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* Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139;
Forsyth Institute, Boston, Massachusetts 02115
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Abstract |
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3 x 107 colony-forming units (CFU) of H. sp. Mice were necropsied at various time points postinoculation (PI). Sham-inoculated mice had no clinical, gross, or histopathological lesions. In contrast, scid mice inoculated IP with H. sp. had severe hemorrhagic diarrhea and decreased weight gain at 2, 7, and 18 weeks postinoculation (PI), with severe proliferative typhlocolitis, phlebothrombosis, and hepatitis. A/J mice had no clinical signs, but had mild to moderate proliferative typhlocolitis and moderate to marked cholangiohepatitis at 7 and 24 weeks PI. A/J mice infected with H. sp. developed robust immune responses of a predominant Th1 type. This report demonstrates that infection with a urease-negative helicobacter can cause inflammatory bowel disease (IBD) and hepatitis in scid and immunocompetent A/J mice. These results provide a new model of IBD and cholangiohepatitis associated with a specific urease-negative, novel H. species.
Key Words: Helicobacter • mouse • typhlocolitis • cholangiohepatitis
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The purpose of this study was to determine the pathogenic potential of a novel urease-negative H. species in both immunocompromised and immunocompetent mice, and to determine if infection with this organism in these mice would provide a suitable model for enterohepatic disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bacteria.
The novel urease-negative H. sp. (MIT 96-1001) was originally isolated from the cecum of a clinically normal feral mouse. It was grown under microaerobic conditions in vented jars containing N2, H2, and CO2 (80:10:10) at 37°C in Brucella broth supplemented with 5% fetal calf serum. The bacteria were harvested after 24 to 48 hr of growth, resuspended in phosphate-buffered saline (PBS), and visualized by gram stain and phase microscopy for purity, morphology, and motility. The optical density (OD600) was adjusted to 1.0 and 0.3 ml of this inoculum (3 x 107 colony-forming units [CFU]) was used for each intraperitoneal (IP) injection or oral dose.
Bacterial Isolation.
Bacteria were cultured from fecal pellets (ante mortem) or from cecal contents or ground liver at necropsy. At scheduled intervals, pooled samples of three to five fresh fecal pellets were obtained from each cage of mice. Fecal pellets were suspended in 1 ml of sterile PBS, and the resulting slurry was plated directly onto CVA plates (cefaperazone, vancomycin, amphotericin B; Remel, Lenexa, KS) for microaerobic isolation of the novel H. sp. At necropsy, cecal contents were applied directly to CVA plates. Aseptically collected liver was ground and applied directly to CVA plates for isolation of H. sp., and to blood agar plates for isolation of aerobic bacteria. Although growth of H. sp. was generally evident within 5 days, plates were maintained for 2 weeks before a determination of no growth was made.
Electron Microscopy.
The novel H.The novel H. sp. was examined by electron microscopy. Cells were grown on blood agar plates (Remel) at 37°C under microaerobic conditions for 48 h. Then they were gently resuspended in 10 mM Tris buffer (pH 7.4) at a concentration of approximately 108 cells per milliliter. Samples were negatively stained with 1% (w/v) phosphotungstic acid (pH 6.5) for 20 to 30 sec and were examined with a JEOL model JEM-1200EX transmission electron microscope operating at 100 kV.
Genomic DNA Extraction for 16S rRNA Gene Sequencing.
Bacteria were cultured on blood agar plates and the cells were harvested and washed twice with 1 ml of double-distilled H2O. The pellets were suspended in STET buffer (8% sucrose, 50 mM EDTA, 0.1% Triton X-100, and 50 mM Tris HCl, pH 8.0) and lysozyme (hen egg white, Boehringer Mannheim Biochemicals, Indianapolis, IN) was added to a final concentration of 3 mg/ml. The suspension was incubated for 12 min at 37°C and was then lysed with sodium dodecyl sulfate. RNAse A (bovine pancreas; Boehringer Mannheim) was added to a final concentration of 0.05 mg/ml and the solution was incubated for 1 hr at 37°C. Then 0.1 volume of a 5% cetyltrimethylammonium bromed-0.5 M NaCl solution (Sigma Chemical, St. Louis, MO) was added and the solution was gently mixed and incubated at 65°C for 10 min. DNA was extracted with an equal volume of phenol-chloroform (1:1, v/v), precipitated overnight in 0.3 M sodium acetate with 2 volumes of absolute ethanol at -20°C, and pelleted by centrifugation at 13,000g for 1 hr at 4°C. The ethanol was decanted and the pellet was air dried and suspended in distilled water.
16S rRNA Gene Sequencing.
The sequence of the 16S rRNA gene of the novel urease-negative helicobacter isolate was determined. For amplification of the 16S rRNA cistrons, 16S rRNA gene sequencing, and 16S rRNA data analysis, we used the methods described by Fox et al. (4). Briefly, primers C60 and B37 (4) were used to amplify the 16S rRNA genes. The amplicons were purified and directly sequenced by using a TAQeunce cycle sequencing kit (US Biochemica, Cleveland, OH). The 16S rRNA gene sequences were entered into a program for analysis of 16S rRNA data in Microsoft QuickBASIC for use on PC-compatible computers and were aligned as previously described (18). The sequence database used contains approximately 100 helicobacter, wolinella, arcobacter, and campylobacter sequences and more than 900 sequences for other bacteria. Similarity matrices were constructed from the aligned sequences by using only those base positions for which 90% of the strains had data and were corrected for multiple base changes by the method of Jukes and Cantor (19). Phylogenetic trees were constructed by using the neighbor-joining method of Saitou and Nei (20).
ELISA for Anti-H. sp. IgG in Serum and IgA in Feces.
An outer membrane antigen preparation (OMP) of H. sp. was obtained by methods previously described for preparing H. hepaticus antigen (21). Briefly, H. sp. was cultured in trypticase soy broth containing 5% fetal bovine serum for 48 hr under microaerobic conditions as detailed above. After three washes in PBS and examination for bacterial contaminants using gram stain and phase microscopy, the pellet was resuspended in 4 ml of 1% N-octyl-beta-glucopyranoside (Sigma) for 30 min at room temperature. Insoluble material was removed by ultracentrifugation at 100,000g for 1 hr. After dialysis against PBS for 24 hr at 4°C, supernatant protein concentration was measured by the Lowry technique (Sigma). For serum IgG measurement, 96-well plates were coated with 100 µl per well of 1 µg/ml of H. sp. OMP protein in carbonate buffer (pH 9.6) overnight at 4°C. Coating concentration of antigen was increased to 10 µg/ml for measurement of IgA in fecal extracts. Biotinylated secondary antibodies included goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL), 
-chain-specific goat anti-mouse IgA (Sigma), and monoclonal rat anti-mouse antibodies produced by clones G1-6.5 and R19-15 (Pharmingen, San Diego, CA) for detecting IgG1 and IgG2a, respectively. Incubation with extravidin peroxidase (Sigma) was followed by ABTS substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) for color development. OD development at 
= 405 mµ was recorded by an ELISA plate reader (Dynatech MR7000, Dynatech Laboratories, Chantilly, VA).
Sample Preparation and Analysis.
Sera were collected from all mice prior to dosing with H. sp. and then again at necropsy, and were stored at -20°C prior to analysis. Serum IgG results are reported as mean OD values at a sample dilution of 1:100, with all samples run in triplicate. Feces were collected from all mice prior to dosing with H. sp. and then monthly until necropsy. Four freshly voided fecal pellets (approximately 100 mg) were suspended in a protease inhibitor cocktail (1 µg/ml aprotonin, 10 µM leupeptin, 3.25 µ M bestatin, and 0.2 mM 4-[2-aminoethyl]-benzene sulfonylfluoride [Sigma] in 5% nonfat dry milk) as previously described (1). The fecal slurry was microcentrifuged at 10,000 rpm (Microcentrifuge 235C, Fisher Scientific, Pittsburgh, PA) for 10 min to yield supernatant for IgA measurement. Fecal extracts were frozen at -70°C pending analysis, and were assayed undiluted. Because of an unknown dilution factor inherent in sample preparation, the OD measurement of IgA specific for H. sp. in fecal extracts was standardized against total IgA concentration of the sample. A standard curve was generated on each ELISA plate by applying known amounts of purified mouse IgA-
(Sigma) precoated with -chain-specific sheep anti-mouse IgA.
Histology.
Sections of liver and gastrointestinal tissues were fixed in neutral buffered 10% formalin and in Carnoy's fixative. Tissues were embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin (H&E) stain for assessment of histopathology, Masson's trichrome stain for assessment of hepatic fibrosis, or Warthin-Starry silver stain to visualize bacteria in tissues (formalin-fixed sections only).
Statistics and Analysis.
Statistics reported throughout the text are based on a two-tailed Student's t test.
Experimental Design.
Pooled feces from the mice were analyzed by PCR and were cultured microaerobically upon arrival to ensure that they were not colonized with any H. sp. After 1 week of acclimatization, baseline blood samples were obtained from each mouse. Mice were randomly assigned to inoculation groups. The mice were inoculated IP with 0.3 ml of PBS (sham-dosed controls) or 0.3 ml of PBS containing 3 x 107 CFU of the novel H. sp. Two uninoculated scid cage-contact mice were housed with IP-inoculated scid mice for the purpose of establishing whether natural fecal-oral transmission of the novel H. sp. occurred. In addition, to determine whether the IP route of infection influenced the severity of clinical signs, five scid mice were inoculated orally with 0.3 ml PBS containing 3 x 107 CFU of H. sp. Mice were observed daily and weighed at scheduled intervals. Mice were necropsied at time points listed in Table I
. Unless otherwise stated, all data presented in the Results section pertains to IP-inoculated mice only.
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Results |
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Ultrastructure.
The novel H. sp. was spiral and it measured 0.15 by 2 to 10 µm with three to seven spiral turns (Fig. 1). The bacterium possessed single bipolar, sheathed flagella, but did not have periplasmic fibers.
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. The novel H. sp. sequence fell in the helicobacter cluster, which includes H. canis, H. bilis, H. cinaedi, H. sp. Group 8, and H. westmeadii. The three sequences were identical and were most closely related to that of H. sp. Flexispira Group 8 (GenBank AF047851), and ``H. ulmiensis'' (GenBank AJ007931). The novel H. sp. strains contain an intervening sequence (IVS) that is essentially identical to that in H. bilis and H. sp. Flexispira Group 8. The novel H. sp. sequence differs by 12 bases from that of the ``F. rappini'' (GenBank AF047851) and differs from the ``H. ulmiensis'' sequence (AJ007931) by seven bases in the ribosomal portion of the sequence (data not shown); however, ``H. ulmiensis'' contains a uniquely different IVS.
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). Control mice had no gross lesions at necropsy at 7 or 24 weeks PI. Multiple 1- to 5-mm pale yellow foci in the liver parenchyma (corresponding to hepatic necrosis) were observed in two of 10 inoculated mice at 7 weeks PI. Translucent nodules and tracts (corresponding to mononuclear cell accumulations seen by light microscopy) were apparent on cut sections of the livers of nearly all (eight of 10) inoculated mice at 24 weeks PI.
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). Control mice had no clinical signs and no grossly apparent lesions at necropsy at 2, 7, 18, or 24 weeks PI. In contrast, all mice inoculated IP with H. sp. developed intermittent severe bloody diarrhea at 2 weeks PI. Diarrhea episodes typically lasted 1 week, with frequent relapses noted. Eight IP-inoculated scid mice died or were euthanized by 18 weeks PI. Multiple 1- to 5-mm pale yellow foci in the liver parenchyma (corresponding to hepatic necrosis) were seen in five of 10 IP-inoculated mice at 7 weeks PI. At 18 weeks PI, there was gross evidence of massive hepatic necrosis and remodeling of the livers of three of four surviving H. sp.-inoculated scid mice. They had abnormally shaped fused liver lobes indicative of prior massive necrosis and subsequent repair.
Orally dosed scid mice.
To determine if the severe clinical signs were due to the IP route of inoculation, five scid mice were orally inoculated with H. sp. These mice developed bloody diarrhea within 1 week of inoculation, an even earlier time point than in the IP inoculated scid mice, indicating that the clinical signs were not an artifact of IP inoculation.
Uninoculated scid cage contact mice.
To determine if fecal-oral transmission could occur, two uninoculated scid cage contacts were co-housed with IP-inoculated infected mice 2 weeks after inoculation. The cage contacts also developed intermittent severe bloody diarrhea within 1 week of exposure.
Colonization by the Novel H. sp.
All of the sham-dosed scid (n = 20) and A/J mice (n = 20) remained culture-negative for the novel H. sp. The H. sp.-dosed scid and A/J mice were culture positive within 2 weeks of dosing by the IP route. H. sp. was also isolated from the feces of orally inoculated scid mice and the uninoculated scid cage contacts within 2 weeks of exposure. The novel H. sp. was readily cultured from the livers of selected IP-inoculated scid or A/J mice with gross liver lesions. A few slender spiral argryophilic organisms consistent with H. sp. were seen in selected livers stained with Warthin-Starry silver stain.
Histopathology.
IP-inoculated scid mice
No lesions were observed in the livers or gastrointestinal tracts of the control scid mice at 7 weeks PI. At 18 weeks PI, mild mucosal hyperplasia and limited focal inflammation were observed in the rectums of two control mice. Mild focal hyperplasia of the gastric antral mucosa was also observed in two control mice at this time point. In contrast, infection with H. sp resulted in moderate-to-marked proliferative typhlocolitis accompanied by hepatic necrosis and inflammation both mice necropsied at 2 weeks PI. At 2 weeks PI, IP-inoculated scid mice had moderate proliferative typhlocolitis with multifocal epithelial necrosis. Hyperplastic crypts were elongated and lined by densely packed, hyperchromic epithelium with diminished goblet cell differentiation (Fig. 4a). The proximal colon and cecum were further characterized by submucosal edema and infiltration by histiocytes, granulocytes, and small mononuclear cells (Fig. 4b). Both of the mice necropsied at the 2-week time point developed phlebothrombosis within the portal circulation, accompanied by extensive, multifocal coagulative necrosis and inflammation (Fig. 4c). Subacute periportal inflammation and multifocal hepatic microabscessation were prominent in the affected livers.
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). Cecal epithelial necrosis was observed in several mice (not shown). The intensity of colonic inflammation was diminished to mild intensity present in only seven out of eight mice at 7 weeks PI, and colitis was manifested as moderate hyperplasia and inflammation polarized toward the distal colon and rectum in four out of four mice at 18 weeks PI. Multifocal hepatic phlebothrombosis was common, associated with moderate-to-marked hepatic necrosis and inflammation. Remodeling of the hepatic tissue was manifested grossly as large, abnormally contoured lobes. Portal inflammation, multifocal necrosis, fibroplasia, organization of thrombi, and multifocal suppurative inflammation were observed in hepatic tissue of eight out of eight mice at 7 weeks PI and three out of four mice at 18 weeks PI (Fig. 4d). Extrahepatic thrombi were also rarely observed in veins of the cecum and pancreas in mice from the 7-weeks PI group.
IP-inoculated A/J mice.
No significant lesions were observed in the livers or gastrointestinal tracts of the control A/J mice at 7 and 24 weeks PI. Infected A/J mice typically developed less intense inflammation and proliferation of the cecum and colon, as compared with scid mice. At 7 weeks PI, proliferative typhlitis (present in 10/10 mice) was mild in intensity, and was characterized by hyperplasia and multifocal or diffuse infiltration by lymphocytes. At 24 weeks PI, typhlitis progressed toward mild (four out of 10 mice) or moderate (five out of 10 mice) intensity (Fig. 5a), and in one case developed a severe inflammation and mucosal hyperplasia with multifocal cecal ulceration. Moderate focal gastric antral hyperplasia and inflammation and mild ileitis were also observed in several mice. Mild-to-moderate colitis was observed infrequently (three out of 10 mice), limited primarily to the distal colon or rectum.
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). Multifocal hepatic phlebitis, characterized by prominent lymphocytic perivascular infiltrates, was also present. At 24 weeks PI, in eight out of 10 mice, the portal inflammation was marked in intensity and was manifested by large lymphoplasmacytic infiltrates that formed thick cuffs around portal structures, especially bile ducts (Fig. 5c). Scattered bile ducts contained suppurative exudate and necrotic cells (Fig. 5d). Hepatic fibrosis was observed as a pattern of mild-to-moderate collagen deposition, extending from the portal areas into the periportal parenchyma.
Serology.
Serum IgG responses.
A/J mice experimentally infected with the novel H. sp. developed significant (P < 0.001) serum IgG responses to the infection by 24 weeks PI (Table II). Using an isotype-specific ELISA for discrimination between IgG2a (Th1-like) and IgG1 (Th2-like) antibody responses, mice infected with the novel H. sp. developed a predominant IgG2a serum response to H. sp. infection. The IgG2a response was significantly higher than the IgG1 response in all mice tested (P < 0.0005) with a mean ratio of IgG2a to IgG1 of 2.8 ± 0.6.
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Discussion |
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Our interpretation is that the clinical signs were due to colonization of the gut by the H. sp., and that this colonization takes place faster when the bacteria are inoculated directly per os than when they have to reach the gut via the IP route. The increased severity of the enterohepatic lesions noted in mice in the current study may be due to a gender predilection or to the presence of other bacterial species in the gut of ``restricted'' rather than ``defined'' flora mice. Finally, there may be bacterial-specific factors that are important determinants of the severity of disease. For instance, strains of H. pylori that elaborate vacuolating cytotoxin have been reported to be more pathogenic than strains without the cytotoxin (25, 26). H. hepaticus has been reported to produce a granulating cytotoxin (27) and a newly reported cytolethal distending toxin (28). Urease is an essential colonization factor for gastric helicobacters, allowing for the organism's survival in an acidic environment. Urease is produced by the established enterohepatic pathogens H. hepaticus and H. bilis, but not by the novel H. sp. reported on here, or by ``H. typhlonicus,'' which has been reported to cause disease in IL10-/- and scid mice (2, 29). Thus, our results confirm that urease is not a required virulence factor for enterohepatic helicobacters to cause hepatic or enteric disease. It remains to be determined whether there are virulence factors unique to the novel H. sp., or strains of the novel H. sp. that are more or less pathogenic.
The liver lesions reported in this study are similar to those seen in aged mice naturally infected with H. hepaticus (8) and to IL-10-/- mice naturally or experimentally infected with another recently described urease-negative H. sp. (H. typhlonicus), which is taxonomically distinct from the novel H. sp used in this study (2, 29). The early onset of these lesions in our study (apparent at 7 weeks) may be due to the route of exposure (IP versus oral) or may indicate that the novel urease negative H. sp. is more virulent. The severe cholangitis and pericholangitis may be the direct result of colonization of the liver and bile ducts with H. sp. or may reflect an autoimmune response directed against an antigen common to intestinal helicobacters and the biliary epithelium (30). Prolonged (1–2 years) infection with H. hepaticus is associated with the development of hepatic tumors (31). Although the development of inflammatory lesions was greatly accelerated compared to infection with H. hepaticus, no hepatic tumors were seen in mice infected with the novel H. sp. The study was of insufficient duration to determine the carcinogenic potential of H. sp. and further studies with longer-term infection are clearly needed. Infection with H. hepaticus also causes a significant hepatic vasculitis (3, 31). It has recently been theorized that H. pylori exerts some pathogenic effects on the vasculature, and it has been epidemiologically and anecdotally linked with vascular disorders such as migraine and Reynaud's syndrome (32).
The significant serum IgG and fecal IgA responses of the A/J mice to the novel H. sp. are consistent with the marked inflammatory response to the infection. Mice infected with the novel H. sp. developed a serum IgG2a antibody response that was greater then the IgG1 response, indicating that a pro-inflammatory Th1 response to the infection predominated over a Th2 response, as previously observed in A/JCr mice infected with H. hepaticus (21) and in C57BL/6 mice infected with H. felis (33).
It has been hypothesized that some human IBD is triggered by bacterial antigens and that the IBD is a dysregulated immune response to unspecified bacterial insult (34–36). Several mouse models of IBD have been reported to result from the elimination or overexpression of cytokines involved in the balance between a Th1 and Th2 response (37, 38). For example, Kullberg et al. (37) showed that H. hepaticus induces chronic colitis in SPF-reared IL-10(-/-) mice, and the disease is accompanied by a type 1 cytokine response (IFN
, TNF, and nitric oxide). In contrast, wild-type C57BL-background animals infected with the same bacteria did not develop disease and they produced IL-10 as the dominant cytokine in response to Helicobacter antigen. In vivo neutralization of IFN or IL-12 resulted in a significant reduction of intestinal inflammation in H. hepaticus-infected IL-10-/- mice, suggesting an important role for these cytokines in the development of colitis in the model. Apparently, in immunocompetent hosts, IL-10 stimulated in response to intestinal flora is important in preventing IBD. The A/J mice in this study exhibited a Th1 response to the novel H. sp. and a robust inflammatory response in the liver. However, the scid mice also developed severe enterohepatic lesions, similar in quality to those in the A/J mice, despite a lack of T cells. The inflammatory infiltrate in the scid mice was primarily mononuclear, predominantly consisting of cells morphologically identified as macrophages. Although macrophage activation is enhanced by T cells, it has recently been shown that H. pylori alone can stimulate macrophages and induce iNOS (39). The severe histiocytic lesions in the intestines and livers of the scid mice in this study certainly suggest that novel H. sp. shares this property, although this needs to be confirmed by in vitro testing.
Von Freeden-Jeffry et al. (38) found that H. hepaticus-infected mice mutant for both IL-7 (a macrophage activator) and RAG-2 (IL-7/RAG-2-/-) did not develop myeloid responses or colitis whereas RAG2-/- did, indicating that IL-7 plays a critical role in exacerbating a non-T cell/non-B cell-mediated chronic inflammatory response. Therapy with recombinant IL-10 protein (which inhibits antigen presentation and macrophage production of IL-1, IL-6, and TNF) was able to prevent the occurrence of colitis in susceptible RAG-2(-/-) mice, suggesting a pivotal role for macrophages (38). The present study, as well as our earlier studies using helicobacter-infected defined flora scid mice, using B and T-cell free scid mice, is additional evidence for an important role for macrophages in IBD (13, 24).
There is some evidence that intestinal helicobacters may play a role in IBD in humans; H. cinaedi (40) and H. fennelliae (41) were first identified as agents causing proctitis and colitis in immunocompromised humans (41). In this study, a novel urease-negative murine H. sp. closely related to a cluster of enteric helicobacters by 16S rRNA sequence analysis produced mild-to-moderate IBD in both outbred scid mice and inbred A/J mice. Importantly, in this study the H. sp. also caused hepatitis with cholangitis. In some human patients with IBD, primary sclerosing cholangitis is a clinically important sequelum of unknown etiology. An animal model with similar pathological changes could prove valuable for investigating hypotheses related to bacterial/autoimmune etiology of this disease process.
Intestinal helicobacters may play an important role as factors or cofactors in the development of IBD in many species. It is particularly important to consider the murine intestinal helicobacters as potential pathogens. Some genetic mouse models of IBD may now be attributable to H. sp. infection (10), and some long-term toxicology studies using B6C3F1 mice have been compromised by H. sp. infection (3, 31). This study both confirms the pathogenicity of H. sp. in immunocompetent and immunocompromised mice, and offers a potential murine model for studies of IBD and associated liver disease.
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Footnotes |
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This work was funded in part by NIH (grants R01CA67529, R01DK52413, P01CA26731, and RR01046).
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