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Animal Models |
From the Department of Comparative Medicine,*
theUniversity of Washington, Seattle; the Department ofImmunobiology,
Immunex Corporation, Seattle;and ZymoGenetics Inc.,
Seattle, Washington
Abstract
mdr1a-deficient mice lack P-glycoprotein and
spontaneously develop colitis with age. Helicobacter
spp. are gram-negative organisms that have been associated with colitis
in certain mouse strains, but Helicobacter spp.
have been excluded as contributing to the spontaneous colitis that
develops in mdr1a-/- mice. We wished to determine
whether infection with either H. bilis or H.
hepaticus would accelerate the development of inflammatory
bowel disease (IBD) in mdr1a-/- mice. We found that
H. bilis infection induced diarrhea, weight
loss, and IBD in mdr1a-/- mice within 6 to 17
weeks post-inoculation and before the expected onset of spontaneous
IBD. Histopathology of H. bilis-induced IBD included
crypt hyperplasia, inflammatory cell infiltrates, crypt
abscesses, and obliteration of normal gut architecture. Reverse
transcription-polymerase chain reaction and Taqman analysis from
colonic tissue showed increased transcripts for interferon-
and
interleukin-10 from H. bilis-infected colitic
mdr1a-/- mice. Additionally, mesenteric lymph
nodes had increased cellularity with expansion of CD4+ and
CD8+ T cells and B cells and increased proliferation to
soluble H. bilis antigens with elaboration of
interferon-, tumor necrosis factor-
and interleukin-10.
In contrast, H. hepaticus infection of
mdr1a-/- mice did not accelerate disease but rather
delayed the onset of spontaneous colitis which was milder in severity.
mdr1a-/- mice infected with
Helicobacter spp. may provide a useful tool to explore
the pathogenesis of microbial-induced IBD in a model with a presumed
epithelial cell "barrier" defect.
Helicobacter are microaerophilic gram-negative spiral
bacterial organisms14
that have been associated with
hepatitis,15
hepatocellular carcinoma,16,17
and IBD in rodents.18
Several species of
Helicobacter infecting rodents have been
described,19,20
including H.
hepaticus21
and H.
bilis,19,22,23
which have been shown to colonize the
liver and intestine and elicit IBD in immunodeficient mice and
rats.24
Experimental infection with either H.
bilis or H. hepaticus induces IBD in mice lacking T and
B lymphocytes (SCID),22,23,25
in mice with cytokine
dysregulation (IL-10)26
and in mice with abnormal T cell
receptor repertoires (TCR-/-).27
To better understand
how bacteria "trigger" IBD in a genetically susceptible host, we
infected mdr1a-/- mice with Helicobacter spp.
We found that H. bilis accelerated development of IBD in
mdr1a-/- mice while H. hepaticus delayed
development of spontaneous disease.
Materials and Methods
Animals
mdr1a-/- and FVB+/+ (FVB) mice were obtained from Taconic Farms (Albany, NY). To determine whether the absence of another membrane transporter was important in a bacterial-induced IBD, we also infected multidrug resistance associated protein null (mrp-/-) mice (Taconic Farms) with H. bilis. For all studies reported, we used 3- to 6-week-old, female FVB (n = 85), mdr1a-/- (n = 129), and mrp-/- (n = 20) mice. Foundation stock for all of these strains are Cesarean-derived and populated with an altered Schaedler’s cocktail; expansion stock are maintained under specific pathogen-free (SPF) conditions before being shipped. All mice were certified free of Helicobacter spp. by the vendor and re-tested on site before each experiment. Cohorts of mice used for experiments were housed at Immunex Corporation (IMNX) and at the University of Washington (UW). Animals were housed in an SPF environment in polycarbonate microisolator cages containing Bed-O-Cob (Andersons, Maumee, OH) and a nestlet. Mice were fed irradiated Picolab Rodent Diet 20 5053 (PMI Nutrition International, Brentwood, MO) and autoclaved, acidified water. All supplies entering animal rooms were autoclaved and rooms were maintained at 70–74°F, 45 to 55% humidity, with 28 air changes/hour 12/12-hour light/dark cycle. To prevent cross contamination of uninfected mice and infected mice, animals were housed in separate cubicles within the same room and cages changed in "uninfected" or "infected" laminar flow hoods (UW) or, uninfected mice and infected animals were housed in separate rooms (IMNX). Sentinel mice 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, quarterly sterile colon samples were screened for Citrobacter rodentium, non-lactose fermenting Escherichia coli, Salmonella spp., Klebsiella spp., and Clostridium spp. (Phoenix Laboratories, Seattle, WA). All animal procedures were approved by the UW Animal Care and Use Committee and the IMNX Animal Care and Use Committee.
Experimental Design
Before inoculation, mice were determined to be negative for Helicobacter spp. by fecal polymerase chain reaction (PCR). mdr1a-/- and FVB mice were given H. bilis or H. hepaticus or broth while mrp-/- mice were given H. bilis or broth. Helicobacter species-specific fecal PCR was done on pooled cage samples taken every 4 to 8 weeks until the end of the study. Mice were given Brucella broth alone or H. hepaticus or H. bilis by oral gavage 3 times over a 1-week period. Mice were infected with 2 x 107 CFU of H. bilis or H. hepaticus in 0.2 ml volume on each occasion. Mice were weighed weekly and monitored for weight loss, dehydration, and diarrhea. Mice were euthanized by CO2 or cervical dislocation when they developed diarrhea or 20% body weight loss and samples were taken for histopathology, RNA isolation, or lymphocyte phenotype and proliferation analysis. Fecal samples from both uninfected and infected mice were tested by PCR for cross-contamination with the other Helicobacter spp. in infected mice or for absence of Helicobacter infection in uninfected animals.
Bacterial Cultures
H. bilis was a natural isolate and kindly provided by L. Riley (University of Missouri, Columbia, MO) and H. hepaticus was obtained from the American Type Culture Collection (ATCC 51448). Organisms were streaked onto Brucella blood agar plates and grown under microaerobic conditions (90% N2, 5% H2, and 5% CO2) in vented jars (Oxoid, Hampshire, England), and kept at 37°C. Bacteria were harvested and inoculated into flasks containing 150 ml of Brucella broth supplemented with 5% fetal bovine serum (Sigma Chemical Co., St. Louis, MO). The flasks were placed on a continuous shaker and incubated for 24 to 48 hours at 37°C in microaerobic conditions. The organisms were centrifuged at 10,000 rpm at 4°C for 20 minutes. The resultant pellet was examined by gram stain and phase microscopy for purity, morphology, and motility. Organisms were confirmed to be catalase, urease and oxidase positive. The pellet was resuspended in Brucella broth and optical density was adjusted to 1.0 (OD600) for an estimated 108 CFU/ml.
Generic Helicobacter and Specific H. hepaticus and H. bilis PCR
Fecal samples were analyzed for Helicobacter spp.,
H. hepaticus, and H. bilis as described
previously28-30
with slight modifications. For generic
Helicobacter PCR, 5 µl of unquantitated fecal DNA was used
as template, and for H. hepaticus and H. bilis
PCR, 10 µl of unquantitated fecal DNA was used as template for all
samples. Primers, their sequences, and PCR conditions used are
summarized in Table 1
.
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Liver, MLN, cecum, colon, and rectum were fixed in 10% buffered
formalin. The colon was prepared in a "Swiss roll"
technique31
to evaluate the entirety of the proximal,
middle, and distal colon on the same section. The 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.W.) to the infection status of the animal. The
cecum, proximal colon, middle and 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 intestine affected in any manner (Extent
1) + extent of intestine affected at level 3 or 4 (Extent
2)].26
The total score for each mouse was derived by
summing the scores from the individual segments and subsequently, the
mean was derived for each treatment group.
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The following antibodies were used for flow cytometric
analyses: rat IgG2a 
(R35–95), anti-CD45R/B220 (RA3–6B2),
anti-Mac-1/CD11b (M1/70), anti-NK1.1 (PK136), anti-CD4 (RM4–5),
anti-CD8 (53–6.7), anti-TCRß (H57–597), anti-TCR
(GL3),
anti-IgD (11–26c.2a), anti-IgM (G155–228). All mAbs were purchased
from Pharmingen, San Diego, CA. The following antibodies were used
for antigen-specific serum antibody analyses: anti-IgG1, anti-IgG2a,
anti-IgG2b, anti-IgG3, anti-IgA, anti-IgG unlabeled and human adsorbed
HRP-labeled antibodies were all purchased from Southern Biotechnology
Associates, Inc., Birmingham, AL. The following antibodies
were used for cytokine analyses: anti-IFN- (R4–6A2 and XMG1.2),
anti-IL-4 (11B11 and BVD6–24G2), anti-IL-10 (JES5–2A5 and SXC-1)
unlabeled and biotin-labeled antibodies were all purchased from
Pharmingen, San Diego, CA. TNF- ELISA kits were purchased from R & D
Systems, Minneapolis, MN.
Preparation of Helicobacter Antigen: Helicobacter Antigens Were Prepared from Cultures of H. bilis or H. hepaticus
The organisms were harvested as for inoculation, washed in PBS, and sonicated at 4°C for 2 minutes, using 50% duty cycle with output setting 7 (Branson Ultrasonics, Danbury, CT). Lysed suspensions were centrifuged at 10,000 x g at 4°C for 20 minutes and the supernatant was 0.22 µm filtered. Protein content was determined by BCA protein assay (Pierce, Rockford, IL) and endotoxin levels were determined by LAL assays. Supernatants were frozen at -20°C until use.
Cell Preparation
MLN or spleens were collected in cold HBSS, 5% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mmol/L glutamine. Single cell suspensions were prepared by teasing tissues apart in collection medium, followed by filtering though nylon mesh. Cells were washed and resuspended in tissue culture medium (RPMI, 5% FBS, PSG as above, 1 mmol/L sodium pyruvate, 0.1 mmol/L non-essential amino acids, 50 µmol/L ß-mercaptoethanol, and 20 mmol/L HEPES) at 107 cells/ml. Prior to final wash, RBCs from spleen were lysed for 5 minutes at room temperature in Tris-ammonium chloride, pH 7.6. Experiments were performed with lymphocyte suspensions pooled from 2 to 4 mice per group.
FACs Staining
All FACS staining was performed in covered ice buckets. Cells (0.25–1 x 105 cells/stain) were washed in PBS-FBS (0.1%) then nonspecific binding sites were blocked by incubating the cells for 10 minutes in PBS-FBS with 2% mouse serum, 10% goat serum, and 10 µg/ml anti-Fc receptor II (2.4G2), followed by another wash in PBS-FBS. Cells were incubated with fluorochrome-labeled antibodies for 30 minutes, washed twice, resuspended in PBS-FBS with 0.5% paraformaldehyde, and stored at 4°C until analysis on a FACScan flow cytometer (Becton Dickenson, San Jose, CA).
Lymphocyte Proliferation
Lymphocyte suspensions at a density of 105 cells/well were cultured in 96-well flat-bottomed plates in a total volume of 0.2 ml/well at 37°C and 5% CO2. All cultures were performed in triplicate. Cells were cultured in medium alone with various concentrations of Helicobacter antigens (Ags), LPS (E. coli 0127:B8, Difco Laboratories, Detroit, MI) at the highest concentration determined for the Ag preparation and 1 µg/ml solid phase anti-CD3 (mAb 500A2). Proliferative responses were measured by [3H]thymidine incorporation (1 µCi/well [3H]thymidine, NEN, Boston, MA) during the final 6 hours of a 72-hour incubation. The amount of radioactivity incorporated into DNA was measured using a Matrix-96 cell harvester (Inotech, Lansing, MI) and a direct ß counter (Packard, Meridian, CT). The data are reported as the mean counts ± SD per minute of triplicate wells.
Cytokine Analysis by ELISA
Lymphocytes were cultured exactly as for proliferation assays. All
cultures were performed in triplicate. After 48 or 72 hours, plates
were centrifuged at 1000 rpm for 10 minutes at 4°C and culture
supernatants were transferred to round-bottomed 96-well plates. Samples
were stored at -20°C until assays were performed. Levels of soluble
mIFN-, mTNF-, mIL-10, and mIL-4 were determined by ELISA.
Purified capture antibodies were coated overnight at 4°C onto 96-well
ELISA plates (Maxisorp, Nunc, Naperville, IL) at a concentration of
1–2 µg/ml in 0.1 mol/L NaHCO3 buffer, pH 8.5.
Plates were washed four times with PBS/0.1% Tween-20 (PBST) and
nonspecific binding sites were blocked with 10% FBS in PBS for 2 hours
at room temperature. Plates were washed four times with PBST, standard
dilutions and samples were loaded either neat (mIL-10, mIL-4, mTNF-)
or diluted 1:3 (mIFN-) with blocking buffer and incubated overnight
at 4°C. Plates were washed four times with PBST, and biotinylated
antibodies specific for mouse cytokines, diluted to 1 µg/ml with 10%
FBS in PBS were added. The plates were allowed to incubate for 2 hours
at room temperature, then were washed six times.
Streptavidin-horseradish peroxidase (SA-HRP) (Zymed Laboratories Inc.,
South San Francisco, CA) was added and the plates incubated for 1 hour
at room temperature followed by six washes with PBST. Enzyme activity
was detected with 3,3',5,5'-tetramethyl-benzidine (TMB) substrate
(Kirkegaard & Perry, Inc., Gaithersburg, MD) and development stopped
with 1 N phosphoric acid. Optical densities were measured at 450 nm
with a microplate reader (Molecular Devices, Sunnyvale, CA) and protein
concentrations were determined by using the Deltasoft Program
(BioMetallics Incorporated, Princeton, NJ).
Analysis of Ag-Specific Serum Antibodies
Blood samples were obtained via cardiac puncture at the same time tissues were collected for analyses. Resulting sera samples were stored at -20°C until assays were performed. Determination of Helicobacter-specific antibodies in sera was performed by ELISA. Helicobacter antigens were coated overnight at 4°C onto 96-well Maxisorp ELISA plates at a concentration of 8 µg/ml in carbonate buffer, pH 9.5. Plates were washed six times with PBST and nonspecific binding sites were blocked with 10% FBS in PBS for 1 hour at room temperature. Plates were washed six times with PBST, samples were diluted with blocking buffer and incubated for one hour at room temperature, followed by six more washes. Peroxidase-conjugated goat antibodies specific for mouse immunoglobulins were diluted with 50% goat serum in PBS, allowed to incubated for 1 hour at room temperature, and the plates then washed six times. Enzyme activity detection was performed as described above.
RNA and cDNA Preparation from Colon Tissue Samples
Fecal contents were removed and the colon was homogenized in guanidinium isothiocyanate buffer (4.5 mol/L guanidinium isothiocyanate, 50 mmol/L sodium citrate, and 0.5% (w/v) sodium sarcosyl) containing 2% 2-ME (Life Technologies). Two to four colons per group were pooled and total RNA was isolated over a cesium chloride cushion and subjected to phenol chloroform extraction. cDNA was prepared from 5 µg of isolated RNA which was reverse transcribed with Superscript II reverse transcriptase (GIBCO BRL, Gaithersburg, MD) in the presence of 0.3 µg of random primers, 5X RT buffer, 0.1 mol/L DTT, 10 mmol/L dNTPs, and water.
Cytokine Analysis by Polymerase Chain Reaction
IL-4, IL-10, and IFN- mRNA was analyzed using the Mouse TH1/TH2
Switch cytokine kit (Biosource International, Camarillo, CA). PCR
reaction tubes contained 5 µl of cDNA and reagents provided by the
kit. The following PCR profile was used for amplification: 96°C for 1
minute, followed by two cycles of 96°C for 1 minute and 59°C for 4
minutes, then 30 cycles of 94°C for 1 minute and 59°C for 2.5
minutes, and a final cycle of 70° for 10 minutes. PCR product and 1
µl of loading buffer were loaded onto 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.
Reverse Transcription and Real-Time Quantitative PCR
cDNA was synthesized from 5 µg of DNA-free treated (AMBION,
Austin, TX) total RNA using mixture of 125 ng of oligodT and 37.5 ng of
random hexamers and Superscript II reverse transcriptase (Lifetech,
Gaithersburg, MD) per manufacturer’s instructions. Real-time
quantitative PCR analyses were performed starting with 250 ng and 125
ng of reverse transcribed total RNA with 10 pmol of both sense and
antisense primers in a final volume of 25 µl using the SYBR Green PCR
Core Reagents on the GeneAmp5700 Sequence Detection System. PCR
reactions were cycled 95°C for 10 minutes followed by 40 cycles of 15
seconds at 95°C, 1 minute at 60°C. PCR reactions were also
subjected to a heat dissociation protocol. Real-time PCR primers were
design with Primer Express (PE Biosystems) software (IFN-: 5'-CTC
ATC CGA GTG GTC CA-3' and 5'-AGC AGC GAC TCC TTT TC-3'; IL-10: 5'-TTC
CAA ATC CTC GGC ATA ATG-3' and 5'-CCA GCG TCG TGA TTA GC-3' and HPRT:
5'-TTC CAA ATC CTC GGC ATA ATG -3' and 5'-GTC CCA GCG TCG TGA TTA
GC-3') A standard curve for each gene (HPRT, IFN-, and IL-10) was
constructed whose concentration was based on the equivalent of the
reverse transcribed total RNA input. Following normalization of the
cDNA concentrations to HPRT, relative concentration of IFN- and
IL-10 was calculated for each experimental sample. The data are
expressed by showing relative concentration of experimental sample to
the standard sample.
Statistical Analyses
Differences between mean total pathology scores and body weight in
uninfected and H. bilis-infected or H.
hepaticus-infected mdr1a-/- mice were evaluated by
one way analysis of variance and Student’s t-test;
significance was set at P 
0.05 (SigmaStat; Jandel
Corp., San Rafael, CA).
Results
Spontaneous IBD in mdr1a-/- Mice
mdr1a-/- mice with spontaneous IBD were assessed
using our pathology scoring system to determine the degree and extent
of disease in mice free of Helicobacter infection. At 4
months of age, mdr1a-/- mice
had severe IBD in all portions of the large bowel (Table 3)
(Figure 1)
. In the colon and rectum, the disease
was characterized by diffuse epithelial cell hyperplasia, goblet cell
loss, and a chronic inflammatory cell infiltrate in the mucosa,
submucosa, and occasionally was transmural. The infiltrate was
comprised predominantly of macrophages and lymphocytes, with fewer
neutrophils (Figure 1A)
. Intestinal crypts were often dilated and
filled with cellular debris (crypt abscesses) while occasional crypt
branching and herniation were also seen (Figure 1A)
. Interestingly,
squamous metaplasia of the rectal epithelium (Figure 1E)
was found in
all mice. Although significant chronic IBD occurred in the cecum of
mdr1a-/- mice (Figure 1C)
, the cecum was involved to a
lesser degree and lesions were segmental rather than diffuse as
compared to the colon (Figure 1A)
. While mice showed mucosal ulceration
throughout the colon (Figure 1, G and H)
, cecal ulcers were infrequent.
Wild-type FVB mice of similar ages showed no evidence of IBD (Figure 1, B, D, and F)
.
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Mdr1a-/ mice infected with H. bilis at 3 to
4 weeks of age had evidence of diarrhea beginning 3 weeks
post-infection (PI) and showed altered weight gains as compared to
uninfected mdr1a-/- mice (Figure 2A)
. In contrast, uninfected and
Helicobacter-infected FVB mice showed no evidence of
diarrhea or differences in body weight curves (Figure 2, B and D)
and
subsequent pathological analysis revealed no evidence of IBD (Tables 4 and 5)
(Figure 3B)
. In H. bilis-infected
mdr1a-/- mice, lesions were found from cecum to rectum and
these were more severe than the minimal disease seen in uninfected
mdr1a-/- mice within the time frame of this study (8
weeks). This was reflected in higher mean total histopathology scores
in H. bilis-infected mdr1a-/- mice (59) than in
uninfected mdr1a-/- mice (4) (Table 4)
(Figure 3)
.
Specifically, H. bilis-induced IBD in mdr1a-/-
mice was characterized by severe chronic-active inflammation with
pronounced accumulations of lymphocytes, macrophage, and neutrophils
within the lamina propria (Figure 3, A and C)
. However, only
occasionally did the inflammatory infiltrates extend into the submucosa
and tunica muscularis. Changes to the colonic mucosa ranged from severe
epithelial hyperplasia to loss of crypts and attenuation of the
overlying epithelium. Ulcers were rarely seen. Crypt abscesses were
present (Figure 3A)
; however, no crypt herniation or branching were
seen. Lesions were similar in the cecum (Figure 3C)
but the presence of
crypt abscesses was significantly less than that seen in the colon. An
additional infection study was done using larger numbers of
mdr1a-/- mice (Table 6)
. By 16 weeks PI, 9 of 10 H.
bilis-infected mdr1a-/- mice had developed
significant IBD, while only 4 of 15 uninfected mdr1a-/-
mice had developed spontaneous IBD; disease pathology was similar to
that seen in the previous study (data not shown).
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Studies with H. hepaticus-infected mdr1a-/- mice were
longer term than H. bilis infection studies since no
diarrhea or body weight changes were noted until 15 weeks PI in either
the H. hepaticus or the uninfected groups. Notably, H.
hepaticus-infected mdr1a-/- mice showed a
significantly elevated body weight gain at 15 weeks PI compared to
uninfected mdr1a-/- mice, which exhibited body weight loss
as spontaneous IBD developed (Figure 2C)
. All mice were 5 months of age
at the time of necropsy. Unexpectedly, H. hepaticus-infected
mice had less severe disease in all portions of the large bowel as
indicated by lower mean total pathology scores (22) compared with
uninfected mdr1a-/- mice with spontaneous disease (63)
(Table 5)
. When disease developed,
IBD in the colon of H. hepaticus-infected
mdr1a-/- mice was characterized by moderate epithelial
hyperplasia and inflammatory cell infiltrate in the lamina propria
which occasionally extended into the submucosa (Figure 3E)
. These
lesions were less extensive than that seen in uninfected
mdr1a-/- mice with spontaneous IBD (Figure 3F)
and ulcers
were infrequently observed in H. hepaticus-infected
mdr1a-/- mice. These results suggest that H.
hepaticus infection delayed or attenuated the development of
spontaneous IBD in mdr1a-/- mice. That H.
hepaticus infection delays onset and severity of colitis in
mdr1a-/- mice was confirmed in two additional infection
studies which compared the development of spontaneous IBD in uninfected
mdr1a-/- mice with disease development in H.
bilis or H. hepaticus infection in
mdr1a-/- mice. In one of these studies, by 27 weeks PI,
only 2 of 16 H. hepaticus-infected mdr1a-/-
mice had developed colitis, while 9 of 15 uninfected
mdr1a-/- mice had developed spontaneous colitis and all 10
H. bilis-infected mdr1a-/- mice had developed
colitis (Table 6)
. Histopathological
analysis confirmed the presence or absence of disease (data not shown).
Ulcer Formation Characterizes Spontaneous IBD but Not Helicobacter-Induced IBD in mdr1a-/- Mice
The spontaneous IBD that develops in mdr1a-/ mice was
characterized by ulcers in cecum, colon, and rectum, while
Helicobacter-induced IBD rarely included ulcer formation.
Combining several groups of uninfected spontaneously colitic
mdr1a-/- mice with total pathology scores of 35 to 72, 24
of 26 animals (92%) had ulcers in multiple sites; their mean age (±
SD) was 14 (± 9.8) weeks at necropsy. However, in a combined group of
H. bilis-infected colitic mdr1a-/- mice with
total pathology scores of 44 to 68, only 1 of 20 mice (5%) had ulcer
formation limited to the proximal colon; their mean age was 13.6 (±
3.6) weeks at necropsy. A large ulcer in the middle colon and rectum of
a mdr1a-/- mouse with spontaneous colitis is shown in
Figure 1, G and H
.
mrp-/- Mice Do Not Develop IBD When Infected with H. bilis
mrp-/- mice lack the multidrug resistance-associated protein, Mrp1, gene which encodes for another ATP-transport protein.32 Mrp1 is expressed ubiquitously including the intestinal tract.33 In contrast to mdr1a-/- mice, H. bilis did not trigger IBD in mrp-/- mice. More than 5 months PI, 10 of 10 H. bilis-infected mrp-/- mice showed no diarrhea or clinical signs suggestive of IBD. This was confirmed histologically in all 10 mice necropsied (data not shown).
Lymphocyte Changes in mdr1a-/- mice Infected with H. bilis or H. hepaticus
MLN from mice with H. bilis-induced IBD had increased
cellularity (3.8x uninfected) which was due to expansion of
CD4+ and CD8+ T cells, B
cells, macrophages, and NK cells (Figure 4, A and C)
. Interestingly, FVB mice
infected with H. bilis but not exhibiting IBD also showed
increased MLN cellularity (2.4x) with similar changes in lymphocyte
subsets. Mice shown in Figure 4, A and C
are approximately 3 months old
and uninfected mdr1a-/- mice are not yet showing
spontaneous IBD. MLN from mdr1a-/- mice infected with
H. hepaticus similarly showed increased cellularity compared
with uninfected mdr1a-/- mice (1.9x) which was also due
to an expansion of CD4+ and
CD8+ T cells, B cells, macrophages, and NK cells
(Figure 4, B and D)
. However, lymphocyte expansion did not correlate
with the presence or severity of IBD since uninfected
mdr1a-/- mice exhibited severe spontaneous IBD while
H. hepaticus-infected mdr1a-/- mice showed less
severe IBD. Mice shown in Figure 4, B and D
are approximately 5 months
old. Similar to H. bilis, FVB mice infected with H.
hepaticus and not exhibiting IBD had increased MLN cellularity
(2x).
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Mdr1a-/ mice with H. bilis-induced colitis
had increased lymphocyte proliferative responses to H. bilis
antigens compared to the lower proliferative responses seen in cells
from non-colitic H. bilis-infected FVB mice (Figure 5, A and C)
. H.
hepaticus-infected mdr1a-/- mice with mild colitis
exhibited lymphocyte proliferative responses that were similar to
non-colitic H. hepaticus-infected FVB mice (Figure 5, B and D)
. Supernatants derived from lymphocyte cultures from H.
bilis-infected mdr1a-/- mice showed elevated levels
(ng/ml) of IFN- and detectable levels (pg/ml) of TNF- and IL-10
with a dose response to H. bilis antigens (Figure 6, A–C)
.
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Mdr1a-/ mice showed increased serum immunoglobulin
levels in response to Helicobacter infection but there were
some differences in responses between H. bilis-infected
colitic mice and H. hepaticus-infected mice with mild to no
colitis. Figure 7
shows serum
immunoglobulin levels from H. bilis-infected mice taken at
58 days post-infection (left panels) compared with H.
hepaticus infected mice taken at 17 weeks post-infection (right
panels). H. bilis infected mdr1a-/- mice showed
elevated serum IgA, IgG1 and particularly IgG2a (left panels). Despite
the absence or development of less severe colitis, H.
hepaticus-infected mdr1a-/- mice also mounted an
H. hepaticus-specific immunoglobulin response which was
primarily of the IgG2a isotype (lower right panel). It is noteworthy
that H. bilis infected FVB mice had minimal to no detectable
H. bilis specific immunoglobulin responses while H.
hepaticus-infected FVB mice had substantial H.
hepaticus-specific IgG1 and IgG2a elevations (right panels). The
lack of serum immunoglobulin responses in H. bilis infected
FVB mice may be related to the duration post-infection which was 58
days with H. bilis infections and 17 weeks with H.
hepaticus infections. H. hepaticus-specific IgG
responses in A/JCr mice are not detectable until 16 weeks
post-infection.16
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Since lymphocyte cultures from lymph nodes draining the bowel of
infected mdr1a-/- mice indicated that cytokines previously
associated with IBD were elaborated, we analyzed colonic tissue for
alterations in pro- and anti-inflammatory cytokine mRNA levels.
H. bilis-infected mdr1a-/- mice with colitis
showed increased transcripts for IFN-, a Th1 cytokine, when compared
with uninfected and infected FVB and uninfected mdr1a-/-
mice not exhibiting colitis. Despite the presence of IBD, H.
bilis-infected mdr1a-/- mice also showed increased
transcripts for the down-regulatory cytokine IL-10 by RTPCR (Figure 8A)
. This was confirmed quantitatively by
Taqman analysis (Figure 8B)
. Since H. bilis and H.
hepaticus induced different pathology in mdr1a-/-
mice, we compared colonic cytokines in H. bilis-infected
colitic mice, H. hepaticus-infected mdr1a-/-
mice with mild to moderate colitis, and mdr1a-/- mice with
spontaneous colitis by RTPCR (Figure 8C)
. Both spontaneous and H.
bilis-infected colitic mdr1a-/- mice showed increases
in IFN- mRNA; in contrast, H. hepaticus-infected
mdr1a-/- mice had low levels of IFN- mRNA. Taqman
analysis further verified and quantitated low levels of IFN- mRNA
and lower levels of IL-10 mRNA in H. hepaticus-infected mice
with mild to moderate colitis compared with uninfected
mdr1a-/- mice with severe spontaneous colitis (Figure 8D)
.
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The mdr1a-/- mouse model of IBD differs from most other mouse models. The majority of other spontaneous colitis models occur in animals bearing a cytokine imbalance5,6,34 or T cell defect35,36 and hence are very susceptible to immunological dysregulation. mdr1a codes for P-glycoprotein, an ATP-membrane efflux transporter that is expressed in the intestinal epithelium and in lymphocytes and is absent in mdr1a-/- mice.12 We have previously shown that it is a defect in epithelial mdr1a-/- expression that leads to IBD susceptibility.13 It is not known why mdr1a-/- mice develop spontaneous colitis as they age. Sequential fecal microbial analyses over time would need to be done to see if disease development could be related to the gradual acquisition of commensal luminal organisms. Rather than examine the development of spontaneous IBD that occurs in response to the acquisition of enteric micoflora, we wished to determine whether Helicobacter infection, known to induce IBD in other mouse models, would accelerate and synchronize development of IBD in mdr1a-/- mice.
The mechanism by which H. bilis accelerates development of
IBD in mdr1a-/- mice is unclear. P-glycoprotein is a
membrane transporter important in drug efflux and its endogenous
substrates are unknown. P-glycoprotein is expressed on the apical
surface of intestinal epithelial cells and could play a role in
protection against bacterial toxins. One possibility is that
Helicobacter-induced IBD may be mediated via a family of
bacterial toxins termed cytolethal distending toxins (CDT) that have
been considered virulence factors in certain Helicobacter
infections.37
Genes encoding for CDT have been identified
in H. bilis38
and sonicates of H.
hepaticus have been shown to cause progressive cell distension and
cell cycle arrest in vitro39
presumably related
to their CDT activity. Further compromise of intestinal epithelial
barrier function by elaboration of IFN-,40
which we
noted in mdr1a-/- mice with H. bilis-induced
colitis, may allow more access of intestinal flora to macrophages,
dendritic cells, and lymphocytes in the lamina propria to incite an
H. bilis specific inflammatory response. Studies by Elson et
al. have shown that the spontaneous colitis that develops in C3H/HeJBir
mice is mediated by CD4+ T cells responding to
enteric bacterial antigens.41
The ability to deal with one
or more organisms capable of triggering IBD may reside in the
intestinal expression of certain critical genes and
mdr1a-/- mice could be lacking in such critical genes.
Recent evidence shows that the introduction of one commensal bacterial
organism into gnotobiotic mice can modulate expression of multiple
genes, including mdr1a.42
Although
mdr1a-/- mice develop IBD independent of
Helicobacter infection, H. bilis infection
induces IBD at a much earlier age, and the phenotypic appearance of
spontaneous disease is similar, but not identical to
Helicobacter-induced IBD. In a group of uninfected
mdr1a-/- mice with spontaneous colitis, 92% showed ulcers
in all areas of the colon and rectum while only 5% of H.
bilis-induced colitic mice, of comparable age and disease
severity, exhibited ulcers in one location (proximal colon). This
difference in IBD phenotype may be a useful model in sorting out
factors associated with ulcer formation in colitis.
The initial characterization of mdr1a-/- mice using bone
marrow chimeras suggested that these animals are immunologically
normal.13
Recent studies have shown that
mdr1a-/- mice have altered intestinal intraepithelial
lymphocyte populations associated with altered proliferation and
cytokine responses43
which may be contributing to their
susceptibility to colitis; however, it may be hard to distinguish if
these alterations in cell number and phenotype are indicative of the
early IBD process. Characterization of colitis studies in germ-free
mdr1a-/- mice would help clarify the role of luminal
bacteria in mucosal lymphocyte changes with or without colitis but we
are unaware of any colitis studies done in germ free
mdr1a-/- mice. We noted an expected expansion of
lymphocyte numbers in draining lymph nodes of mdr1a-/-
mice but this did not necessarily correlate with colitis.
Both uninfected and infected mdr1a-/- mice
developed expansion of all major lymphocyte subsets in lymph nodes
draining the bowel. However, H. hepaticus-infected
mdr1a-/- mice had the greatest expansion of lymphocytes
accompanied by the mildest colitis (Figure 4)
.
Mounting an immunological response to the inciting enteric bacterial
antigen correlated with the development of disease in
mdr1a-/- mice as has been noted in other bacterial-related
IBD mouse models21,44
as well as in human patients with
IBD.45
Lymphocytes from H. bilis-infected
mdr1a-/- mice (showing colitis) proliferated in response
to H. bilis antigens while lymphocytes from H.
hepaticus-infected mdr1a-/- mice (showing mild
colitis) did not (Figure 5)
. mdr1a-/- mice with H.
bilis-induced colitis produced elevated serum levels of H.
bilis-specific IgA, IgG1, and Ig2A (relative to infected FVB mice)
at 58 days post-infection while serum levels of H.
hepaticus-specific IgA, IgG1, and Ig2A of mdr1a-/-
mice infected with H. hepaticus (mild colitis) were similar
to or lower than H. hepaticus-infected FVB mice at 17 weeks
post-infection (Figure 7)
.
Up-regulation of Th1-mediated cytokines like IFN- and TNF- have
been noted in other bacterial models of IBD46
and have
been reported to characterize Helicobacter-induced
disease.21,26,47
Hence, it is not surprising that IFN-
is similarly up-regulated in H. bilis-induced colitis in
mdr1a-/- mice. The significance of decreased IFN-
transcripts from colonic tissue of H. hepaticus- infected
mdr1a-/- mice is unclear. IFN-, generally associated
with inflammatory processes, may be absent simply because there is
minimal disease in H. hepaticus-infected
mdr1a-/- mice. Alternatively, H. hepaticus
infection may act to down-regulate this inflammatory cytokine and
its role in delaying or preventing colitis in this susceptible mouse
strain may be important. Determining which of these hypotheses may be
true is the focus of ongoing studies. IL-10 is an important cytokine in
modulating inflammatory responses in IBD presumably through regulatory
T cells as suggested by animal studies.48-50
However,
human therapeutic trials have had variable success51,52
possibly because IL-10 may play different roles depending on the phase
of disease development.53
We found that
mdr1a-/- mice exhibited severe IBD, with or without
H. bilis infection, that was accompanied by increased
colonic IL-10. On the other hand, the apparent delay in IBD development
in H. hepaticus-infected mdr1a-/- mice was not
accompanied by similar increases in IL-10, suggesting that this
cytokine was not critical in this particular phase of disease
development or protection. Future studies are aimed at evaluating the
kinetics of IL-10 levels particularly early in H. hepaticus
infection of mdr1a-/- mice.
It is well known that bacterial flora play a role in IBD pathogenesis.1,54 Mouse models raised under germ-free conditions or treated with antibiotics confirm the necessity for microbial flora for development of colitis.13,55,56 T cell lines activated with enteric bacterial antigens induce colitis57 and the presence of a particular bacterial species can prevent disease.58 In our studies, the ability of H. hepaticus infection to delay the development of IBD in mdr1a-/- mice was an unexpected finding and raises questions about the nature of the commensal populations capable of inciting an inflammatory and proliferative response in a genetically susceptible animal. We consider it unlikely that the H. hepaticus isolate used was not as pathogenic as the H. bilis isolate since we have repeatedly induced severe IBD in IL-10-/- mice using the same H. hepaticus and H. bilis isolates.26 Studies are underway to determine a) whether H. hepaticus infection postpones or prevents the development of spontaneous or H. bilis-induced IBD in mdr1a-/- mice and b) the nature of the microbial populations that are associated with severe versus mild versus no IBD in mdr1a-/- mice. The use of a mouse model, with a presumed intestinal "barrier" defect due to the absence of intestinal P-glycoprotein, and whose colitis can be modulated with a particular bacterium may have relevance to studying the human disorder59,60 and offers a unique opportunity to further dissect IBD pathogenesis.
Acknowledgements
We thank Lela Riley for providing the H. bilis isolate, Loida Torres and Scott Hauff for their help in the care of these mice, and Deanna Hill, Shin-e Lin and Jacque Wilk for their technical assistance.
Footnotes
Address reprint requests to Lillian Maggio-Price, Department of Comparative Medicine, School of Medicine, Box 357190, Seattle, WA 98195.
Supported in part by National Center for Research Resources (NCRR) Grant T32 RR07019 (to A.B.).
Accepted for publication October 17, 2001.
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