whereas H. hepaticus-infected IL-10
/
mice showed normal
growth curves (data not shown). It is important to note that there were
no significant weight change differences in H. bilis-infected wild-type mice (Fig. 1B). Diarrhea was
not observed in Helicobacter spp-infected wild-type mice or
in uninfected wild-type and IL-10
/
mice.

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Fig. 1.
Helicobacter bilis infection induces altered
weight gain in interleukin (IL)-10 / mice. Change in
body weight over time is expressed as percentage of original body
weight before Helicobacter infection. Data are means ± SE of 9 mice/infected group and 3 mice/uninfected group unless
otherwise stated. A: growth curve was altered in H. bilis-infected IL-10 / mice. B: growth
curves for Helicobacter-infected and uninfected wild-type
mice were similar. aMice remaining after histopathological
analysis at 3, 7, and 12 wk postinfection (PI). bOne animal
in group was euthanized because of severe weight loss. Results are
representative of 3 experiments.
|
|
Both H. bilis and H. hepaticus induced IBD in
IL-10
/
mice (Fig.
2B and data not
shown), whereas no IBD was observed in uninfected IL-10
/
mice (Fig. 2A) or
Helicobacter-infected and uninfected wild-type C57BL/10J
mice (Fig. 2C). Generally, both H. hepaticus and
H. bilis produced chronic proliferative typhlocolitis and
proctitis beginning at 3 wk PI. The mean total pathology scores are
summarized in Table 3. Although
H. hepaticus produced its most severe lesions in the cecum,
proximal colon, and rectum, H. bilis induced significant disease in all portions of the large bowel.

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Fig. 2.
IL-10 / mice infected with H. bilis or
H. hepaticus developed severe inflammatory bowel disease
(IBD) beginning 3 wk PI. However, H. bilis-induced disease
may be more severe than that induced by H. hepaticus in
middle and distal colon of IL-10 / mice. A:
distal (bottom) and proximal (top) colon of
uninfected IL-10 / mouse 12 wk after treatment.
B: distal colon of IL-10 / mouse infected
with H. bilis 12 wk PI. Note severe mucosal inflammation and
hyperplasia with crypt herniation into the submucosa. Similarly, at
this time point, the distal colon of H. hepaticus-infected
IL-10 / mice showed moderate mucosal inflammation and
epithelial hyperplasia. C: distal colon of C57BL/10J mouse
infected with H. bilis 12 wk PI. There is no evidence of
inflammation. Segments stained with hematoxylin and eosin; original
magnification, ×10.
|
|
With H. hepaticus infection, the chronic colitis was
characterized by varying degrees of mucosal thickening resulting from epithelial hyperplasia, with elongation and occasional branching of
crypts (data not shown). Crypt penetration into the submucosa was
observed in the rectum of two of three mice 12 wk PI (data not shown).
An inflammatory cell infiltrate was present that consisted primarily of
lymphocytes and macrophages with varying numbers of plasma cells and
neutrophils. The infiltrate was most often confined to the lamina
propria and submucosa but was occasionally transmural. Crypt abscesses
and mucosal erosions were infrequently present. The typhlitis was
characterized by severe mucosal hyperplasia and scattered erosions,
goblet cell loss, and a predominantly lymphoplasmacytic infiltrate
(Fig. 3, A and B).

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Fig. 3.
IL-10 / mice infected with H. hepaticus or H. bilis developed proliferative typhlitis
and proctitis beginning 3 wk PI. A: cecum of uninfected
IL-10 / mouse 12 wk after treatment. B: cecum
of IL-10 / mouse infected with H. hepaticus
12 wk PI. Note severe mucosal hyperplasia, inflammation, and focal
erosion. Lumens of occasional crypts are dilated and contain debris.
Ceca of H. bilis-infected mice at this time point had a
similar appearance. C: rectum of uninfected
IL-10 / mouse 12 wk after treatment. D:
rectum of IL-10 / infected with H. bilis 12 wk PI. Note crypt branching and herniation. Similar lesions were
present at 3 and 7 wk PI. Segments stained with hematoxylin and eosin;
original magnification, ×10.
|
|
Microscopic changes in H. bilis-infected
IL-10
/
mice were similar to those observed in H. hepaticus animals but tended to be more severe, especially in the
middle and distal colon 7 and 12 wk PI (Table 3). Specifically, severe
dilation of the submucosal lymphatics and submucosal edema were found
at 3 and 7 wk PI. Transmural inflammation, crypt abscesses, and mucosal
erosions were frequently observed at all time points, which differed
from findings with H. hepaticus infection. Crypt penetration
into the submucosa, observed at 12 wk PI in two of three mice, extended
into the distal colon of H. bilis-infected mice (Fig.
2B), and the rectum was similarly affected (Fig. 3,
C and D).
H. hepaticus- and H. bilis-induced IBD in
IL-10
/
mice was associated with a marked increase in
MHC class II expression in both the proximal and distal colon. The
staining was evident on both epithelial cells and cells within the
lamina propria (Fig.
4A), with scores
ranging from 2 to 3 (Table 4). Uninfected
IL-10
/
mice without colitis showed a minimal to
moderate increase in MHC class II expression (Fig. 4B; Table
4). H. hepaticus- and H. bilis-infected and
uninfected wild-type mice had minimal MHC class II expression that was
primarily localized to the surface colonic epithelial cells (Fig.
4C). As a scoring reference, colonic tissue from MHC
II
/
mice was stained and showed no MHC class II
expression (Fig. 4D).

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Fig. 4.
Major histocompatibility complex (MHC) class II
expression was elevated in both the proximal and distal colon of
H. hepaticus- and H. bilis-infected
IL-10 / mice. Sections were stained with
3,3'-diaminobenzidine and counterstained with methyl green. Shown are
samples of distal colon (A-C) obtained from H. bilis-infected and uninfected mice 12 wk after treatment. Similar
results were seen in the proximal colon. A:
IL-10 / mouse infected with H. bilis 12 wk
PI. Note intense MHC class II staining of lamina propria and epithelial
cells. Both surface and crypt epithelial cells are stained.
B: uninfected IL-10 / mouse 12 wk after
treatment. Note decreased intensity of MHC class II staining, present
primarily on surface epithelial cells and a few lamina propria cells.
C: C57BL/10J mouse infected with H. bilis 12 wk
PI. Decreased intensity of MHC class II staining is present primarily
on surface epithelial cells and random lamina propria cells.
D: colonic tissue section from an MHC class
II / mouse. No expression of MHC class II can be seen.
Original magnification, ×20 for all. Data are representative of 2 or 3 mice/infected group.
|
|
Our initial observations suggested that H. bilis infection
in IL-10
/
mice may produce more extensive and severe
IBD than H. hepaticus (Table 3). Therefore, in study
2, a larger group of IL-10
/
mice were infected
with H. bilis or H. hepaticus to determine whether this difference was statistically significant. It was necessary
to euthanize all mice at 4 wk PI because of weight loss observed in the
IL-10
/
mice infected with H. bilis. This
study, consisting of mixed ages of mice, showed no statistically
significant differences between H. bilis- and H. hepaticus-induced IBD pathology (Fig. 5, A and B). Mean
total pathology scores were 43 and 44 for IL-10
/
mice
infected with H. bilis or H. hepaticus,
respectively (P = 0.605; Table 3). A low level of mild
spontaneous IBD (mean total pathology score of 2) was observed in 4 of
15 uninfected IL-10
/
mice (data not shown), whereas all
others remained disease free (Fig. 5C). Interestingly, when
mean total pathology scores from mice that were older (6-7 wk of
age; data not shown) at the time of H. bilis or H. hepaticus infection were analyzed and compared, we found
a trend towards significance (P = 0.076). Based on this observation, as well as the altered growth curves seen in H. bilis-infected compared with H. hepaticus-infected
IL-10
/
mice, it is our impression that H. bilis-induced IBD in IL-10
/
mice tends to be more
severe than H. hepaticus-induced disease, especially in mice
that are older at the time of Helicobacter infection.
Clearly, the data support the hypothesis that both species of
Helicobacter can induce severe IBD in this model.

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Fig. 5.
IL-10 / mice infected with H. bilis or
H. hepaticus developed IBD but the disease was not
significantly different in severity. A: distal colon of
IL-10 / mouse infected with H. bilis 4 wk PI.
Note severe epithelial hyperplasia with a mixed inflammatory cell
infiltrate into the lamina propria, extending somewhat into the
submucosa. B: distal colon of IL-10 / mouse
infected with H. hepaticus 4 wk PI. Disease is similar to
that seen in IL-10 / mice infected with H. bilis, with severe epithelial hyperplasia and mucosal
inflammation. However, rarely did the inflammatory cells infiltrate the
submucosa. C: distal colon of uninfected
IL-10 / mouse 4 wk after treatment. This uninfected
mouse shows no evidence of inflammation, although 4 of 15 uninfected
IL-10 / mice did show very mild spontaneous IBD.
Segments stained with hematoxylin and eosin; original magnification,
×10.
|
|
Susceptibility of Rag1
/
mice to Helicobacter spp
infection.
Rag1
/
mice infected with either H. hepaticus
or H. bilis did not develop the clinical or typical
histological features of IBD that were seen in the
IL-10
/
mice in studies extending to 12 wk PI.
Similarly, uninfected Rag1
/
mice showed no evidence of
intestinal inflammation. However, one of three mice from the H. hepaticus and H. bilis groups developed an acute,
focal, mild inflammation of the colon and/or cecum at the 3 or 12 wk
time points, respectively. Although Helicobacter spp-infected Rag1
/
mice did not develop IBD, MHC class
II expression was moderately increased (score of 2) in the proximal
colon of H. hepaticus-infected Rag1
/
mice at
3 and 7 wk PI (Table 4) compared with uninfected controls. Similarly,
H. bilis-infected Rag1
/
mice showed mild to
moderate (score of 1.3-2.0) MHC class II staining in the proximal
and distal colon at 3 and 7 wk PI. At all time points examined,
uninfected Rag1
/
mice showed mild MHC class II staining
intensity of the proximal and distal colon (score of 1.0-1.5;
Table 4).
To determine whether long-term infection with Helicobacter
could initiate IBD in Rag1
/
mice, both wild-type and
Rag1
/
mice were infected with either H. hepaticus or H. bilis and their progress was followed
for 30-36 wk. Significant morbidity occurred in our
Rag1
/
mice as a result of Pneumocystis
carinii infection and precipitated the end of the study at these
time points. Approximately 9 mo after Helicobacter
infection, there were no clinical indications of IBD (diarrhea, rectal
prolapse) in Rag1
/
or wild-type mice chronically
infected with Helicobacter. This is in marked contrast to
the rapid onset of disease in similarly infected IL-10
/
mice (1.5-3 wk PI). Additionally, no histological evidence of IBD
was found in Helicobacter-infected and uninfected wild-type mice or uninfected Rag1
/
mice (Fig.
6, A and C; data
not shown). However, two of five Rag1
/
mice in both the
H. hepaticus and H. bilis groups developed mild focal mucosal inflammation characterized by minimal to mild epithelial hyperplasia and low numbers of a predominantly neutrophilic infiltrate (Table 5; Fig. 6, B and
D). Interestingly, lesions in H. bilis-infected Rag1
/
mice were found solely in the proximal colon,
whereas those of H. hepaticus-infected Rag1
/
mice were found in the cecum (Fig. 6, B and D).
It is important to note that typical IBD lesions such as those seen in
similarly infected IL-10
/
mice did not develop in
Helicobacter-infected Rag1
/
mice.

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|
Fig. 6.
Recombinase-activating gene (Rag1 / ) mice infected
long-term with either H. bilis or H. hepaticus
developed an acute mild form of IBD that differed from the severe
chronic form of IBD seen in IL-10 / mice infected with
Helicobacter. A: proximal colon of uninfected
Rag1 / mouse 32 wk after treatment. B:
proximal colon of Rag1 / mouse infected with H. bilis 33 wk PI. Note the mild accumulations of (predominantly)
neutrophils in the lamina propria and minimal epithelial hyperplasia.
C: cecum of uninfected Rag1 / mouse 33 wk
after treatment. D: cecum of Rag1 / mouse
infected with H. hepaticus 30 wk PI. There are mild
accumulations of inflammatory cells in the lamina propria, and there is
minimal epithelial hyperplasia. Segments stained with hematoxylin and
eosin; original magnification, ×20.
|
|
Susceptibility of T cell-deficient mice to H. bilis infection.
Because we were unable to induce significant IBD with
Helicobacter spp in a T cell-deficient mouse
(Rag1
/
) but did obtain severe intestinal inflammation
with Helicobacter in a model having T cells
(IL-10
/
), we wanted to determine the importance of T
cell populations in the initiation of IBD. Therefore, we infected
wild-type, TCR-
/
, and TCR-
/
mice
with H. bilis in the same manner previously described. We chose H. bilis for these experiments based on our clinical
experience with this organism in eliciting profound rapid inflammation
in the IL-10
/
mouse. Two separate experiments were
performed, extending to 17.5 (study 1) and 29 (study
2) wk PI. Helicobacter-infected
TCR-
/
mice were only included in the initial 17.5-wk
study. Of these two mutant strains, we were only able to induce IBD in
TCR-
/
mice in the time frame of these studies. The
onset of disease in the TCR-
/
mice was more delayed
than that historically seen in the IL-10
/
mice infected
under a similar protocol. Clinically, diarrhea was evident in the
TCR-
/
mice in these two studies beginning
10-12.5 wk PI. However, diarrhea was not evident in all
Helicobacter-infected TCR-
/
mice. This
was much later than what we had observed in H. bilis-infected IL-10
/
mice (3 wk PI). Furthermore,
growth curves for H. bilis-infected and uninfected
TCR-
/
mice were similar (data not shown).
At 17.5 wk PI, three of five H. bilis-infected
TCR-
/
mice had lesions in the cecum, colon, and
rectum that were similar to but milder than lesions observed in the
corresponding H. bilis-infected IL-10
/
mice
(see Clinical and histopathological findings associated with
Helicobacter spp infection in IL-10
/
mice).
Pathology scores are summarized in Table 5. Although lesions in the
TCR-
/
mice were found in all areas of the large
bowel, they were segmental within each area. Generally, the lesions
were characterized by moderate crypt epithelial hyperplasia with
inflammatory cell infiltrates limited to the mucosa and submucosa (Fig.
7). One of four uninfected TCR-
/
and one of four uninfected
TCR-
/
mice had mild colitis limited to a focal area
in the middle colon. These focal lesions were similar to but milder
than those in the H. bilis-infected TCR-
/
mice and were probably the result of the
non-Helicobacter-associated colitis reported to occur
spontaneously in these strains (25).

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Fig. 7.
T-cell receptor (TCR)- / mice infected
with H. bilis developed IBD that was milder than that seen
in Helicobacter-infected IL-10 / mice.
A: distal colon of uninfected TCR- / mouse
17.5 wk after treatment. B: distal colon of
TCR- / infected with H. bilis 17.5 wk PI.
There is crypt hyperplasia and moderate mucosal inflammation. Segments
mouse stained with hematoxylin and eosin; original magnification,
×10.
|
|
Study 2 was initiated to correlate histopathology and
cytokine regulation during Helicobacter-induced IBD in
TCR-
/
mice (see Proinflammatory cytokine
production in the colon of H. bilis-infected mice for cytokine
results). In mice evaluated histologically at 29 wk PI, four of eight
H. bilis-infected TCR-
/
mice showed IBD
lesions similar to those seen in our initial study (Table 5).
Interestingly, the IBD in these diseased TCR-
/
mice
was completely subclinical, with no evidence of diarrhea or weight loss
(data not shown). Again, we saw evidence of mild, focal, spontaneous
IBD in uninfected TCR-
/
mice (2 of 8).
Proinflammatory cytokine production in the colon of H. bilis-infected mice.
Proinflammatory cytokine expression correlated with the presence of
intestinal inflammation in IL-10
/
and
TCR-
/
mice. With RT-PCR, IL-10
/
mice
infected with H. bilis showed elevated levels of
interferon-
mRNA in the colon compared with uninfected
IL-10
/
mice at all time points analyzed (Fig.
8A). Our results agree with
previous studies that reported interferon-
release on stimulation of
mesenteric lymph node cells from H. hepaticus-infected
IL-10
/
mice (19). Surprisingly, elevated
levels of interferon-
message were also seen in colonic tissue
harvested from diseased H. bilis-infected TCR-
/
mice compared with uninfected
TCR-
/
mice (Fig. 8B). Although this model
is recognized as a predominantly IL-4-driven model of IBD, we did not
see elevated levels of IL-4 message in either H. bilis-infected or uninfected TCR-
/
mice. In
contrast, colonic tissue samples from H. bilis-infected Rag1
/
, TCR-
/
,
and wild-type mice (Fig. 8A and data not shown) showed
greatly reduced amounts of interferon-
message compared with
H. bilis-infected IL-10
/
mice with IBD.
Using an RNase protection assay, we found that H. bilis
upregulated expression of IL-1
(~3-fold), IL-1
(~15- to
26-fold), and IL-1RA (~3-fold) in IL-10
/
mice with IBD (Fig. 9). As an
indicator of dysregulation of proinflammatory cytokine activity, the
IL-1RA-to-IL-1
ratio was determined for infected versus uninfected
IL-10
/
mice. H. bilis-infected
IL-10
/
mice had a lower ratio (0.626) than uninfected
IL-10
/
mice (5.921). Expression of IL-1
, IL-1
,
and IL-1RA in infected and uninfected wild-type and
Rag1
/
mice was not significantly different (data not
shown).

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Fig. 8.
H. bilis induced increased mRNA
expression of interferon (IFN)- in the colon of
IL-10 / and TCR- / mice with IBD
analyzed by RT-PCR. A: low levels of IFN- are seen in
IL-10 / uninfected, H. bilis-infected
C57BL/6J, and H. bilis-infected and uninfected
Rag1 / mice. Additionally, low levels of IL-4 were seen
in both H. bilis-infected and uninfected
Rag1 / mice. RNA samples (3-5 sets) were prepared
from each infected and uninfected strain 6-17 wk PI. For each
sample, the PCR reaction was performed 1-3 times.
IL-10 / mice were 6 wk PI, whereas the C57BL/6J and
Rag1 / mice were 17 wk PI. B: IFN- levels
were significantly elevated in diseased H. bilis-infected
TCR- / mice compared with uninfected controls. Note
the absence of IL-4 message in diseased TCR- / mice.
RNA samples were prepared from infected and uninfected
TCR- / mice 13 wk PI, and the PCR reaction was
performed twice on each sample. GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
|
|

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Fig. 9.
H. bilis induced increased mRNA expression of
IL-1 (B), IL-1 (C), and IL-1 receptor
antagonist (RA; D) in the colon of IL-10 /
mice as detected by RNase protection assay. No such elevations were
seen in H. bilis-infected C57BL/6J mice
(A-D). IL-10 / and wild-type mice were 9 and 7 wk post-H. bilis infection, respectively. All values
are normalized to GAPDH.
|
|
 |
DISCUSSION |
Previous studies (9, 18, 29, 33, 37) have shown that
luminal bacteria are an important factor contributing to the development of IBD in mice and humans. Several laboratories have implicated infection with Helicobacter species in the
development of mucosal inflammation and IBD in various mouse models
(4, 6, 13, 14, 39). By comparing the effects of the less widely studied H. bilis with H. hepaticus in the
IL-10
/
mouse, a well-established model of Th1-driven
IBD (3, 7), we present data that both confirm and extend
these results that suggest a role for Helicobacter in murine
IBD. In addition, because of emerging literature suggesting
that Helicobacter-induced IBD can occur in profoundly
immunodeficient mice, we attempted to address the role of T cells in
the pathogenesis of Helicobacter-induced IBD. Using the
combination of a detailed histopathological scoring system, MHC class
II immunohistochemistry, and cytokine analysis, we demonstrate herein
that both H. bilis and H. hepaticus consistently produce severe IBD in IL-10
/
mice, whereas the ability
of Helicobacter to induce intestinal inflammation in mice
with absent T cells (Rag1
/
and TCR-
/
mice) or significantly altered T cell subsets (TCR-
/
mice) is attenuated. Furthermore, increases in MHC class II expression and colonic proinflammatory cytokine levels correlated with the presence of IBD in Helicobacter-infected
IL-10
/
mice. Surprisingly, despite the fact that the
TCR-
/
mouse has been described as a "Th2" model
of murine IBD (23, 24, 40), Helicobacter
infection of these mice resulted in colitis with elevated levels of
interferon-
without significant induction of IL-4. Taken together,
the current studies strongly support the role of murine
Helicobacter in the development of colitis and highlight the
importance of, but not strict dependence on, T cells in the development
of bacterially driven mucosal inflammation in mouse strains genetically
susceptible to the development of IBD.
Numerous studies have investigated Helicobacter spp as model
organisms in microbial-induced IBD. It is fair to say that the observations made in these studies have varied widely. For example, at
one end of the spectrum, Kullberg et al. (19) have
observed chronic colitis in SPF-reared IL-10
/
mice
after infection with H. hepaticus. In an independent series of experiments, Chin et al. (6) described the ability of
H. hepaticus to induce significant mucosal inflammation in
the cecum and colon of TCR-
mutant mice. In marked contrast,
recent studies have reported that Helicobacter does not
induce IBD in germ-free IL-10
/
mice or potentiate IBD
in germ-free IL-10
/
mice that have been reconstituted
with SPF flora (8, 37). It is important to note that the
published studies have used different protocols in varied mouse
facilities and, in so doing, have not resulted in a consensus regarding
the role of Helicobacter as a potential pathogen in
conventional mouse facilities or its utility as a model organism to
study IBD. How do the data presented in this report fit into the
context of these previous studies? Clearly, we have shown that
Helicobacter can be an important determinant in the
induction of murine colitis in an SPF mouse facility. In our
experience, IBD-prone strains such as IL-10
/
and
TCR-
/
mice rarely develop mucosal inflammation in a
Helicobacter-free, SPF environment, whereas monoinfection
with H. bilis or H. hepaticus induces a
significantly greater incidence and severity of disease in these mice.
A further conclusion from these studies is that the disease induced by
Helicobacter varied significantly among the different IBD
models. Foremost, both H. bilis and H. hepaticus reliably induced severe chronic IBD in the IL-10
/
mouse model of IBD. Disease was evident clinically 1.5-3 wk PI, and inflammation was evident from cecum to rectum. Intermediate in
disease severity was the intestinal inflammation found in our TCR-
/
mice infected with H. bilis.
Lesions were similar to but milder than the inflammation seen in
similarly infected IL-10
/
mice. Additionally, disease
incidence in the Helicobacter-infected TCR-
/
mice was less than that seen in
Helicobacter-infected IL-10
/
mice. In
contrast, minimal mucosal inflammation was found in Rag1
/
mice, which do not have T or B cells. This
inflammation, when present, was characterized by minimal to mild focal
epithelial hyperplasia and had a primarily neutrophilic infiltrate. Our
observations support those of others that show Helicobacter
can induce fulminant disease in certain immunodeficient strains of mice.
As mentioned, there are important differences among all these studies
that may explain the varied results. In our opinion, the major
variables include the unique bacterial flora present in geographically
different mouse colonies and the contribution of genetic background to
IBD susceptibility. Regarding the importance of mouse strain variation
in the development of IBD, Berg et al. (3) have shown that
IL-10
/
mice on a 129/SvEv or BALB/c background are
inherently more susceptible to spontaneous IBD compared with
IL-10
/
mice on a C57BL/6J background. Strain
differences may explain why in our studies with
Helicobacter-infected TCR-
/
and
TCR-
/
mice, we saw less severe disease than that
reported by Chin et al. (6). Specifically, our mice were
on a C57BL/6J background, whereas those of Chin et al. were on a
C57BL/6 × 129-Ola background. Moreover, it is nearly impossible
to characterize the subtle yet potentially important differences in
intestinal microbial flora between different colonies of mice. We and
others (8) assume that in the complex ecosystem of the
colonic microbiota, Helicobacter alone is not sufficient
(even in a highly susceptible model like the IL-10
/
mouse) to induce mucosal inflammation. This may explain the findings that germ-free IL-10
/
mice infected with H. hepaticus do not develop IBD (8). Therefore, the
profound inflammation seen in germ-free mice reconstituted with SPF
flora, with or without Helicobacter (8, 37),
does not discount the potential role of Helicobacter in
murine colitis. Instead, in our opinion, it reinforces the hypothesis
that multiple bacterial species can contribute to the establishment of
IBD, particularly in a setting where mice without natural tolerance to
luminal bacterial (as in germ-free mice not exposed to normal flora
during development) are exposed to a wide range of bacterial flora.
Numerous studies have underlined a prominent role for T cells in the
initiation and maintenance of IBD (3, 7, 17, 25, 28, 30).
Therefore, it is somewhat surprising that several studies have
suggested that Helicobacter can induce IBD in animal models
devoid of T cells (6, 14, 15, 21, 39, 41). We tested this
question directly by infecting Rag1
/
mice, which lack T
and B cells, with H. bilis or H. hepaticus. In
contrast to the consistent induction of severe IBD in
IL-10
/
mice infected with Helicobacter spp,
we observed mild acute inflammation in Rag1
/
mice. It
is also important to note that the mucosal inflammation observed in
Helicobacter-infected Rag1
/
mice was more
focal than that seen in similarly infected IL-10
/
and
TCR-
/
mice and was particularly less consistent in
its appearance. Still, the intestinal inflammation seen in
Rag1
/
mice infected with Helicobacter
suggests that the development of bacterial-driven mucosal
inflammation is not strictly dependent on T cells. It is tempting to
speculate that Helicobacter may trigger the innate immune
system resulting in the induction of cytokines and/or other chemical
mediators that may eventually produce inflammation. Whether it is
through the modulation of intestinal barrier function and/or induction
of proinflammatory cytokines via lipopolysaccharide-mediated activation
of Toll-like receptors on dendritic cells, monocytes, or epithelial
cells or via recently discovered virulence factors in certain
Helicobacter spp (5, 45), this innate immune
response may contribute to the establishment of mucosal inflammation
and IBD.
In summary, our results in a variety of mouse models suggest that
Helicobacter infection plays an important role in the
induction of IBD in SPF mice. In our experience, T cells appear to be
required for the development of chronic, diffuse, and severe
inflammation associated with Helicobacter infection. Still,
the observations in Rag1
/
mice presented here, along
with a recent report from another group (6), demonstrate
that T cells are not strictly required for the induction of mucosal
inflammation by Helicobacter. In addition, we show that
Helicobacter may harbor the ability to skew the cytokine
responses toward a Th1 phenotype, as shown by the elevated levels of
interferon-
in the colons of Helicobacter-infected mice. Again, we suggest that Helicobacter is likely to be
one of a group of bacteria that, in mice, can tip the balance toward mucosal inflammation. Whether there are unifying and/or overlapping features between these microbial triggers for IBD (predilection for
skewing toward Th1 responses, for example) remains to be determined. However, a recent report has shown the ability of Citrobacter rodentium to elicit a Th1 cytokine phenotype associated with IBD, suggesting that Th1 responses may be a stereotypical response to
certain luminal microbes (16). We believe organisms like Helicobacter can serve as useful tools to study
microbial-induced IBD and may be able to both assist in refining the
hypotheses regarding the molecular pathogenesis of IBD and to test
novel therapeutic interventions that may be relevant to the treatment of human IBD. Although there are currently no data to suggest that Helicobacter spp are implicated in human Crohn's
disease or ulcerative colitis, we are intrigued by recent observations of novel Helicobacter species in both the cotton-top tamarin
(36) and in humans with diarrhea (10).
 |
ACKNOWLEDGEMENTS |
We thank Craig Labenz for expert graphics assistance and Loida
Torres for excellent animal care.
 |
FOOTNOTES |
This work was supported, in part, by the Biomedical Research Training
Grant for Veterinary Scientists T32-RR07019 (to A. Burich).
Address reprint requests to Dr. L. Maggio-Price, Dept. of
Comparative Medicine, Box 357190, School of Medicine, Univ. of
Washington, Seattle, WA 98195 (E-mail:
lmprice{at}u.washington.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 30 August 2000; accepted in final form 1 May 2001.
 |
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