Helicobacter Hepaticus Infection Research Articles

MHC class II antigen presentation by intestinal epithelial cells fine-tunes bacteria-reactive CD4 T-cell responses

Although intestinal epithelial cells (IECs) can express major histocompatibility complex class II (MHC II), especially during intestinal inflammation, it remains unclear if antigen presentation by IECs favors pro- or anti-inflammatory CD4+ T-cell responses. Using selective gene ablation of MHC II in IECs and IEC organoid cultures, we assessed the impact of MHC II expression by IECs on CD4+ T-cell responses and disease outcomes in response to enteric bacterial pathogens. We found that intestinal bacterial infections elicit inflammatory cues that greatly increase expression of MHC II processing and presentation molecules in colonic IECs. Whilst IEC MHC II expression had little impact on disease severity following Citrobacter rodentium or Helicobacter hepaticus infection, using a colonic IEC organoid-CD4+ T cell co-culture system, we demonstrate that IECs can activate antigen-specific CD4+ T cells in an MHC II-dependent manner, modulating both regulatory and effector Th cell subsets. Furthermore, we assessed adoptively transferred H. hepaticus-specific CD4+ T cells during intestinal inflammation in vivo and report that IEC MHC II expression dampens pro-inflammatory effector Th cells. Our findings indicate that IECs can function as non-conventional antigen-presenting cells and that IEC MHC II expression fine-tunes local effector CD4+ T-cell responses during intestinal inflammation.

The IL-10 receptor inhibits cell extrinsic signals necessary for STAT1-dependent macrophage accumulation during colitis

The loss of IL-10R function leads to severe early onset colitis and, in murine models, is associated with the accumulation of immature inflammatory colonic macrophages. We have shown that IL-10R-deficient colonic macrophages exhibit increased STAT1-dependent gene expression, suggesting that IL-10R-mediated inhibition of STAT1 signaling in newly recruited colonic macrophages might interfere with the development of an inflammatory phenotype. Indeed, STAT1-/- mice exhibit defects in colonic macrophage accumulation after Helicobacter hepaticus infection and IL-10R blockade, and this was phenocopied in mice lacking IFNγR, an inducer of STAT1 activation. Radiation chimeras demonstrated that reduced accumulation of STAT1-deficient macrophages was based on a cell-intrinsic defect. Unexpectedly, mixed radiation chimeras generated with both wild-type and IL-10R-deficient bone marrow indicated that rather than directly interfering with STAT1 function, IL-10R inhibits the generation of cell extrinsic signals that promote the accumulation of immature macrophages. These results define the essential mechanisms controlling the inflammatory macrophage accumulation in inflammatory bowel diseases.

The Deletion of IL-17A Enhances Helicobacter hepaticus Colonization and Triggers Colitis.

ObjectiveIL-17 is a key regulator of the inflammatory response, and as such, it is involved in the constraint and clearance of pathogens. The mechanism of IL-17 in the pathogenesis of inflammatory bowel disease (IBD) caused by microbial infection is still unclear. Helicobacter hepaticus infection can induce colitis in many mouse strains, and thus, it has been widely used in the study of IBD pathogenesis.MethodsIn this study, male C57BL/6, BALB/c, Il-10−/−, and Il-17a−/− mice were infected with H. hepaticus for several weeks. Histopathology, H. hepaticus colonization and distribution, expression of inflammatory cytokines and lysozyme, and distribution of mucus in proximal colon were examined.ResultsThe colonic colonization of H. hepaticus was abnormally high in Il-17a−/− mice. H. hepaticus infection caused only mild to moderate colitis symptoms in Il-17a−/− mice, including low levels of lymphocyte infiltration, epithelial cell defects, goblet cell reduction, and crypt atrophy without obvious hyperplasia in the later stage of infection. Furthermore, many inflammatory genes were significantly increased in the proximal colon of H. hepaticus-infected Il-17a−/− mice compared with C57BL/6 mice. In addition, the reduction of colonic mucus and the down-regulation of ZO-1, Claudin-1, and IL-22 were observed in Il-17a−/− mice compared with C57BL/6 mice post H. hepaticus infection.ConclusionThese results demonstrated that the deletion of IL-17A impaired the integrity of the intestinal epithelium, weakened the secretion of mucus, attenuated colonic mucosal regeneration, reduced the ability to resist microbial infection, and finally led to colitis caused by H. hepaticus.

Apoptosis triggered by cytolethal distending toxin B subunit of Helicobacter hepaticus is aggravated by autophagy inhibition in mouse hepatocytes

Hepatocytes injury caused by cytolethal distending toxin (CDT) are major events during helicobacter hepaticus (H.hepaticus) infection. Recent study showed that pre-survival autophagy was promoted against CdtB subunit induced DNA damage. In the present study, we demonstrated that inflammatory cytokines IL-6, IL-1β, TNF-α, IFN-α, IFN-γ expression and STAT phosphorylation were promoted by CdtB. Besides, CdtB decreased cell viability while promote apoptosis in mouse liver (AML12) cells. Especially, apoptotic protein caspase-9, caspase-3 and PARP were activated while the ratio of Bcl-2/Bax was decreased after CdtB treatment. Moreover, apoptosis induced by CdtB was inhibited due to Erk/p38 MAPK signaling pathway suppression performed with SB203580 or U0126. Meanwhile, we found that CdtB increased autophagic marker levels accompanied by Akt/mTOR/P70S6K signaling pathway in a dose dependent manner. To assess the correlation between autophagy and apoptosis induced by H.hepaticus, chloroquine (CQ, 50 μM) was employed to inhibit autophagy. The result showed that inhibition of autophagy with CQ treatment promoted apoptosis induced by CdtB. Altogether, all these results suggest that CdtB triggers apoptosis via MAPK/Erk/p38 signaling pathway in caspase dependent manner, which was prevented by autophagy in AML12 cells. Collectively, our findings provide new insights into the virulence potential of CdtB on the molecular pathogenesis throughout H.hepaticus infection.

Helicobacter hepaticus Infection Promotes the Progression of Liver Preneoplasia in BALB/c Mice via the Activation and Accumulation of High-Mobility Group Box-1

It has been documented that Helicobacter hepaticus (H. hepaticus) infection is linked to chronic hepatitis and fibrosis in male BALB/c mice. However, the mechanism underlying the mice model of H. hepaticus–induced hepatocellular carcinoma is not fully known. In this study, male BALB/c mice were infected with H. hepaticus for 3, 6, 12, and 18 months. H. hepaticus colonization, histopathology, expression of proinflammatory cytokines, key signaling pathways, and protein downstream high-mobility group box-1 (HMGB1) in the liver were examined. Our data suggested that the H. hepaticus colonization level in the colon and liver progressively increased over the duration of the infection. H. hepaticus–induced hepatic inflammation and fibrosis were aggravated during the infection, and hepatic preneoplasia developed in the liver of infected mice at 12 and 18 months post-inoculation (MPI). H. hepaticus infection increased the levels of alanine aminotransferase and aspartate aminotransferase in the infected mice. In addition, the mRNA levels of IL-6, Tnf-α, Tgf-β, and HMGB1 were significantly elevated in the liver of H. hepaticus–infected mice from 3 to 18 MPI as compared to the controls. In addition, Ki67 was increased throughout the duration of the infection. Furthermore, HMGB1 protein was activated and translocated from the nucleus to the cytoplasm in the hepatocytes and activated the proteins of signal transducers and activators of transcription 3 (Stat3) and mitogen-activated protein kinase (MAPK) [extracellular regulated protein kinases 1/2 (Erk1/2) and mitogen-activated protein kinase p38 (p38)] upon H. hepaticus infection. In conclusions, these data demonstrated that male BALB/c mice infected with H. hepaticus are prone to suffering hepatitis and developing into hepatic preneoplasia. To verify the effect of HMGB1 in the progression of liver preneoplasia, mice were infected by H. hepaticus for 2 months before additional HMGB1 recombinant adenovirus treatment. All mice were sacrificed at 4 MPI, and the sera and liver tissues from all of the mice were collected. Immunology and histopathology evaluation showed that HMGB1 knockdown attenuated the H. hepaticus–induced hepatic and fibrosis at 4 MPI. Therefore, we showed that H. hepaticus–induced liver preneoplasia is closely correlated with the activation and accumulation of HMGB1.

Helicobacter hepaticus infection-induced IL-33 promotes hepatic inflammation and fibrosis through ST2 signaling pathways in BALB/c mice

It has been documented that Helicobacter hepaticus (H. hepaticus) infection is linked to hepatic inflammation and fibrosis. Interleukin 33 (IL-33) is a cytokine involved in inflammatory and fibrotic diseases, but its relevance to H. hepaticus infection-induced liver inflammation and fibrosis is unknown. In this study, we found that the expression of IL-33 in mice liver was significantly induced by H. hepaticus infection at 24 weeks post infection (WPI). Immunohistochemistry analysis revealed that IL-33 was transferred from the nucleus to the cytoplasm due to infection. The quantitation of inflammatory cytokine and histopathology evaluation showed that IL-33 knockdown attenuated the H. hepaticus-induced hepatic inflammation and fibrosis. More importantly, H. hepaticus promoted the expression of the IL-33 receptor ST2 on cell surfaces, and the expression of ST2 then activated the expression nuclear factor-κB (p65), α-SMA, and Erk1/2. These observations provide novel insights into the pathogenic mechanism of hepatic inflammation and fibrosis during H. hepaticus infection.

Helicobacter hepaticus infection induces chronic hepatitis and fibrosis in male BALB/c mice via the activation of NF-κB, Stat3, and MAPK signaling pathways.

It has been documented that Helicobacter hepaticus (Hhepaticus) infection is linked to chronic hepatitis and liver cancer. However, our understanding of the molecular mechanisms underlying progression of the Hhepaticus-induced hepatic inflammation to cellular hepatocarcinoma is still limited. In our study, male BALB/c mice were infected by Hhepaticus for 8, 12, 16, 20, and 24weeks. Histopathology, Hhepaticus colonization dynamics, select signaling pathways, and expression of key inflammatory cytokines in the liver were examined. We found that Hhepaticus was detectible in feces of mice at 7days postinfection (DPI) by PCR, but it was not detected in the livers by PCR until 8weeks postinfection (WPI). In addition, abundance of colonic and hepatic Hhepaticus was progressively increased over the infection duration. Hhepaticus-induced hepatic inflammation and fibrosis were aggravated over the infection duration, and necrosis or cirrhosis developed in the infected liver at 24 WPI Hhepaticus infection increased levels of alanine aminotransferase and aspartate aminotransferase. Moreover, mRNA levels of Il-6 and Tnf-α were significantly elevated in the livers of Hhepaticus-infected mice compared to uninfected control from 8 WPI to 24 WPI. Furthermore, Stat3, nuclear factor-κB (p65), and MAPK (Erk1/2 and p38) were activated by Hhepaticus infection. These data demonstrated that male BALB/c mice can be used as a new mouse model of Hhepaticus-induced liver diseases and that the Hhepaticus-induced liver injury is triggered by NF-κB, Jak-Stat, and MAPK signaling pathways.

Mutagenicity of Helicobacter hepaticus infection in the lower bowel mucosa of 129/SvEv Rag2-/- Il10-/- gpt delta mice is influenced by sex.

Inflammatory bowel disease and colonic tumors induced by Helicobacter hepaticus (Hh) infection in susceptible mouse strains are utilized to dissect the mechanisms underlying similar human diseases. In our study, infection with genotoxic cytolethal distending toxin-producing Hh in 129/SvEv Rag2-/- Il10-/- gpt delta (RagIl10gpt) mice of both sexes for 21 weeks induced significantly more severe cecal and colonic pathology compared to uninfected controls. The mutation frequencies in the infected RagIl10gpt males were 2.1-fold higher for the cecum and 1.7-fold higher for the colon than male RagIl10gpt controls. In addition, there was a 12.5-fold increase of G:C-to-T:A transversions in the colon of Hh-infected males compared to controls. In contrast, there was no statistical significance in mutation frequencies between infected female Rag2Il10gpt mice and controls. Moreover, Hh infection in RagIl10gpt males significantly up-regulated transcription of Tnfα and iNos, and decreased mRNA levels of cecal Atm compared to the infected females; there was no significant difference in mRNA levels of Il-22, Il-17A, Ifnγ and Atr between the infected males and females. Significantly higher levels of cecal and colonic iNos expression and γH2AX-positive epithelial cells (a biomarker for double-strand DNA breaks [DSB]) in Hh-infected Rag2Il10gpt males vs. Hh-infected females were noted. Finally, Hh infection and associated inflammation increased levels of intestinal mucosa-associated genotoxic colibactin-producing pks+ Escherichia coli. Elevated Tnfα and iNos responses and bacterial genotoxins, in concert with suppression of the DSB repair responses, may have promoted mutagenesis in the lower bowel mucosa of Hh-infected male RagIl10gpt mice.

Helicobacter hepaticus infection in BALB/c mice abolishes subunit-vaccine-induced protection against M. tuberculosis

BCG, the only licensed vaccine against tuberculosis (TB), provides geographically variable protection, an effect ascribed to exposure to environmental mycobacteria (EM). Here we show that altering the intestinal microbiota of mice by early-life infection with the commensal bacterium Helicobacter hepaticus (Hh) increases their susceptibility to challenge with Mycobacterium tuberculosis (Mtb). Furthermore Hh-infected mice immunised parenterally with the recombinant subunit vaccine, human adenovirus type 5 expressing the immunodominant antigen 85A of Mtb (Ad85A), display a reduced lung immune response and protection against Mtb challenge is also reduced. Expression of interleukin 10 (IL10) messenger RNA is increased in the colon of Hh infected mice. Treatment of Hh-infected Ad85A-immunised mice with anti-IL10 receptor antibody, following challenge with Mtb, restores the protective effect of the vaccine. These data show for the first time that alteration of the intestinal microbiota by addition of a single commensal organism can profoundly influence protection induced by a TB subunit vaccine via an IL10-dependent mechanism, a result with implications for the deployment of such vaccines in the field.

Helicobacter hepaticus infection promotes hepatitis and preneoplastic foci in farnesoid X receptor (FXR) deficient mice.

Farnesoid X receptor (FXR) is a nuclear receptor that regulates bile acid metabolism and transport. Mice lacking expression of FXR (FXR KO) have a high incidence of foci of cellular alterations (FCA) and liver tumors. Here, we report that Helicobacter hepaticus infection is necessary for the development of increased hepatitis scores and FCA in previously Helicobacter-free FXR KO mice. FXR KO and wild-type (WT) mice were sham-treated or orally inoculated with H. hepaticus. At 12 months post-infection, mice were euthanized and liver pathology, gene expression, and the cecal microbiome were analyzed. H. hepaticus induced significant increases hepatitis scores and FCA numbers in FXR KO mice (P<0.01 and P<0.05, respectively). H. hepaticus altered the beta diversity of cecal microbiome in both WT and FXR KO mice compared to uninfected mice (P<0.05). Significant upregulation of β-catenin, Rela, Slc10a1, Tlr2, Nos2, Vdr, and Cyp3a11 was observed in all FXR KO mice compared to controls (P<0.05). Importantly, H. hepaticus and FXR deficiency were necessary to significantly upregulate Cyp2b10 (P<0.01). FXR deficiency was also a potent modulator of the cecal microbiota, as observed by a strong decrease in alpha diversity. A significant decrease in Firmicutes, particularly members of the order Clostridiales, was observed in FXR KO mice (P<0.05 and FDR<5%, ANOVA). While FXR deficiency strongly affects expression of genes related to immunity and bile acid metabolism, as well as the composition of the microbiome; however, its deficiency was not able to produce significant histopathological changes in the absence of H. hepaticus infection.

Helicobacter hepaticus infection in primary hepatocellular carcinoma tissue

Helicobacter (H.) hepaticus infection causes chronic active hepatitis and induces hepatocellular tumours in A/JCr mice, but evidence of this in humans is scarce. This study aimed to demonstrate the correlation between H. hepaticus and human primary hepatocellular carcinoma (HCC). The sera of 50 patients with primary HCC were tested for the presence of anti-H. pylori and anti-H. hepaticus immunoglobulin G (IgG) antibodies. The liver tissues of patients who tested positive for serum antibody were analysed for H. hepaticus-specific 16S rRNA, H. hepaticus cdtB, H. pylori cagA, H. pylori vacA and H. pylori ureC genes using polymerase chain reaction. After the anti-H. pylori antibodies in the serum samples were absorbed by H. pylori antigen, the anti-H. hepaticus IgG serum antibody detection rate was 50.0% in patients with primary HCC. This was significantly higher (p < 0.001) than the detection rate in the benign liver tumour (7.7%) and normal liver tissue (6.3%) groups. Of the 25 primary HCC samples that tested positive for anti-H. hepaticus IgG serum antibody, the H. hepaticus-specific 16S rRNA gene was detected in nine (36.0%) samples. Sequencing showed that the polymerase chain reaction-amplified product exhibited 95.5%-100% homology to the H. hepaticus-specific 16S rRNA gene. Among these nine primary HCC tissue samples, the H. hepaticus cdtB gene was detected in four (44.4%) samples, while no such expression was observed in the benign liver tumour or normal liver tissue groups. The present study identified the presence of H. hepaticus infection in patients with primary HCC using serological and molecular biological detection, suggesting that H. hepaticus infection may be involved in the progression of HCC.

Histopathological Studies on Cases of Chronic Mouse Hepatitis by Natural &lt;i&gt;Helicobacter&lt;/i&gt; Infections

It is known that Helicobacter hepaticus or Helicobacter bilis infection causes chronic inflammation of the colon and liver. Chronic active hepatitis was found in radiation exposure experiments using male C3H/HeNrs mice at our institute. Histopathologically, 103 cases among 978 mice (64-91 weeks of age at autopsy) had hepatic lesions regardless of irradiation exposure. Mild lesions showed only focal necrosis and focal inflammation in the liver. Severe cases were accompanied by hepatocytomegaly, bile duct hyperplasia, hypertrophy and activation of Kupffer cells, cholangitis, pleomorphic hepatocytes and/or tumor. Helical-shaped bacteria were detected between hepatocytes by Warthin-Starry silver stain and immunohistochemistry (IHC) with an antibody against Helicobacter pylori. It was suggested that these cases of chronic hepatitis were caused by Helicobacter spp. Although chronic hepatitis occurred frequently in mice exposed high-dose irradiation compared with nonirradiated mice in one lot, it was not concluded that radiation might influence the incidence or degree of hepatitis. Our report suggested that natural Helicobacter spp. infection in mice can occur in an experimental animal facility. Therefore, it is suggested that monitoring of Helicobacter infection is very important for quality control of animal experiments.

Identification of a genetic locus controlling bacteria-driven colitis and associated cancer through effects on innate inflammation.

Chronic inflammation of the intestine has been associated with an elevated risk of developing colorectal cancer. Recent association studies have highlighted the role of genetic predisposition in the etiology of colitis and started to unravel its complexity. However, the genetic factors influencing the progression from colon inflammation to tumorigenesis are not known. We report the identification of a genetic interval Hiccs that regulates Helicobacter hepaticus-induced colitis and associated cancer susceptibility in a 129.RAG(-/-) mouse model. The 1.7-Mb congenic interval on chromosome 3, containing eight genes and five microRNAs, renders susceptible mice resistant to colitis and reduces tumor incidence and multiplicity. Bone marrow chimera experiments showed that resistance is conferred by the hematopoietic compartment. Moreover, the Hiccs locus controls the induction of the innate inflammatory response by regulating cytokine expression and granulocyte recruitment by Thy1(+) innate lymphoid cells. Using a tumor-promoting model combining chronic Helicobacter hepaticus infection and the carcinogen azoxymethane, we found that Hiccs also regulates the frequency of colitis-associated neoplasia. Our study highlights the importance of innate immune cells and their genetic configuration in driving progression from inflammation toward cancer and opens the door for analysis of these pathways in human inflammatory disorders and associated cancers.

Helicobacter species and liver disease

We read with great interest the recent article by Murakami et al. [1]. The study reported in the article evaluated the pathological relationship between Helicobacter hepaticus infection and gastrointestinal diseases, by measurements of serum anti-H. hepaticus antibody levels in patients with various gastrointestinal diseases. In the literature it has been reported that the presence of abnormal intestinal permeability in patients with chronic liver disease can result in the increased exposure of gut-derived antigens, including microbial antigens, to the immune system and specifically to the liver [2]. In this way Helicobacter species have also been proposed to be implicated in the pathogenesis of primary biliary cirrhosis (PBC), because their DNA has been found in liver tissue, and antibodies to the microbe have been found in the serum and the bile of patients with PBC [3]. In addition, PBC is frequently associated with other autoimmune disorders; in particular, with celiac disease (CD). There are several common pathogenetic mechanisms that occur in these diseases and they include increased intestinal permeability, which is an early abnormality in CD and has also been demonstrated in PBC. Did Murakami et al. [1]detect CD blood markers in their PBC patients?

Strain-specific colitis susceptibility in IL10-deficient mice depends on complex gut microbiota–host interactions

Colitis susceptibility in Il10(-/-) mice depends on genetic background and microbiota composition. A major genetic locus mediating colitis susceptibility, Cdcs1, was transferred from susceptible C3Bir-Il10(-/-) to resistant B6-Il10(-/-) mice, resulting in susceptible congenic BC-R3-Il10(-/-) mice. The aim of this study was to determine the impact of microbiota on this differential colitis susceptibility using a Helicobacter hepaticus infection model. Parental C3Bir-Il10(-/-) , B6-Il10(-/-) , and congenic BC-R3-Il10(-/-) mice were inoculated with H. hepaticus and analyzed for inflammation. In parental Il10(-/-) mice, microbiota composition was determined by terminal restriction fragment length polymorphism (T-RFLP) and quantitative polymerase chain reaction (qPCR). Most severe inflammation was observed in C3Bir-Il10(-/-) in the cecum, in BC-R3-Il10(-/-) in cecum and colon, and, unexpectedly, in B6-Il10(-/-) in the colon. C3Bir-Il10(-/-) and BC-R3-Il10(-/-) secreted significantly more interferon-gamma (IFNγ) and interleukin (IL)17 than B6-Il10(-/-) . T-RFLP analyses in C3Bir-Il10(-/-) and B6-Il10(-/-) mice revealed 1) a significant impact of H. hepaticus infection on species richness and diversity, and 2) strain differences in microbiota composition only after H. hepaticus infection. qPCR revealed higher numbers of Clostridia leptum and Bacteroides spp. in the cecum of infected C3Bir-Il10(-/-) mice, and Lactobacillus spp. in B6-Il10(-/-) mice. Cdcs1 modifies the response to H. hepaticus infection. However, this infection alone does not reflect the original response to a complex colitogenic biota. H. hepaticus-induced inflammation altered intestinal microbiota in a mouse strain-specific manner. Bacteroides spp. became more abundant in susceptible C3Bir-Il10(-/-) , lactobacilli in B6-Il10(-/-) mice. Therefore, both host immune response and differential compositional changes of microbiota play a role in strain-specific colitis susceptibility in Il10(-/-) mice.

Serodiagnosis of Helicobacter hepaticus infection in patients with liver and gastrointestinal diseases: western blot analysis and ELISA using a highly specific monoclonal antibody for H. hepaticus antigen

Helicobacter hepaticus infection might be associated with liver and biliary tract diseases. To investigate its pathogenic role, the properties of anti-H. hepaticus serum antibody in patients with liver and diseases were elucidated. Serum samples were collected from 166 patients-69 with liver diseases, 38 with upper gastrointestinal diseases, 17 with lower gastrointestinal diseases, 26 with biliary tract diseases, and 16 with pancreas diseases; 30 control sera were obtained from 30 healthy blood donors. Serum samples were analyzed by enzyme-linked immunosorbent assay (ELISA) and western blot using the new monoclonal antibody HR II-51. Anti-H. hepaticus serum antibody concentrations in patients with liver disease (n=69) were significantly increased compared with those in other disease groups (p=0.014 to <0.001). Particularly, liver cirrhosis (n=19) showed a significantly higher antibody level compared with other liver diseases (n=50, p=0.005) and healthy donors (n=30, p=0.0005), as well as a higher seroprevalence (68.4%) compared with other liver diseases (p=0.05) and healthy donors (p=0.004). Furthermore, the ELISA value in liver cirrhosis (n=19) was significantly higher than that in patients with hepatitis B virus (HBV)-and/or hepatitis C virus (HCV)-infected chronic hepatitis (n=15) (0.389±0.084 vs. 0.350±0.084, p=0.029). However, there was no relationship between the total immunoglobulin concentration and the anti-H. hepaticus antibody level in each liver disease (Spearman's rank correlation coefficient [rs]<0.225). H. hepaticus infection might play a role in the development of liver diseases; in particular, it might increase the risk of the development of HBV- and/or HCV-infected liver diseases.

SMAD3 deficiency causes immune dysregulation, colitis and colon tumorigenesis (66.13)

Abstract Introduction: TGFβ plays important role in T cell development, differentiation, tolerance and autoimmune diseases. However, recent studies have shown an important role for TGF-β1 and β3 in pathogenic helper T-cell (Th17) differentiation in the presence of IL-1β, IL-6 and IL-23. SMAD3 is mediates TGFβ signaling in many cell types including lymphocytes. Smad3 /- mice develop inflammation-induced colon cancer upon Helicobacter hepaticus infection suggesting that TGFβ1 signaling through SMAD3 is essential to prevent inflammation and progression to colon cancer. In this study we investigated the role of SMAD3 in inflammation using mice deficient in Smad3. Methods: Splenocytes are cultured in the presence of anti-CD3/CD28 beads, TGFβ1 and IL-2 for 3 days. Cytokines are determined by ELISA, CBA and MSD methods. Cells are stained with antibodies for CD4, CD25, CD62L and intracellular FOXP3 and Helios. Results: Smad3-/- mice exhibit inflammatory response upon infection with commensal bacterium H. hepaticus. We find that while SMAD3 signaling is important for inducible Treg cell generation and for their maintenance, deficiency of SMAD3 increases IL-6 and IL-17 levels suggesting a regulatory role for SMAD3 in Th17 cell differentiation. Conclusions: While SMAD3 signaling is important for iTreg cell generation and maintenance, it is not required for Th17 cell differentiation. In addition, we find that SMAD3 signaling negatively regulates Th17 differentiation through induction of FOXP3.

Natural Helicobacter infection modulates mouse intestinal muscularis macrophage responses

Helicobacter species are common laboratory pathogens which induce intestinal inflammation and disease in susceptible mice. Since in vitro studies indicate that Helicobacter products activate macrophages, we hypothesized that in vivo Helicobacter infection regulates the inflammatory response of intestinal muscularis macrophages from C57Bl/6 mice. Helicobacter hepaticus infection increased surface expression of macrophage markers F4/80, CD11b and MHC-II within whole intestinal muscle mounts. However, constitutive cytokine and chemokine production by macrophages isolated from infected mice significantly decreased compared to macrophages from uninfected mice despite no detectable bacterial products in the cultures. In addition, muscularis macrophages from infected mice up-regulated FIZZ-1 and SK-1 gene expression, suggesting the macrophages had an anti-inflammatory phenotype. Corresponding with increased anti-inflammatory gene expression, macrophages from infected mice were more phagocytic but did not produce cytokines after stimulation with LPS and IFN-γ or immune complexes and IL-4. Therefore, the presence of Helicobacter infection matures intestinal muscularis macrophages, modulating the constitutive macrophage response to become more anti-inflammatory and resistant to secondary stimulation.

Hemorrhage-induced intestinal damage is complement-independent in Helicobacter hepaticus-infected mice.

With more than half of the world population infected, Helicobacter infection is an important public health issue associated with gastrointestinal cancers and inflammatory bowel disease. Animal studies indicate that complement and oxidative stress play a role in Helicobacter infections. Hemorrhage (HS) induces tissue damage that is attenuated by blockade of either complement activation or oxidative stress products. Therefore, we hypothesized that chronic Helicobacter hepaticus infection would modulate HS-induced intestinal damage and inflammation. To test this hypothesis, we examined HS-induced jejunal damage and inflammation in uninfected and H. hepaticus-infected mice. Helicobacter hepaticus infection increased HS-induced midjejunal mucosal damage despite attenuating complement activation. In addition, infection alone increased chemokine secretion, changing the HS-induced neutrophil infiltration to a macrophage-mediated inflammatory response. The HS-induced macrophage infiltration correlated with increased secretion of tumor necrosis factor-α and nitric oxide in the infected mice. Together, these data indicate that Helicobacter infection modulates the mechanism of HS-induced intestinal damage and inflammation from a complement-mediated response to a macrophage response with elevated tumor necrosis factor-α and nitric oxide. These data indicate that chronic low-level infections change the response to trauma and should be considered when designing and administering therapeutics.

Helicobacter hepaticus infection in mice: models for understanding lower bowel inflammation and cancer.

Pioneering work in the 1990s first linked a novel microaerobic bacterium, Helicobacter hepaticus, with chronic active hepatitis and inflammatory bowel disease in several murine models. Targeted H. hepaticus infection experiments subsequently demonstrated its ability to induce colitis, colorectal cancer, and extraintestinal diseases in a number of mouse strains with defects in immune function and/or regulation. H. hepaticus is now widely utilized as a model system to dissect how intestinal microbiota interact with the host to produce both inflammatory and tolerogenic responses. This model has been used to make important advances in understanding factors that regulate both acquired and innate immune response within the intestine. Further, it has been an effective tool to help define the function of regulatory T cells, including their ability to directly inhibit the innate inflammatory response to gut microbiota. The complete genomic sequence of H. hepaticus has advanced the identification of several virulence factors and aided in the elucidation of H. hepaticus pathogenesis. Delineating targets of H. hepaticus virulence factors could facilitate novel approaches to treating microbially induced lower bowel inflammatory diseases.