Kathryn A. Eaton

The Role of T Cell Subsets and Cytokines in the Pathogenesis of Helicobacter pylori Gastritis in Mice

Gastritis due to Helicobacter pylori in mice and humans is considered a Th1-mediated disease, but the specific cell subsets and cytokines involved are still not well understood. The goal of this study was to investigate the immunopathogenesis of H. pylori-induced gastritis and delayed-type hypersensitivity (DTH) in mice. C57BL/6-Prkdcscid mice were infected with H. pylori and reconstituted with CD4+, CD4-depleted, CD4+CD45RBhigh, or CD4+CD45RBlow splenocytes from wild-type C57BL/6 mice or with splenocytes from C57BL/6IFN-γ−/− or C57BL/6IL-10−/− mice. Four or eight weeks after transfer, DTH to H. pylori Ags was determined by footpad injection; gastritis and bacterial colonization were quantified; and IFN-γ secretion by splenocytes in response to H. pylori Ag was determined. Gastritis and DTH were present in recipients of unfractionated splenocytes, CD4+ splenocytes, and CD4+CD45RBhigh splenocytes, but absent in the other groups. IFN-γ secretion in response to H. pylori Ags was correlated with gastritis, although splenocytes from all groups of mice secreted some IFN-γ. Gastritis was most severe in recipients of splenocytes from IL-10-deficient mice, and least severe in those given IFN-γ-deficient splenocytes. Bacterial colonization in all groups was inversely correlated with gastritis. These data indicate that 1) CD4+ T cells are both necessary and sufficient for gastritis and DTH due to H. pylori in mice; 2) high expression of CD45RB is a marker for gastritis-inducing CD4+ cells; and 3) IFN-γ contributes to gastritis and IL-10 suppresses it, but IFN-γ secretion alone is not sufficient to induce gastritis. The results support the assertion that H. pylori is mediated by a Th1-biased cellular immune response.

Motility as a factor in the colonisation of gnotobiotic piglets by Helicobacter pylori

Non-motile variants of Helicobacter pylori (strain 26695) occurred with a frequency of 1.6 (SD 0.4) x 10(-4) variants/cell/division cycle, and reversion to the motile form occurred with a frequency of less than 10(-7) variants/cell/division cycle. The two forms remained greater than 90% pure for up to 50 cell divisions and differed only in the presence or absence of motility and flagella. Bacteria were recovered from nine of 10 gnotobiotic piglets inoculated orally with motile H. pylori, but from only two of eight inoculated with the non-motile variant. The motile form survived for 21 days in infected piglets, but the non-motile variant survived for only 6 days. Bacteria recovered from piglets inoculated with the non-motile variant were non-motile. These data support the hypothesis that motility is a colonisation factor for H. pylori.

Chronic gastritis in the hypochlorhydric gastrin-deficient mouse progresses to adenocarcinoma.

The current study tests the hypothesis that chronic atrophic gastritis from hypochlorhydria in the gastrin-deficient mouse predisposes the stomach to gastric cancer. Gross morphology and histology of 12-month-old wild-type (WT), gastrin-deficient (G−/−) and somatostatin-deficient (SOM−/−) mice were examined. Parietal and G cells, Ki67, TUNEL, villin and MUC2 expression were analysed by immunohistochemistry. RUNX3 and STAT3 expression was analysed by Western blot. Anchorage-independent growth was determined by cell cluster formation in soft agar. Compared to the WT and SOM−/− mice, hypochlorhydric G−/− mice developed parietal cell atrophy, significant antral inflammation and intestinal metaplasia. Areas of metaplasia within the G−/− mouse stomach showed decreased RUNX3 expression with elevated MUC2 and villin expression. Cells isolated from the tumor grew in soft agar. However, the cells isolated from WT, nontransformed G−/− and SOM−/− gastric tissue did not form colonies in soft agar. Consistent with elevated antral proliferation, tumor tissue isolated from the G−/− mice showed elevated phosphorylated STAT3 expression. We then examined the mechanism by which STAT3 was constitutively expressed in the tumor tissue of the G−/− mice. We found that IFNγ expression was also significantly higher in the tumor tissue of G−/− mice compared to WT and SOM−/− animals. To determine whether STAT3 was regulated by IFNγ, MKN45 cells were cocultured with IFNγ or gastrin. IFNγ significantly stimulated phosphorylation of STAT3 in the MKN45 cell line, but not gastrin. Therefore, we show here that in the hypochlorhydric mouse stomach, the chronic gastritis, atrophy, metaplasia, dysplasia paradigm can be recapitulated in mice. Moreover, neoplastic transformation of the antral gastric mucosa does not require gastrin.

Candida albicans and Bacterial Microbiota Interactions in the Cecum during Recolonization following Broad-Spectrum Antibiotic Therapy

ABSTRACT Candida albicans is a normal member of the gastrointestinal (GI) tract microbiota of healthy humans, but during host immunosuppression or alterations in the bacterial microbiota, C. albicans can disseminate and cause life-threatening illness. The bacterial microbiome of the GI tract, including lactic acid bacteria (LAB), plays a vital role in preventing fungal invasion. However, little is known about the role of C. albicans in shaping the bacterial microbiota during antibiotic recovery. We investigated the fungal burdens in the GI tracts of germfree mice and mice with a disturbed microbiome to demonstrate the role of the microbiota in preventing C. albicans colonization. Histological analysis demonstrated that colonization with C. albicans during antibiotic treatment does not trigger overt inflammation in the murine cecum. Bacterial diversity is reduced long term following cefoperazone treatment, but the presence of C. albicans during antibiotic recovery promoted the recovery of bacterial diversity. Cefoperazone diminishes Bacteroidetes populations long term in the ceca of mice, but the presence of C. albicans during cefoperazone recovery promoted Bacteroidetes population recovery. However, the presence of C. albicans resulted in a long-term reduction in Lactobacillus spp. and promoted Enterococcus faecalis populations. Previous studies have focused on the ability of bacteria to alter C. albicans; this study addresses the ability of C. albicans to alter the bacterial microbiota during nonpathogenic colonization.

The Unfolded Protein Response and Chemical Chaperones Reduce Protein Misfolding and Colitis in Mice

BACKGROUND & AIMS Endoplasmic reticulum (ER) stress has been associated with development of inflammatory bowel disease. We examined the effects of ER stress-induced chaperone response and the orally active chemical chaperones tauroursodeoxycholate (TUDCA) and 4-phenylbutyrate (PBA), which facilitate protein folding and reduce ER stress, in mice with colitis. METHODS We used dextran sulfate sodium (DSS) to induce colitis in mice that do not express the transcription factor ATF6α or the protein chaperone P58(IPK). We examined the effects of TUDCA and PBA in cultured intestinal epithelial cells (IECs); in wild-type, P58(IPK-/-), and Atf6α(-/-) mice with colitis; and in Il10(-/-) mice. RESULTS P58(IPK-/-) and Atf6α(-/-) mice developed more severe colitis following administration of DSS than wild-type mice. IECs from P58(IPK-/-) mice had excessive ER stress, and apoptotic signaling was activated in IECs from Atf6α(-/-) mice. Inflammatory stimuli induced ER stress signals in cultured IECs, which were reduced by incubation with TUDCA or PBA. Oral administration of either PBA or TUDCA reduced features of DSS-induced acute and chronic colitis in wild-type mice, the colitis that develops in Il10(-/-) mice, and DSS-induced colitis in P58(IPK-/-) and Atf6α(-/-) mice. Reduced signs of colonic inflammation in these mice were associated with significantly decreased ER stress in colonic epithelial cells. CONCLUSIONS The unfolded protein response induces expression of genes that encode chaperones involved in ER protein folding; these factors prevent induction of colitis in mice. Chemical chaperones such as TUDCA and PBA alleviate different forms of colitis in mice and might be developed for treatment of inflammatory bowel diseases.

Switching of Flagellar Motility in Helicobacter pylori by Reversible Length Variation of a Short Homopolymeric Sequence Repeat in fliP, a Gene Encoding a Basal Body Protein

ABSTRACT The genome of Helicobacter pylori contains numerous simple nucleotide repeats that have been proposed to have regulatory functions and to compensate for the conspicuous dearth of master regulatory pathways in this highly host-adapted bacterium. H. pylori strain 26695, whose genomic sequence was determined by The Institute for Genomic Research (TIGR), contains a repeat of nine cytidines in the fliP flagellar basal body gene that splits the open reading frame in two parts. In this work, we demonstrate that the 26695C9 strain with a split fliP gene as sequenced by TIGR was nonflagellated and nonmotile. In contrast, earlier isolates of strain 26695 selected by positive motility testing as well as pig-passaged derivatives of 26695 were all flagellated and highly motile. All of these motile strains had a C8 repeat and consequently a contiguous fliP reading frame. By screening approximately 50,000 colonies of 26695C9 for motility in soft agar, a motile revertant with a C8 repeat could be isolated, proving that the described switch is reversible. ThefliP genes of 20 motile clinical H. pyloriisolates from different geographic regions possessed intactfliP genes with repeats of eight cytidines or the sequence CCCCACCC in its place. Isogenic fliP mutants of a motile, C8 repeat isolate of strain 26695 were constructed by allelic exchange mutagenesis and found to be defective in flagellum biogenesis. Mutants produced only small amounts of flagellins, while the transcription of flagellin genes appeared unchanged. These results strongly suggest a unique mechanism regulating motility in H. pylori which relies on slipped-strand mispairing-mediated mutagenesis of fliP.

Role of Helicobacter felis in Chronic Canine Gastritis

Five gnotobiotic Beagle dogs were orally inoculated with a pure culture of Helicobacter felis. The remaining two littermates served as contact controls. Thirty days after infection, all animals were euthanatized and specimens were collected for evaluation. In infected dogs, H. felis was recovered from all areas of the stomach. Colonization was heaviest in the fundus and antrum. H. felis was not cultured from any segment of the gastrointestinal tract distal to the duodenum. Two weeks after infection, all five infected dogs had detectable IgM and IgG serum antibody to H. felis, whereas control dogs had no measurable H. felis serum antibody throughout the study. Histopathologic changes in the stomachs of infected dogs included large numbers of lymphoid nodules throughout all regions of the gastric mucosa and were most numerous in the fundus and body. A mild, diffuse lymphocytic infiltrate with small numbers of plasma cells and eosinophils was also present in the subglandular region of all portions of the gastric mucosa. Electron microscopic examination revealed large numbers of spiral-shaped H. felis in gastric mucus adjacent to or superimposed over the areas of inflammation. Occasionally, however, H. felis was observed within the canaliculi of gastric parietal cells. Histopathologic changes in the stomachs of the contact control dogs were limited to focal infiltrates of eosinophils and small aggregates of lymphocytes in the subglandular portions of the gastric mucosa in one animal. Infection with H. felis is a likely cause of naturally occurring lymphofollicular gastritis.

Interleukin-1β Promotes Gastric Atrophy Through Suppression of Sonic Hedgehog

BACKGROUND & AIMS In both human subjects and rodent models, Helicobacter infection leads to a decrease in Shh expression in the stomach. Sonic Hedgehog (Shh) is highly expressed in the gastric corpus and its loss correlates with gastric atrophy. Therefore, we tested the hypothesis that proinflammatory cytokines induce gastric atrophy by inhibiting Shh expression. METHODS Shh-LacZ reporter mice were infected with Helicobacter felis for 3 and 8 weeks. Changes in Shh expression were monitored using beta-galactosidase staining and immunohistochemistry. Gastric acidity was measured after infection, and interleukin (IL)-1beta was quantified by quantitative reverse-transcription polymerase chain reaction. Mice were injected with either IL-1beta or omeprazole before measuring Shh mRNA expression and acid secretion. Organ cultures of gastric glands from wild-type or IL-1R1 null mice were treated with IL-1beta then Shh expression was measured. Primary canine parietal or mucous cells were treated with IL-1beta. Shh protein was determined by immunoblot analysis. Changes in intracellular calcium were measured by Fura-2. RESULTS All major cell lineages of the corpus including surface pit, mucous neck, zymogenic, and parietal cells expressed Shh. Helicobacter infection reduced gastric acidity and inhibited Shh expression in parietal cells by 3 weeks. IL-1beta produced during Helicobacter infection inhibited gastric acid, intracellular calcium, and Shh expression through the IL-1 receptor. Suppression of parietal cell Shh expression by IL-1beta and omeprazole was additive. IL-1beta did not suppress Shh expression in primary gastric mucous cells. CONCLUSIONS IL-1beta suppresses Shh gene expression in parietal cells by inhibiting acid secretion and subsequently the release of intracellular calcium.

Autosomal Dominant Polycystic Kidney Disease in Persian and Persian-cross Cats:

A form of autosomal dominant polycystic kidney disease (ADPKD) similar in clinical features to human ADPKD occurs in the Persian cat. We characterized the morphologic and immunohistochemical features of this disease in a colony of affected cats. Complete postmortem examinations were performed on 11 normal and 22 affected cats ranging in age from 3 months to 10 years. Kidneys were evaluated by gross and histologic examination, ultrastructure, lectin staining, bromodeoxyuridine immunochemistry for labeling index, and immunochemistry for distribution of Na/K ATPase. Feline ADPKD was characterized by variable numbers of cysts in the renal cortex and medulla. Ultrastructural examination and lectin staining suggested that cysts arose from proximal and distal nephron segments. Bromodeoxyuridine labeling demonstrated increased proliferation of epithelium lining some cysts in young cats. Immunohistochemical staining showed variable translocation of Na/K ATPase from the basolateral membranes of cyst-lining cells to the cytoplasm or luminal membranes. Cystic renal disease commonly was associated with chronic tubulointerstitial nephritis and hepatobiliary hyperplasia and fibrosis. Focal hyperplasia of renal tubular epithelium, hepatic cysts, and cardiac lesions were present in some cats. Feline ADPKD shares many morphologic and pathogenetic features with human ADPKD.

Role of Helicobacter pylori cag Region Genes in Colonization and Gastritis in Two Animal Models

ABSTRACT The Helicobacter pylori chromosomal region known as the cytotoxin-gene associated pathogenicity island (cag PAI) is associated with severe disease and encodes proteins that are believed to induce interleukin (IL-8) secretion by cultured epithelial cells. The objective of this study was to evaluate the relationship between the cag PAI, induction of IL-8, and induction of neutrophilic gastric inflammation. Germ-free neonatal piglets and conventional C57BL/6 mice were given wild-type or cagdeficient mutant derivatives of H. pylori strain 26695 or SS1. Bacterial colonization was determined by plate count, gastritis and neutrophilic inflammation were quantified, and IL-8 induction in AGS cells was determined by enzyme-linked immunosorbent assay. Deletion of the entire cag region or interruption of thevirB10 or virB11 homolog had no effect on bacterial colonization, gastritis, or neutrophilic inflammation. In contrast, these mutations had variable effects on IL-8 induction, depending on the H. pylori strain. In the piglet-adapated strain 26695, which induced IL-8 secretion by AGS cells, deletion of the cag PAI decreased induction. In the mouse-adapted strain SS1, which did not induce IL-8 secretion, deletion of thecagII region or interruption of any of threecag region genes increased IL-8 induction. These results indicate that in mice and piglets (i) neither the cag PAI nor the ability to induce IL-8 in vitro is essential for colonization or neutrophilic inflammation and (ii) there is no direct relationship between the presence of the cag PAI, IL-8 induction, and neutrophilic gastritis.