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Infection and Immunity, September 2000, p. 5107-5113, Vol. 68, No. 9
Center for Gastrointestinal Biology and
Disease, University of North Carolina at Chapel
Hill,1 and Department of Microbiology,
University of North Carolina Hospitals,3 Chapel
Hill, and College of Veterinary Medicine, North Carolina
State University, Raleigh,2 North
Carolina, and Departments of Surgery and Microbiology,
University of Wisconsin, Madison, Wisconsin4
Received 15 October 1999/Returned for modification 22 December
1999/Accepted 6 June 2000
Helicobacter hepaticus has been reported to induce
colitis, hepatitis, and hepatocellular carcinoma in several different
murine models. The aim of this study was to determine if H. hepaticus will cause colitis in monoassociated mice lacking the
interleukin-10 gene (IL-10 The pathogenesis of chronic
inflammatory bowel diseases is still poorly understood. In the last
decade experimental models of chronic intestinal inflammation have
clarified the concept that a combination of genetic, environmental, and
immunologic factors are necessary to induce and perpetuate these
chronic, spontaneously relapsing diseases (10, 25).
Results in a number of rodent models have conclusively documented that
luminal bacteria are required for chronic intestinal inflammation
(27). For example, mice that lack the interleukin-10 gene
(IL-10 Although these findings suggest that normal intestinal flora provide
the constant antigenic drive for chronic intestinal inflammation in
susceptible hosts, the possibility of a persistent pathogenic microbial
infection, or an unconventional pathogen which is difficult to detect,
has not been excluded in either human inflammatory bowel disease
or rodent models (3). Among those pathogens considered potential candidates to induce chronic intestinal inflammation are
Helicobacter species (2). Helicobacter
hepaticus is a widespread contaminant of murine colonies
(31), including genetically engineered mice with colitis,
such as our IL-10 The aim of the present studies was to investigate whether H. hepaticus plays an essential or potentiating pathogenetic role in
the development of colitis in susceptible mice with functioning T
cells. Colitis was quantified by clinical, histologic, and immunologic parameters in germfree IL-10 Animals.
Two sublines of germfree IL-10 Study design.
We report here the results of two separate
studies designed to evaluate the role of H. hepaticus in
development of colitis in IL-10
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Helicobacter hepaticus Does Not Induce
or Potentiate Colitis in Interleukin-10-Deficient Mice
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
/ mice) and potentiate
colitis in specific-pathogen-free (SPF) IL-10/ mice.
Germfree IL-10/ mice on either a mixed (C57BL/6 × 129/Ola) or inbred (129/SvEv) genetic background were monoassociated
with H. hepaticus ATCC 51448 by oral feeding and rectal
enemas. In a second experiment, germfree IL-10/ mice
were colonized with stool from SPF mice that harbored or did not harbor
endogenous H. hepaticus. After 7 to 9 weeks of colonization, weight loss and mortality were assessed, the colon was
isolated for histology and IL-12 secretion, and mesenteric lymph node
cells were assessed for T-cell activation markers. It was found that
IL-10/ mice monoassociated with H. hepaticus for up to 16 weeks showed almost no histologic colitis
or increased IL-12 production. SPF IL-10-knockout mice had no
significant difference in weight loss, mortality rate, histologic
scores, colonic IL-12 secretion, or T-cell activation with or without
H. hepaticus. We conclude that H. hepaticus
does not induce or potentiate disease in our IL-10/
mice and therefore is not required to induce colitis in genetically susceptible hosts.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
/ mice) develop a spontaneous, lethal
enterocolitis when housed in a conventional environment
(18). However, when these animals are kept under
specific-pathogen-free (SPF) conditions, they develop nonlethal
inflammation restricted to the colon (18, 30), and in a
germfree (sterile) state, IL-10/ mice fail to develop
colitis or mucosal immune activation (30). These findings
elegantly demonstrate how commensal intestinal bacteria can induce and
perpetuate colitis in a genetically susceptible host. This concept has
been confirmed in several other genetically engineered colitis models,
such as HLA-B27 transgenic rats (22, 33)
IL-2/ mice (24, 29), mice lacking the T-cell
receptor 
gene (8), and Tg
26 transgenic mice
(34), as well as by the absence of chronic small intestinal
ulceration in Lewis rats exposed to indomethacin (26).
/ mice (30). H. hepaticus causes chronic active hepatitis, hepatocellular adenoma,
and carcinoma, especially in A/JCr mice (13, 37). A/JCr mice
experimentally infected with H. hepaticus sporadically develop cecal inflammation (typhlitis) (15, 38), as do CD45 RBhi reconstituted SCID mice (3). Also, mice
which lack functional T lymphocytes, such as athymic nude mice
(36), SCID/NCr mice (20), and
RAG-2/ mice (35), develop colitis after
colonization with H. hepaticus. Other
Helicobacter species are also associated with colitis in SCID mice, such as Helicobacter bilis, resulting in a
proliferative typhlitis and colitis (17, 32). Of
considerable interest is that a novel urease-negative intestinal
Helicobacter species was cultivated from cotton-top tamarins
with chronic colitis (28), whereas recently another
urease-negative Helicobacter species was isolated and
associated with colitis in IL-10/ mice (14).
/ mice,
IL-10/ mice monoassociated with H. hepaticus
ATCC 51448, or IL-10/ mice colonized with SPF luminal
bacteria with or without endogenous H. hepaticus recovered
from IL-10/ mice (30). All bacterial
colonizations were performed by the physiologic route of several oral
feedings plus rectal swabs.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
/
mice were used in these studies. One subline is on a mixed
(C57BL/6 × 129/Ola) background. The other is on the 129/SvEv
inbred background. Both sublines were provided by D. Rennick, DNAX,
Palo Alto, Calif., and were rederived into germfree conditions by E. Balish. The genotype was confirmed by analysis of tail tip digests
using the PCR as described previously (30).
/ mice. In the first
study, 2-month-old germfree C57BL/6 × 129/Ola IL-10/ mice were transferred to SPF housing conditions.
We have previously determined that colonization of mice at this age
with SPF bacteria induced optimal colitis (30). Transferred
mice were maintained for 4 to 5 days in cages that contained sterile
bedding and were fed autoclaved food and water. Afterwards, bedding
from Helicobacter-free SPF mice was added to each cage in
order to colonize all mice with the same SPF flora. Two days later,
each mouse received fecal contents from either endogenous H. hepaticus-positive or H. hepaticus-negative mice. The
fecal pellets were solubilized in sterile phosphate-buffered saline
(PBS) and administered to each recipient mouse by oral feeding and
rectal swabs. This procedure was repeated three times during a 1-week
period. The donors of H. hepaticus-positive stool were
C57BL/6 × 129 Ola IL-10/ mice housed in SPF
conditions that were found to harbor endogenous H. hepaticus
as detected by PCR. This H. hepaticus strain was the
strain originally found in our IL-10/ mice
(30). Mice were sacrificed 1, 3, or 8 weeks after PCR detection of H. hepaticus DNA in fecal pellets. Tissue were
collected for histology, intestinal fragment culture, and flow
cytometry of mesenteric lymph node cells.
/
mice on the mixed (C57BL/6 × 129 Ola) background and germfree
IL-10/ inbred 129/SvEv mice 2 months of age were
colonized with a well-characterized murine H. hepaticus
strain (ATCC 51448) (15), which was kindly provided by D. Schauer, Massachusetts Institute of Technology, Cambridge. The animals
were colonized by oral gavage twice using 1 ml of an H. hepaticus suspension prepared to a McFarland turbidity standard of
1.0 in PBS. The presence of H. hepaticus in the stool was
documented at the start of as well as during the study and at necropsy
by culture as well as by PCR, as described below. The mice were
sacrificed 9 weeks after successful colonization as documented by PCR
and culture of fecal pellets. Control littermates remained
germfree. Also included in the study were IL-10/
129/SvEv mice which were colonized with H. hepaticus
at birth as the offspring of IL-10/ mice that were
previously monoassociated with H. hepaticus (ATCC 51448) and
which were kept in a separate isolator. These animals were killed at 16 weeks of age. Studies were approved by the University of North Carolina
at Chapel Hill Institutional Animal Care and Use Committee.
Cecal bacterial culture. The cecal contents of each mouse were homogenized in 1 ml of sterile PBS, after which the slurry was passed through a 0.45-µm syringe tip filter. The filtrate was then cultured for H. hepaticus at 37°C on blood agar supplemented with trimethoprim, vancomycin, and polymyxin (Remel Labs, Lenexa, Kans.) under microaerophilic conditions as described previously (13).
PCR of stool and cecal content.
Helicobacter DNA was
identified in stool or in cecal contents at necropsy by PCR
amplification using primers B38 and B39 as described by Shames et al.
(31). The presence or absence of other
Helicobacter species was determined by PCR using consensus primers C97 and C98, recognizing a broad panel of
Helicobacter species as described by Fox et al.
(12). To confirm the presence of bacterial DNA in all
specimens, a PCR using primers PC5 and P3, detecting conserved
bacterial 16S ribosomal DNA, was performed as described previously
(39). All primers sequences used are shown in Table
1.
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Histopathology. Sections of stomach, duodenum, ileum, various parts of the colon (representing cecum and proximal and distal colon), and liver were fixed in 10% neutral buffered formalin and stained with hematoxylin and eosin for histologic scoring. Scoring was conducted in a blinded fashion on a validated scale of 0 to 4, with 0 representing no inflammation and 4 representing severe inflammation characterized by widespread infiltration with inflammatory cells, crypt hyperplasia, distorsion of architecture, and the presence of ulcers and crypt abscesses as previously described and validated (22, 30). The total colonic histology score was determined by adding the scores for each section of the large intestine divided by the total number of sections examined.
Colon cultures.
Cultures of colon fragments were prepared
according to published methods (4) as described previously
(30). Between 50 and 100 mg of tissue per well of a 24-well
tissue culture plate (Costar) was cultured in duplicate or triplicate,
as tissue permitted, and cultured in 1 ml of complete RPMI medium as
described previously (30). The cultures were incubated at
37°C for 18 h. Supernatants were collected and stored at
20°C until assayed.
IL-12 assay. Immunoreactive IL-12 p40 in colon culture supernatants was measured by ELISA, using the commercially available antibodies C15.6 and biotinylated C17.8 (Pharmingen, San Diego, Calif.) for capture and detection, respectively of the IL-12 p40 subunit, as previously described (30).
Lymphoid cell preparations and flow cytometry. Mesenteric lymph nodes (MLN) were isolated and single-cell preparations were prepared by gentle teasing, as previously described (5). Cells were washed and resuspended in complete medium RPMI 1640 (Tissue Culture Facility, University of North Carolina Lineberger Cancer Center, Chapel Hill) supplemented with 5% heat-inactivated fetal calf serum (Irvine Scientific, Santa Ana, Calif.), 2 mM L-glutamine, 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol, and 50 µg of gentamicin (Sigma, St. Louis, Mo.) per ml. The proportion of CD4+ MLN cells that display an activated and/or memory phenotype was analyzed by two-color flow cytometry using rat monoclonal anti-mouse CD44, anti-mouse CD45RB, and anti-mouse CD62L (L-selectin), as described previously (5), as well as anti-CD69 (H1.2F3; Pharmingen).
Statistics. Statistical analysis was performed using a commercial software package (InStat; GraphPad Software, San Diego, Calif.). Normally distributed data are expressed as the mean ± standard deviation, and differences among groups were identified by the Student t test; significance was set at a P value of <0.05. For nonnormally distributed categorical data, comparison of median values was achieved using a Kruskal-Wallis one-way analysis of variance on ranks, and group differences were identified by Dunn's multiple comparison procedure.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Germfree mice transferred to SPF conditions colonized with or
without H. hepaticus.
Germfree IL-10/ mice
were transferred into SPF housing conditions and then colonized with
intestinal microorganisms from mice that harbor endogenous H. hepaticus or from mice found to be free of this organism as
determined by PCR. Within 1 week of administering the third dose of
intestinal microorganisms by oral feeding and rectal swabs, H. hepaticus DNA was detectable in stool samples of the transferred
mice. H. hepaticus DNA remained in fecal pellets of these
mice throughout the study period and was also documented in cecal
contents when the mice were sacrificed 8 to 9 weeks after first
confirmation of H. hepaticus DNA in fecal pellets (Fig. 1). In addition, fecal pellets and cecal
contents of SPF mice colonized with bacteria free of H. hepaticus remained H. hepaticus negative by PCR. We
excluded other known Helicobacter species by performing PCR
using primers that recognize consensus sequences of a panel of
Helicobacter species (12).
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/ mice developed a significant but moderate
mucosal inflammation, especially prominent in the cecum (median
of 2.25 in SPF versus 0 in germfree IL-10/ mice;
P < 0.001) (Fig. 2A),
which progressively increased to submucosal involvement by 3 weeks
(median score of 3.0) and severe pancolitis with transmural
inflammation of the cecum by 8 weeks of bacterial colonization (median
of 4.0) (Fig. 2A and 3). H. hepaticus-infected SPF IL-10/ mice had almost
identical histologic scores (cecal medians: 1 week, 2.25; 3 weeks, 2.5;
8 weeks, 4.0); there were no significantly different inflammatory
scores in the H. hepaticus-negative group at any time point
(Fig. 2A). There was a similar rapid increase in total colonic
inflammation after 1 week of colonization with H. hepaticus-negative SPF flora. Total colonic histology scores from
these mice also did not differ from those for SPF
IL-10/ mice colonized with H. hepaticus at
any time point (Fig. 2B). By 8 weeks after SPF exposure, all
IL-10/ mice showed similar degrees of weight loss
(2.4 ± 1.5 g in H. hepaticus-negative mice versus
2.7 ± 2.2 g in H. hepaticus-positive mice). Of
note, two of five mice in the 8-week H. hepaticus-negative group died, versus no mortality in the H. hepaticus-positive
group. Liver abnormalities were not found by histologic assessment in either group. These results indicate that endogenous H. hepaticus colonization did not potentiate or accelerate the
progressive colitis that develops in germfree IL-10/
mice after colonization with SPF bacteria.
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Monoassociation with H. hepaticus.
Germfree
IL-10/ mice were successfully monoassociated with
H. hepaticus (ATCC 51448) by oral feeding and rectal
swabbing, as confirmed by PCR analysis of the stool during the study
and of cecal contents at necropsy (Fig. 1). The persistence of H. hepaticus colonization was also confirmed by anaerobic culture in
all animals at the end of the study period. The absence of
contamination with other organisms was confirmed by Gram staining and
aerobic and anaerobic culture. C57BL/6 × 129/Ola
IL-10/ mice monoassociated with H. hepaticus
for 9 weeks developed no evidence of colitis (Fig. 2C and 3). Other
organs, including the liver and stomach, also did not show any
histologic abnormalities (data not shown). These results were confirmed
with gnotobiotic IL-10/ mice on an inbred 129SvEv
genetic background, which did not develop colitis at 9 weeks after
H. hepaticus monoassociation (cecal median score of 0) and
which showed only minimal inflammation of the cecum at 16 weeks of age
after monoassociation with H. hepaticus since birth (cecal
median score of 0.25) (Fig. 2C and 3) (this result was not
significantly different from that for germfree controls).
Colonic IL-12 secretion.
IL-12 has been shown to play a
pivotal role in the intestinal inflammation that develops in several
different murine models of colitis, including the colitis that occurs
spontaneously in IL-10/ mice (7, 9, 21). In
a previous study designed to determine if luminal bacteria are required
for development of colitis in IL-10/ mice, we
identified IL-12 in supernatants of colon fragment cultures from SPF
but not germfree IL-10/ mice (30). In the
present studies, we asked whether or not H. hepaticus alone
or H. hepaticus as one of the luminal bacterial species
influenced the production of colonic IL-12 in IL-10/
mice. The results in Fig. 4A demonstrate
detection of significantly higher levels of colonic IL-12 in SPF
IL-10/ mice than in SPF IL-10+/+ mice, in
both the presence and absence of H. hepaticus colonization. H. hepaticus colonization of SPF IL-10/ mice
did not increase colonic IL-12 levels. Colonic IL-12 in SPF
IL-10/ mice did not show significant differences at the
various time points, and at no time were differences seen in mice with
or without H. hepaticus (data not shown). Colonic fragment
culture supernatants from IL-10/ mice monoassociated
with H. hepaticus in both strains of IL-10/
mice contained only low levels of IL-12, which were not significantly different from the amounts of IL-12 detected in colonic supernatants from germfree IL-10/ mice (Fig. 4B). IL-12 levels in
colon culture supernatants from IL-10/ mice
monoassociated with H. hepaticus for 16 weeks since birth also did not significantly differ from those of germfree littermates (data not shown).
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T-cell activation of CD4+ MLN cells.
As shown in
Table 2, we detected statistically
significant albeit limited differences in the expression of some but
not all T-cell activation markers among gnotobiotic
IL-10/ mice on the 129/SvEv background. We observed
higher proportions of CD69+ CD4+ T cells and of
CD4+ T cells that express high levels of CD44 and low
levels of CD45RB in MLN of some H. hepaticus-monoassociated
IL-10/ mice than in IL-10/ germfree
mice. We did not observe significant differences when we compared the
expression of these cell surface molecules on CD4+ MLN T
cells from H. hepaticus-monoassociated
IL-10/ mice on a C57BL/6 × 129/Ola background
with those in germfree IL-10/ on the same background
(data not shown). It is interesting, however, that MLN from germfree
129/SvEv IL-10/ mice contain a significantly higher
proportion of CD4+ T cells (42.7% ± 1.2%) than do
germfree IL-10/ mice on a C57BL/6 × 129/Ola
background (30.9% ± 4.9%) (P < 0.005). In SPF
IL-10/ mice there was not a significant increase in
T-cell activation with H. hepaticus colonization. However,
there were fewer CD44hi T cells in SPF
IL-10/ mice colonized with H. hepaticus.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Spontaneous H. hepaticus infection has been detected in
IL-10/ mice with colitis, including our conventional
colony of C57BL/6 × 129/Ola IL-10/ mice
(30). To establish if H. hepaticus that is
endogenous to our colony could induce and/or potentiate colitis in
IL-10/ mice, we compared the time course of development
of colitis in germfree IL-10/ mice colonized with the
endogenous H. hepaticus 2 days after transfer to an SPF
environment with that in littermates similarly conventionalized but
remaining free of H. hepaticus. Our results show that
adolescent and young adult IL-10/ mice develop a rapid
onset of colitis within 1 week after colonization with SPF flora, with
no influence on either the aggressiveness of inflammation or the rate
of progression when endogenous H. hepaticus was persistently
present. The lack of a potentiating or accelerating effect of native
H. hepaticus on colonic inflammation is most definitely
evident at 1 week, when cecal inflammation is relatively mild, so that
a potentiating effect could be more easily recognized. Since
progressive, severe typhlitis and colitis developed in the absence of
H. hepaticus, as determined by PCR, we conclude that the
endogenous H. hepaticus in our IL-10/
mice is not required to induce colitis, confirming earlier
observations (30). Novel features of the present
study which advance our previous observations that SPF bacteria devoid
of H. hepaticus can induce aggressive colitis in
C57BL/6 × 129 Ola IL-10/ mice (30) are
a direct comparison of IL-10/ mice colonized with SPF
bacteria with and without H. hepaticus, monoassociation with
a defined H. hepaticus strain, and the failure to
detect other Helicobacter species in the SPF flora by PCR
using Helicobacter consensus primers (12). We can
therefore also conclude that colitis in our colony of an SPF
IL-10/ mouse strain is not caused by (known)
Helicobacter species but is more likely the result of an
interplay between commensal, nonpathogenic intestinal bacteria in a
susceptible host which has a defect in immune homeostasis. This
hypothesis is consistent with observations in a number of models,
including HLA-B27 transgenic rats (22), IL-2/ mice (5, 29), and mice lacking the
T-cell receptor gene (8). We have documented by PCR the
absence of Helicobacter species in our SPF HLA-B27
transgenic rats (R. K. Sellon, unpublished data) and in SPF
IL-2/ and Tg26 transgenic mice, all of which develop
active Th1-mediated colitis (22, 29, 34).
Our monoassociation results indicate that at least the strain of
H. hepaticus that we investigated is not able to induce
colitis in our IL-10/ mice in the absence of other
bacteria, since selective colonization with H. hepaticus for
at least 9 to 16 weeks did not induce aggressive experimental colitis
in gnotobiotic IL-10/ mice either on a mixed
C57BL/6 × 129/Ola background or on an inbred 129/SvEv background.
Monoassociated IL-10/ mice had no increased colonic
histologic scores or IL-12 production relative to SPF
IL-10+/+ or germfree IL-10/ controls.
Persistent H. hepaticus colonization was documented by PCR
and culture. It should be noted that in IL-10/ mice, no
single bacterial species has been shown to induce colitis, although
HLA-B27 transgenic rats monoassociated with Bacteroides vulgatus develop colonic inflammation, establishing a precedent for a single organism to induce immune-mediated colitis in genetically engineered rodents (23).
Our results concerning the role of H. hepaticus in
IL-10/ mice are different from previous reports. Fox et
al. (16) colonized germfree Swiss Webster mice with the same
strain of H. hepaticus used in this study and reported
persistent hepatitis and enterocolitis. These differences could be
explained by genetic host factors. Another reason for the divergent
results is the longer duration of H. hepaticus
colonization in Swiss Webster mice, in which minimal to mild colitis
developed only after 28 to 33 weeks of colonization. In the present
study, monoassociated IL-10/ mice were examined after 9 weeks of colonization with H. hepaticus. Although the
kinetics of colitis development can be different in monoassociated
mice, we wanted to explore the effect of H. hepaticus
within the time frame that colitis develops in IL-10/
mice moved from germfree to SPF conditions. However, only minimal inflammation of the cecum was observed in 16-week-old
IL-10/ mice that were monoassociated with H. hepaticus at birth.
Even if H. hepaticus is not required to induce colitis
in IL-10/ mice, this organism could potentiate
inflammation induced by nonpathogenic commensal bacteria. We therefore
compared histologic and immunologic parameters between
IL-10/ mice at several times after transfer to a SPF
environment in the presence or absence of endogenous H. hepaticus. Longer exposure of IL-10/ mice to SPF
flora worsened the severity of colitis, especially in the cecum.
However, our results consistently reveal that the persistent presence
of H. hepaticus did not potentiate typhlitis and
colitis at the onset or advanced stage of disease in these mice as
determined by blinded histologic scores for different regions of the
cecum and colon and levels of colonic IL-12.
Our results are in contrast to a recent study by Kullberg et al.
(19), who reported that H. hepaticus
triggered colitis in SPF IL-10/ mice through IL-12- and
gamma interferon-dependent mechanisms. Several differences in the
experimental designs of the two studies could explain these discrepant
results. One potential explanation for the different outcomes is that
we colonized our IL-10/ mice with an endogenous
H. hepaticus strain, whereas Kullberg et al.
(19) used the original Frederick isolate (ATCC 51488) derived from A/JCr mice (13, 37). However, we clearly
demonstrated that monoassociation of our IL-10/ mice
with the same H. hepaticus strain (ATCC 51488) used by
Fox et al. (16) and Kullberg et al. (19) did not
result in significant colitis. An additional difference in the two
studies is the method of H. hepaticus colonization. In
the study of Kullberg et al., IL-10/ mice were
inoculated with H. hepaticus intraperitoneally and intragastrically (19), whereas in our study, oral feeding
and enemas containing SPF enteric flora with H. hepaticus were used. We chose the oral-fecal colonization method
because this mode of transmission more accurately reflects physiologic
transfer of this organism than does a parenteral route (16,
19). An important methodologic difference which could explain the
discrepancy between our results and those of Kullberg et al.
(19) is that we colonized germfree IL-10/
mice with SPF bacteria with or without H. hepaticus at
2 months of age, whereas the IL-10/ mice in the
previous study were born in an SPF environment and colonized with
H. hepaticus at 6 to 9 weeks of age. We have
demonstrated that older germfree mice colonized with SPF bacteria
develop more aggressive cecal inflammation and colitis than mice born
into an SPF environment (30). Another reason for the
divergent results with the study of Kullberg et al. could be
differences in the composition of the intestinal flora, other than
H. hepaticus, in the animal facilities of the two
institutions. However, to date specific inducing organisms in
IL-10/ mice still need to be clarified.
It is also possible that failure to induce and potentiate colitis in
our IL-10/ mice, in contrast to the results of Kullberg
et al. (19), is due to genetic differences between the
strains of IL-10/ mice used in the two studies. We
investigated IL-10/ mice on a mixed C57BL/6 × 129 Ola background as well as inbred 129/SvEv IL-10/ mice,
whereas Kullberg et al. used inbred C57BL/10SgSnAi
IL-10/ mice (19). Berg et al. (1)
have demonstrated important differences in the susceptibilities of
inbred IL-10/ mice on different genetic backgrounds to
colitis and colon cancer. In the studies 129/SvEv
IL-10/ mice developed aggressive inflammation, whereas
C57/BL6 IL-10/ mice had mild, delayed-onset disease.
IL-10/ mice on a mixed background developed
intermediate disease (1). Differences in genetic background
as well as other host factors mentioned above could explain why the SPF
C57BL/10 IL-10/ mice without H. hepaticus used by Kullberg et al. had very little colitis. In
support of our observations, a preliminary report by Czinn et al.
showed no potentiation of colitis in SPF IL-10/ mice on
a C57BL/10 background by colonization with H. hepaticus strain ATCC 51488 (6).
Except for the development of colitis in older Swiss Webster
(16) and A/JCr mice (15, 37), most of the initial
studies reporting H. hepaticus-induced colitis and
hepatitis focused on immunodeficient mice, such as nude (11,
36) and SCID (20) mice, which lack functional T cells,
demonstrating that H. hepaticus can induce disease in
the absence of T cells. However, the worsening of colitis in SCID mice
reconstituted with CD45 RBhi CD4+ T cells
(3) and the findings of Kullberg et al. in C57BL/10 IL-10/ mice (19) indicate not only that
immunocompetent mice can develop colitis in the presence of this
organism but also that disease can occur in animals that have
functional T lymphocytes. In immunocompetent mice, H. hepaticus-induced colitis or chronic active hepatitis is
accompanied by a Th1 cytokine response (19, 38). Not only did H. hepaticus infection not induce or potentiate
colitis in our IL-10/ mice, but it also did not
significantly affect the levels of mucosal Th1-associated cytokines.
Colonic culture IL-12 concentrations were no different in SPF mice with
or without H. hepaticus infection and were not
increased by monoassociation of germfree IL-10/ mice
with H. hepaticus. Increased colonic culture IL-12
concentrations in SPF IL-10/ mice indicate a vigorous
Th1 cytokine response following SPF colonization with normal resident
bacteria in the absence of H. hepaticus. However, this
Th1 response in SPF IL-10/ mice was not influenced by
the presence of H. hepaticus at any time during the
development of colitis.
In conclusion, oral-fecal colonization with H. hepaticus (ATCC 51448) did not induce significant colitis in
monoassociated IL-10/ mice, nor was colitis potentiated
in SPF IL-10/ mice cocolonized with endogenous
H. hepaticus, when mice were monitored to the
aggressive stage of disease. This study indicates that H. hepaticus is not required for induction of colitis in IL-10/ mice and suggests that the ability of
H. hepaticus to induce colitis depends on the
interaction between this organism and genetic or immunologic host factors.
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ACKNOWLEDGMENTS |
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We thank Lisa Holt for her help and technical guidance during the PCR analysis. We are also grateful for the technical support of Julie Kwan with MLN isolation and flow cytometry analysis.
This work was supported by NIH grants KO8 553773, DK40249, DK533047, DK
34987, and AI 01122 and by the Crohn's and Colitis Foundation of
America. Support for derivation of the germfree IL-10/
mice on a 129/SvEv background was provided by NIH grant DK 50980 (Peter
Ernst, principal investigator).
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Digestive Diseases, Glaxo Bldg., Room 146, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7080. Phone: (919) 966-7886. Fax: (919) 966-6842. E-mail: ldiele{at}med.unc.edu.
Editor: J. D. Clements
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Infection and Immunity, September 2000, p. 5107-5113, Vol. 68, No. 9
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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