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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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Intestinal immune responses to microbes are controlled by the cytokine IL-10 to avoid immune pathology. Here, we use single-cell RNA sequencing of colon lamina propria leukocytes (LPLs) along with RNA-seq and ATAC-seq of purified CD4 + T cells to show that the transcription factors Blimp-1 (encoded by Prdm1) and c-Maf co-dominantly regulate Il10 while negatively regulating proinflammatory cytokines in effector T cells. Double-deficient Prdm1 fl/fl Maf fl/fl Cd4 Cre mice infected with Helicobacter hepaticus developed severe colitis with an increase in T H 1/NK/ILC1 effector genes in LPLs, while Prdm1 fl/fl Cd4 Cre and Maf fl/fl Cd4 Cre mice exhibited moderate pathology and a less-marked type 1 effector response. LPLs from infected Maf fl/fl Cd4 Cre mice had increased type 17 responses with increased Il17a and Il22 expression and an increase in granulocytes and myeloid cell numbers, resulting in increased T cell-myeloid-neutrophil interactions. Genes over-expressed in human inflammatory bowel disease showed differential expression in LPLs from infected mice in the absence of Prdm1 or Maf, revealing potential mechanisms of human disease., (© 2024. The Author(s).)

The intestinal immune system is highly adapted to maintaining tolerance to the commensal microbiota and self-antigens while defending against invading pathogens 1,2 . Recognizing how the diverse network of local cells establish homeostasis and maintains it in the complex immune environment of the gut is critical to understanding how tolerance can be re-established following dysfunction, such as in inflammatory disorders. Although cell and molecular interactions that control T regulatory (T reg ) cell development and function have been identified 3,4 , less is known about the cellular neighbourhoods and spatial compartmentalization that shapes microorganism-reactive T reg cell function. Here we used in vivo live imaging, photo-activation-guided single-cell RNA sequencing 5-7 and spatial transcriptomics to follow the natural history of T cells that are reactive towards Helicobacter hepaticus through space and time in the settings of tolerance and inflammation. Although antigen stimulation can occur anywhere in the tissue, the lamina propria-but not embedded lymphoid aggregates-is the key microniche that supports effector T reg (eT reg ) cell function. eT reg cells are stable once their niche is established; however, unleashing inflammation breaks down compartmentalization, leading to dominance of CD103 + SIRPα + dendritic cells in the lamina propria. We identify and validate the putative tolerogenic interaction between CD206 + macrophages and eT reg cells in the lamina propria and identify receptor-ligand pairs that are likely to govern the interaction. Our results reveal a spatial mechanism of tolerance in the lamina propria and demonstrate how knowledge of local interactions may contribute to the next generation of tolerance-inducing therapies., (© 2024. The Author(s).)

In recent years, an increasing number of studies have reported that intestinal microbiota have an important effect on tumour immunity by affecting the tumour microenvironment (TME). The intestinal microbiota are closely associated with various immune cells, such as T lymphocytes, natural killer cells (NK cells) and macrophages. Some bacteria, such as Akkermansia muciniphila (A. muciniphila) and Lactobacillus reuteri (L. reuteri), have been shown to improve the effect of tumour immunity. Furthermore, microbial imbalance, such as the increased abundance of Fusobacterium nucleatum (F. nucleatum) and Helicobacter hepaticus (H. hepaticus), generally causes tumour formation and progression. In addition, some microbiota also play important roles in tumour immunotherapy, especially PD-L1-related therapies. Therefore, what is the relationship between these processes and how do they affect each other? In this review, we summarize the interactions and corresponding mechanisms among the intestinal microbiota, immune system and TME to facilitate the research and development of new targeted drugs and provide new approaches to tumour therapy., (© 2021 The Authors. Immunology published by John Wiley & Sons Ltd.)

Gut microbiota and the immune system are in constant exchange shaping both host immunity and microbial communities. Here, improper immune regulation can cause inflammatory bowel disease (IBD) and colitis. Antibody therapies blocking signaling through the CD40-CD40L axis showed promising results as these molecules are deregulated in certain IBD patients. To better understand the mechanism, we used transgenic DC-LMP1/CD40 animals with a constitutive CD40-signal in CD11c + cells, causing a lack of intestinal CD103 + dendritic cells (DCs) and failure to induce regulatory T (iTreg) cells. These mice rapidly develop spontaneous fatal colitis, accompanied by dysbiosis and increased inflammatory IL-17 + IFN-γ + Th17/Th1 and IFN-γ + Th1 cells. In the present study, we analyzed the impact of the microbiota on disease development and detected elevated IgA- and IgG-levels in sera from DC-LMP1/CD40 animals. Their serum antibodies specifically bound intestinal bacteria, and by proteome analysis, we identified a 60 kDa chaperonin GroEL (Hsp60) from Helicobacter hepaticus ( Hh ) as the main specific antigen targeted in the absence of iTregs. When re-derived to a different Hh -free specific-pathogen-free (SPF) microbiota, mice showed few signs of disease, normal microbiota, and no fatality. Upon recolonization of mice with Hh , the disease developed rapidly. Thus, the present work identifies GroEL/Hsp60 as a major Hh -antigen and its role in disease onset, progression, and outcome in this colitis model. Our results highlight the importance of CD103 + DC- and iTreg-mediated immune tolerance to specific pathobionts to maintain healthy intestinal balance.

Mononuclear phagocytes (MNPs) are vital for maintaining intestinal homeostasis but, in response to acute microbial stimulation, can also trigger immunopathology, accelerating recruitment of Ly6C hi monocytes to the gut. The regulators that control monocyte tissue adaptation in the gut remain poorly understood. Interferon regulatory factor 5 (IRF5) is a transcription factor previously shown to play a key role in maintaining the inflammatory phenotype of macrophages. Here, we investigate the impact of IRF5 on the MNP system and physiology of the gut at homeostasis and during inflammation. We demonstrate that IRF5 deficiency has a limited impact on colon physiology at steady state but ameliorates immunopathology during Helicobacter hepaticus -induced colitis. Inhibition of IRF5 activity in MNPs phenocopies global IRF5 deficiency. Using a combination of bone marrow chimera and single-cell RNA-sequencing approaches, we examined the intrinsic role of IRF5 in controlling colonic MNP development. We demonstrate that IRF5 promotes differentiation of Ly6C hi monocytes into CD11c + macrophages and controls the production of antimicrobial and inflammatory mediators by these cells. Thus, we identify IRF5 as a key transcriptional regulator of the colonic MNP system during intestinal inflammation., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Study Question: Does the genotype of the surrogate mother modulate the body composition and immunity of her offspring?, Summary Answer: C57BL/6J (B6) progenies carried by immunodeficient NOD SCID (NS) mothers had increased adaptive but decreased innate, immune responsiveness in comparison with the same genotype offspring carried by immunocompetent mothers, B6 and BALB/c (C); the B6 progenies carried by the same genotype mothers also showed higher body fat than the others., What Is Known Already: Differences in the major histocompatibility complex (MHC) genes between mother and foetus is considered as an important factor in prenatal embryo development, whereas the impact of such dissimilarity on the phenotype of the mature progeny is unclear., Study Design, Size, Duration: Transplantation of two-cell mouse embryos into recipient females of the different MHC (H2) genotypes was used as an approach to simulate three variants of the immunogenic mother-foetus interaction: (i) bidirectional immunogenic dialogue between B6 (H2b haplotype) embryos and C (H2d haplotype) surrogate mother; (ii) one-way immunogenic interaction between B6 embryos and immunodeficient NS (H2g7 haplotype) surrogate mother and (iii) reduced immunogenetic dialogue between embryos and surrogate mother of the same H2b haplotype resulting in only a maternal response to HY antigens of male foetuses. Delivered by Caesarean section, pups were fostered by lactating B6 females and weighed after weaning (n = 171). Body mass and composition and innate and adaptive immunity were assessed in selected progeny groups at 9-11 weeks of age., Participants/materials, Setting, Methods: The study was performed on the specific pathogen-free mouse, inbred strains C57BL/6J, NOD SCID and BALB/c. Plasma progesterone in pregnant females was measured by enzyme-linked immunosorbent assay (ELISA). Body composition was determined by magnetic resonance spectroscopy using a low-field NMR spectrometer (EchoMRI, USA). To assess peritoneal macrophage responses (innate immunity) to anthrax, lactate dehydrogenase (LDH) and interleukin-1 (IL-1β) were measured in a culture medium 24 h after the addition of both anthrax-lethal factor and anthrax-protective antigen. To assess adaptive immunity, 9-10 males in experimental groups were infected with Helicobacter hepaticus. Faeces collected 2 and 4 weeks after infection was used for quantitative assessment of the H. hepaticus DNA by real-time polymerase chain reaction. IgA, interferon (IFN-γ), tumour necrosis factor (TNFα), interleukin-17 (IL-17) and interleukin-10 (IL-10) in colon tissue and IgG in serum were determined in samples collected 4 weeks after gavage with H. hepaticus using ELISA. For statistical analyses, ANCOVA, post hoc least significant difference (LSD) test, Student's t-test, Spearman rank correlations and χ2 test were performed. P-value <0.05 was considered as a statistically significant difference., Main Results and the Role of Chance: ANCOVA with litter size and age as covariates revealed significant effects of the surrogate mother genotype on body mass and percent of fat in their adult progeny (F2149 = 15.60, P < 0.001 and F2149 = 5.02, P = 0.007, respectively). Adult B6 mice carried by B6 surrogate mothers were characterized by a higher percentage of body fat in comparison with offspring that were carried by NS and C females. In comparison with the male offspring carried by the B6 and C mothers, male B6 progenies carried by immunodeficient NS mothers had a higher humoral immune response (serum IgG) against oral infection with H. hepaticus, but lower in vitro macrophage IL-1β reaction to the anthrax. Four weeks after the infection of offspring, concentrations of serum IgG and colon IL-10 correlated positively with maternal progesterone on Day 4 after embryo transfer and negatively with DNA of H. hepaticus. One-way ANOVA confirmed a statistically significant impact of surrogate mother genotype on adaptive (IgG) and innate (IL-1β) immunity (F2.26 = 26.39, P < 0.001 and F2.27 = 5.89, P = 0.008, respectively)., Large Scale Data: N/A., Limitations, Reasons for Caution: The main limitation of our study is the number of combinations of mother and foetus interactions, in particular, transfer of only one embryo genotype was used. Also, it is a descriptive study, which requires further analysis of the epigenetic mechanisms of the observed phenotypic effects of surrogate mother genotype., Wider Implications of the Findings: Our experimental data demonstrate that the transfer of inbred embryos to surrogate mothers of the different genotypes is a prospective experimental model for the study of epigenetic effects of the immunogenetic interactions between mother and foetus. The experimental approach tested in our study will be in demand for the development of criteria for choosing surrogate mothers. In particular, immunocompetence of the surrogate mother along with genetic distance of her MHC alleles to the transferred embryos have a significant impact on offspring development., Study Funding/competing Interest(s): This work was supported by the Russian FPI (6/099/2017), budget projects (0324-2016-0002 and 0324-2018-0016) and implemented using the equipment of the Centre for Genetic Resources of Laboratory Animals at ICG SB RAS, supported by the Ministry of Education and Science of Russia (Unique project identifier RFMEFI62117X0015). The authors report no conflicts of interest.

Inflammatory bowel disease (IBD) are heterogenous disorders of the gastrointestinal tract caused by a spectrum of genetic and environmental factors. In mice, overlapping regions of chromosome 3 have been associated with susceptibility to IBD-like pathology, including a locus called Hiccs. However, the specific gene that controls disease susceptibility remains unknown. Here we identify a Hiccs locus gene, Alpk1 (encoding alpha kinase 1), as a potent regulator of intestinal inflammation. In response to infection with the commensal pathobiont Helicobacter hepaticus (Hh), Alpk1-deficient mice display exacerbated interleukin (IL)-12/IL-23 dependent colitis characterized by an enhanced Th1/interferon(IFN)-γ response. Alpk1 controls intestinal immunity via the hematopoietic system and is highly expressed by mononuclear phagocytes. In response to Hh, Alpk1 -/- macrophages produce abnormally high amounts of IL-12, but not IL-23. This study demonstrates that Alpk1 promotes intestinal homoeostasis by regulating the balance of type 1/type 17 immunity following microbial challenge.

Chronic inflammation contributes to tumor initiation in colitis-associated colorectal cancer (CRC). Indeed, inflammatory bowel disease (IBD) patients show an increased risk of developing CRC. Cancer immune evasion is a major issue in CRC and preclinical and clinical evidence has defined a critical role for myeloid-derived suppressor cells (MDSCs) that contribute to tumor growth and progression by suppressing T-cells and modulating innate immune responses. MDSCs comprise a heterogeneous population of immature myeloid cells that can be distinct in two subtypes: CD11b + Ly6G + Ly6C low with granulocytic phenotype (G-MDSCs) and CD11b + Ly6G - Ly6C high with monocytic phenotype (M-MDSCs). Hydrogen sulfide (H 2 S) is an endogenous gaseous signaling molecule that regulates various physiological and pathophysiological functions. In particular, several studies support its anti-inflammatory activity in experimental colitis and ulcer. However, the role of the H 2 S pathway in innate immune-mediated IBD has not yet been elucidated. To better define a possible link between MDSCs and H 2 S pathway in colitis-associated CRC development, we used an innate immune-mediated IBD model induced by infection with the bacterium Helicobacter hepaticus ( Hh ), closely resembling human IBD. Here, we demonstrated an involvement of MDSCs in colitis development. A significant time-dependent increase of both G-MDSCs and M-MDSCs was observed in the colon and in the spleen of Hh -infected mice. Following, we observed that chronic oral administration of the H 2 S donor DATS reduced colon inflammation by limiting the recruitment of G-MDSCs in the colon of Hh -infected mice. Thus, we identify the metabolic pathway l-cysteine/H 2 S as a possible new player in the immunosuppressive mechanism responsible for the MDSCs-promoted colitis-associated cancer development.

Both microbial and host genetic factors contribute to the pathogenesis of autoimmune diseases. There is accumulating evidence that microbial species that potentiate chronic inflammation, as in inflammatory bowel disease, often also colonize healthy individuals. These microorganisms, including the Helicobacter species, can induce pathogenic T cells and are collectively referred to as pathobionts. However, how such T cells are constrained in healthy individuals is not yet understood. Here we report that host tolerance to a potentially pathogenic bacterium, Helicobacter hepaticus, is mediated by the induction of RORγt + FOXP3 + regulatory T (iT reg ) cells that selectively restrain pro-inflammatory T helper 17 (T H 17) cells and whose function is dependent on the transcription factor c-MAF. Whereas colonization of wild-type mice by H. hepaticus promoted differentiation of RORγt-expressing microorganism-specific iT reg cells in the large intestine, in disease-susceptible IL-10-deficient mice, there was instead expansion of colitogenic T H 17 cells. Inactivation of c-MAF in the T reg cell compartment impaired differentiation and function, including IL-10 production, of bacteria-specific iT reg cells, and resulted in the accumulation of H. hepaticus-specific inflammatory T H 17 cells and spontaneous colitis. By contrast, RORγt inactivation in T reg cells had only a minor effect on the bacteria-specific T reg and T H 17 cell balance, and did not result in inflammation. Our results suggest that pathobiont-dependent inflammatory bowel disease is driven by microbiota-reactive T cells that have escaped this c-MAF-dependent mechanism of iT reg -T H 17 homeostasis.

Interactions between the host and its microbiota are of mutual benefit and promote health. Complex molecular pathways underlie this dialog, but the identity of microbe-derived molecules that mediate the mutualistic state remains elusive. Helicobacter hepaticus is a member of the mouse intestinal microbiota that is tolerated by the host. In the absence of an intact IL-10 signaling, H. hepaticus induces an IL-23-driven inflammatory response in the intestine. Here we investigate the interactions between H. hepaticus and host immune cells that may promote mutualism, and the microbe-derived molecule(s) involved. Our results show that H. hepaticus triggers early IL-10 induction in intestinal macrophages and produces a large soluble polysaccharide that activates a specific MSK/CREB-dependent anti-inflammatory and repair gene signature via the receptor TLR2. These data identify a host-bacterial interaction that promotes mutualistic mechanisms at the intestinal interface. Further understanding of this pathway may provide novel prevention and treatment strategies for inflammatory bowel disease., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The risk of colon cancer is increased in patients with Crohn's disease and ulcerative colitis. Inflammation-induced DNA damage could be an important link between inflammation and cancer, although the pathways that link inflammation and DNA damage are incompletely defined. RAG2-deficient mice infected with Helicobacter hepaticus (Hh) develop colitis that progresses to lower bowel cancer. This process depends on nitric oxide (NO), a molecule with known mutagenic potential. We have previously hypothesized that production of NO by macrophages could be essential for Hh-driven carcinogenesis, however, whether Hh infection induces DNA damage in this model and whether this depends on NO has not been determined. Here we demonstrate that Hh infection of RAG2-deficient mice rapidly induces expression of iNOS and the development of DNA double-stranded breaks (DSBs) specifically in proliferating crypt epithelial cells. Generation of DSBs depended on iNOS activity, and further, induction of iNOS, the generation of DSBs, and the subsequent development of dysplasia were inhibited by depletion of the Hh-induced cytokine IL-22. These results demonstrate a strong association between Hh-induced DNA damage and the development of dysplasia, and further suggest that IL-22-dependent induction of iNOS within crypt epithelial cells rather than macrophages is a driving force in this process.

Objectives: Although n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFAs) are used widely in the treatment of chronic inflammatory diseases, their effect in infectious disease requires a particular attention., Methods: The present article discusses their anti-inflammatory and immune properties involved in the host defence and presents a systematic review of the effects of their oral administration on the prevention and outcome of experimental and clinical infections., Results: At a dose corresponding to an human dose of 500 mg/day, n-3 LC-PUFAs intake is beneficial against experimental infections caused by extracellular pathogens including Streptococcus pneumoniae, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus by reducing inflammation, and reduces the incidence of pneumococcal infections in the elderly, but at 2-4-fold higher doses as occurs in some human intervention and/or during long-term it becomes detrimental in intestinal infections with Citrobacter rodentium or Helicobacter hepaticus by exacerbating anti-inflammatory response. They are also harmful against infections caused by intracellular pathogens as Mycobacterium tuberculosis, Salmonella, Influenza virus and Herpes simplex virus by affecting the immune cell response., Conclusion: The effects of n-3-LC-PUFAs on infections depend on the pathogen and the n-3 LC-PUFA dose and timing. Caution should be recommended for high-dose and long-term supplementation in humans., (Copyright © 2016 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

The discovery of Th17 cell plasticity, in which CD4(+) IL-17-producing Th17 cells give rise to IL-17/IFN-γ double-producing cells and Th1-like IFNγ(+) ex-Th17 lymphocytes, has raised questions regarding which of these cell types contribute to immunopathology during inflammatory diseases. In this study, we show using Helicobacter hepaticus-induced intestinal inflammation that IL-17A(Cre)- or Rag1(Cre)-mediated deletion of Tbx21 has no effect on the generation of IL-17/IFN-γ double-producing cells, but leads to a marked absence of Th1-like IFNγ(+) ex-Th17 cells. Despite the lack of Th1-like ex-Th17 cells, the degree of H. hepaticus-triggered intestinal inflammation in mice in which Tbx21 was excised in IL-17-producing or Rag1-expressing cells is indistinguishable from that observed in control mice. In stark contrast, using experimental autoimmune encephalomyelitis, we show that IL-17A(Cre)-mediated deletion of Tbx21 prevents the conversion of Th17 cells to IL-17A/IFN-γ double-producing cells as well as Th1-like IFN-γ(+) ex-Th17 cells. However, IL-17A(Cre)-mediated deletion of Tbx21 has only limited effects on disease course in this model and is not compensated by Ag-specific Th1 cells. IL-17A(Cre)-mediated deletion of Rorc reveals that RORγt is essential for the maintenance of the Th17 cell lineage, but not immunopathology during experimental autoimmune encephalomyelitis. These results show that neither the single Th17 subset, nor its progeny, is solely responsible for immunopathology or autoimmunity., (Copyright © 2016 The Authors.)

Although IL-10 promotes a regulatory phenotype of CD11c+ dendritic cells and macrophages in vitro, the role of IL-10 signaling in CD11c+ cells to maintain intestinal tolerance in vivo remains elusive. To this aim, we generated mice with a CD11c-specific deletion of the IL-10 receptor alpha (Cd11ccreIl10rafl/fl). In contrast to the colon, the small intestine of Cd11ccreIl10rafl/fl mice exhibited spontaneous crypt hyperplasia, increased numbers of intraepithelial lymphocytes and lamina propria T cells, associated with elevated levels of T cell-derived IFNγ and IL-17A. Whereas naive mucosal T-cell priming was not affected and oral tolerance to ovalbumin was intact, augmented T-cell function in the lamina propria was associated with elevated numbers of locally dividing T cells, expression of T-cell attracting chemokines and reduced T-cell apoptosis. Upon stimulation, intestinal IL-10Rα deficient CD11c+ cells exhibited increased activation associated with enhanced IL-6 and TNFα production. Following colonization with Helicobacter hepaticus Cd11ccreIl10rafl/fl mice developed severe large intestinal inflammation characterized by infiltrating T cells and increased levels of Il17a, Ifng, and Il12p40. Altogether these findings demonstrate a critical role of IL-10 signaling in CD11c+ cells to control small intestinal immune homeostasis by limiting reactivation of local memory T cells and to protect against Helicobacter hepaticus-induced colitis., Competing Interests: The authors declare no conflicts of interests.

In inflammatory bowel diseases, a breakdown in host microbial interactions accompanies sustained activation of immune cells in the gut. Functional studies suggest a key role for interleukin-23 (IL-23) in orchestrating intestinal inflammation. IL-23 can be produced by various mononuclear phagocytes (MNPs) following acute microbial stimulation, but little is known about the key cellular sources of IL-23 that drive chronic intestinal inflammation. Here we have addressed this question using a physiological model of bacteria-driven colitis. By combining conditional gene ablation and gene expression profiling, we found that IL-23 production by CD11c(+) MNPs was essential to trigger intestinal immunopathology and identified MHCII(+) monocytes and macrophages as the major source of IL-23. Expression of IL-23 by monocytes was acquired during their differentiation in the intestine and correlated with the expression of major histocompatibility complex class II (MHCII) and CD64. In contrast, Batf3-dependent CD103(+) CD11b(-) dendritic cells were dispensable for bacteria-induced colitis in this model. These studies reinforce the pathogenic role of monocytes in dysregulated responses to intestinal bacteria and identify production of IL-23 as a key component of this response. Further understanding of the functional sources of IL-23 in diverse forms of intestinal inflammation may lead to novel therapeutic strategies aimed at interrupting IL-23-driven immune pathology.

In space, the lifestyle, relative sterility of spaceship and extreme environmental stresses, such as microgravity and cosmic radiation, can compromise the balance between human body and human microbiome. An astronaut's body during spaceflight encounters increased risk for microbial infections and conditions because of immune dysregulation and altered microbiome, i.e. dysbiosis. This risk is further heightened by increase in virulence of pathogens in microgravity. Health status of astronauts might potentially benefit from maintaining a healthy microbiome by specifically managing their diet on space in addition to probiotic therapies. This review focuses on the current knowledge/understanding of how spaceflight affects human immunity and microbiome.

It is clear that IL-10 plays an essential role in maintaining homeostasis in the gut in response to the microbiome. However, it is unknown whether IL-10 also facilitates immune homeostasis at distal sites. To address this question, we asked whether splenic immune populations were altered in IL-10-deficient (Il10(-/-)) mice in which differences in animal husbandry history were associated with susceptibility to spontaneous enterocolitis that is microbiome dependent. The susceptible mice exhibited a significant increase in splenic macrophages, neutrophils, and marginal zone (MZ) B cells that was inhibited by IL-10 signaling in myeloid, but not B cells. The increase in macrophages was due to increased proliferation that correlated with a subsequent enhancement in MZ B cell differentiation. Cohousing and antibiotic treatment studies suggested that the alteration in immune homeostasis in the spleen was microbiome dependent. The 16S rRNA sequencing revealed that susceptible mice harbored a different microbiome with a significant increase in the abundance of the bacterial genus Helicobacter. The introduction of Helicobacter hepaticus to the gut of nonsusceptible mice was sufficient to drive macrophage expansion and MZ B cell development. Given that myeloid cells and MZ B cells are part of the first line of defense against blood-borne pathogens, their increase following a breach in the gut epithelial barrier would be protective. Thus, IL-10 is an essential gatekeeper that maintains immune homeostasis at distal sites that can become functionally imbalanced upon the introduction of specific pathogenic bacteria to the intestinal track., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Recent studies suggest that gastrointestinal tract microbiota modulate cancer development in distant non-intestinal tissues. Here we tested mechanistic hypotheses using a targeted pathogenic gut microbial infection animal model with a predilection to breast cancer. FVB-Tg(C3-1-TAg)cJeg/JegJ female mice were infected by gastric gavage with Helicobacter hepaticus at three-months-of-age putting them at increased risk for mammary tumor development. Tumorigenesis was multifocal and characterized by extensive infiltrates of myeloperoxidase-positive neutrophils otherwise implicated in cancer progression in humans and animal models. To test whether neutrophils were important in etiopathogenesis in this bacteria-triggered model system, we next systemically depleted mice of neutrophils using thrice weekly intraperitoneal injections with anti-Ly-6G antibody. We found that antibody depletion entirely inhibited tumor development in this H. hepaticus-infected model. These data demonstrate that host neutrophil-associated immune responses to intestinal tract microbes significantly impact cancer progression in distal tissues such as mammary glands, and identify gut microbes as novel targets for extra-intestinal cancer therapy.

Inflammatory bowel diseases (IBD) represent idiopathic chronic inflammatory disorders of the intestinal tract that are associated with aberrant immune responses against intestinal bacteria. Here, we describe two T cell-dependent models of experimental murine IBD. In the "T cell transfer" model, lymphopenic (scid or Rag (-/-) ) mice develop colitis upon adoptive transfer of naïve CD4(+) T cells. This model has also been extensively employed to identify mechanisms through which CD4(+)CD25(+) regulatory T cells suppress intestinal inflammation in vivo. We also describe a model of T cell-dependent IBD in immunocompetent mice, induced by infection with the intestinal bacterium Helicobacter hepaticus and concomitant treatment with a blocking αIL-10R mAb, which leads to the development of chronic inflammation of the caecum and colon (typhlocolitis). Both models reproduce many facets of human IBD pathology, including epithelial hyperplasia, goblet cell depletion, and leukocyte infiltration. These models provide reliable and tractable systems for the analyses of the induction and regulation of chronic inflammation in the gut.

Bacterial-induced intestinal inflammation is crucially dependent on interleukin (IL)-23 and is associated with CD4(+) T helper type 1 (Th1) and Th17 responses. However, the relative contributions of these subsets during the induction and resolution of colitis in T-cell-sufficient hosts remain unknown. We report that Helicobacter hepaticus-induced typhlocolitis in specific pathogen-free IL-10(-/-) mice is associated with elevated frequencies and numbers of large intestinal interferon (IFN)-γ(+) and IFN-γ(+)IL-17A(+) CD4(+) T cells. By assessing histone modifications and transcript levels in IFN-γ(+), IFN-γ(+)IL-17A(+), and IL-17A(+) CD4(+) T cells isolated from the inflamed intestine, we show that Th17 cells are predisposed to upregulate the Th1 program and that they express IL-23R but not IL-12R. Using IL-17A fate-reporter mice, we further demonstrate that H. hepaticus infection gives rise to Th17 cells that extinguish IL-17A secretion and turn on IFN-γ within 10 days post bacterial inoculation. Together, our results suggest that bacterial-induced Th17 cells arising in disease-susceptible hosts contribute to intestinal pathology by switching phenotype, transitioning via an IFN-γ(+)IL-17A(+) stage, to become IFN-γ(+) ex-Th17 cells.

Objective: To prepare the polyclonal antibody against methyl-accepting chemotaxis signal transduction protein (MCP) of Helicobacter hepaticus (H.hepaticus)., Methods: The recombinant plasmid pET22b+/MCP was transformed into E.coli BL2l(DE3) to express the fusion protein His-rhMCP under the induction of IPTG. The fusion protein was purified and the antibody was obtained by immunizing rabbits. The titer of the polyclonal antibody was tested by indirect ELISA, and the specificity of the antibody was identified based on Western blotting using the prepared cell surface proteins (CSPs) of the bacteria., Results: The fusion protein was successfully expressed, and the titer of the antibody reached 1:32 000. Western blotting indicated that the antibody could specifically bind to CSPs and His-rhMCP., Conclusion: The antibody with a high titer and specificity was prepared to facilitate further study of the pathogenicity and epidemiology of H.hepaticus in human.

Helicobacter hepaticus-infected Rag2(-/-) mice emulate many aspects of human inflammatory bowel disease, including the development of colitis and colon cancer. To elucidate mechanisms of inflammation-induced carcinogenesis, we undertook a comprehensive analysis of histopathology, molecular damage, and gene expression changes during disease progression in these mice. Infected mice developed severe colitis and hepatitis by 10 wk post-infection, progressing into colon carcinoma by 20 wk post-infection, with pronounced pathology in the cecum and proximal colon marked by infiltration of neutrophils and macrophages. Transcriptional profiling revealed decreased expression of DNA repair and oxidative stress response genes in colon, but not in liver. Mass spectrometric analysis revealed higher levels of DNA and RNA damage products in liver compared to colon and infection-induced increases in 5-chlorocytosine in DNA and RNA and hypoxanthine in DNA. Paradoxically, infection was associated with decreased levels of DNA etheno adducts. Levels of nucleic acid damage from the same chemical class were strongly correlated in both liver and colon. The results support a model of inflammation-mediated carcinogenesis involving infiltration of phagocytes and generation of reactive species that cause local molecular damage leading to cell dysfunction, mutation, and cell death. There are strong correlations among histopathology, phagocyte infiltration, and damage chemistry that suggest a major role for neutrophils in inflammation-associated cancer progression. Further, paradoxical changes in nucleic acid damage were observed in tissue- and chemistry-specific patterns. The results also reveal features of cell stress response that point to microbial pathophysiology and mechanisms of cell senescence as important mechanistic links to cancer.

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.

Cistanche deserticola has commonly been used in traditional Chinese medicine to treat many health problems including irritable bowel syndrome or constipation. This study was designed to test the efficacy of a water-extract of C. deserticola in the prevention of colorectal cancer in a mouse model. Polysaccharide-rich water-extract of C. deserticola was prepared by boiling its stem powder in distilled water. Tgfb1Rag2 null mice were used as an experimental model. Here we showed that feeding of water-extract of C. deserticola significantly reduced the number of mucosal hyperplasia and intestinal helicobacter infection in mice. This beneficial effect correlated with significant stimulation of the immune system, evidenced by the enlargement of the spleens with increased number of splenic macrophage and natural killer cells, and with more potent cytotoxicity of splenocytes. In vitro water-extract of C. deserticola enhanced the cytotoxicity of naïve splenocytes against a human colon cancer cell line, and in macrophage cultures up-regulated nitric oxide synthase II expression and stimulated phagocytosis. In conclusion, our data indicate that oral administration of C. deserticola extract reduces inflammatory hyperplastic polyps and helicobacter infection in mice by its immune-stimulatory activity, suggesting that C. deserticola extract may have potential in preventing intestinal inflammation disorders including colorectal cancer., (Copyright © 2011 John Wiley & Sons, Ltd.)

Aim: To identify and characterize drosophila mothers against decapentaplegic (SMAD)3-dependent changes in immune cell populations following infection with Helicobacter hepaticus (H. hepaticus)., Methods: SMAD3(-/-) (n = 19) and colitis-resistant SMAD3(+/-) (n = 24) mice (8-10 wk of age) were infected with H. hepaticus and changes in immune cell populations [T lymphocytes, natural killer (NK) cells, T regulatory cells] were measured in the spleen and mesenteric lymph nodes (MsLNs) at 0 d, 3 d, 7 d and 28 d post-infection using flow cytometry. Genotype-dependent changes in T lymphocytes and granzyme B(+) cells were also assessed after 28 d in proximal colon tissue using immunohistochemistry., Results: As previously observed, SMAD3(-/-), but not SMAD3(+/-) mice, developed colitis, peaking at 4 wk post-infection. No significant changes in T cell subsets were observed in the spleen or in the MsLNs between genotypes at any time point. However, CD4(+) and CD8(+)/CD62L(lo) cells, an effector T lymphocyte population, as well as NK cells (NKp46/DX5(+)) were significantly higher in the MsLNs of SMAD3(-/-) mice at 7 d and 28 d post-infection. In the colon, a higher number of CD3(+) cells were present in SMAD3(-/-) compared to SMAD3(+/-) mice at baseline, which did not significantly change during infection. However, the number of granzyme B(+) cells, a marker of cytolytic lymphocytes, significantly increased in SMAD3(-/-) mice 28 d post-infection compared to both SMAD3(+/-) mice and to baseline values. This was consistent with more severe colitis development in these animals., Conclusion: Data suggest that defects in SMAD3 signaling increase susceptibility to H. hepaticus-induced colitis through aberrant activation and/or dysregulation of effector lymphocytes.

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.

Mutations in the NOD2 gene are strong genetic risk factors for ileal Crohn's disease. However, the mechanism by which these mutations predispose to intestinal inflammation remains a subject of controversy. We report that Nod2-deficient mice inoculated with Helicobacter hepaticus, an opportunistic pathogenic bacterium, developed granulomatous inflammation of the ileum, characterized by an increased expression of Th1-related genes and inflammatory cytokines. The Peyer's patches and mesenteric lymph nodes were markedly enlarged with expansion of IFN-gamma-producing CD4 and CD8 T cells. Rip2-deficient mice exhibited a similar phenotype, suggesting that Nod2 function likely depends on the Rip2 kinase in this model. Transferring wild-type bone marrow cells into irradiated Nod2-deficient mice did not rescue the phenotype. However, restoring crypt antimicrobial function of Nod2-deficient mice by transgenic expression of alpha-defensin in Paneth cells rescued the Th1 inflammatory phenotype. Therefore, through the regulation of intestinal microbes, Nod2 function in nonhematopoietic cells of the small intestinal crypts is critical for protecting mice from a Th1-driven granulomatous inflammation in the ileum. The model may provide insight into Nod2 function relevant to inflammation of ileal Crohn's disease.

Background and Aims: Infection with Helicobacter hepaticus has been associated with development of hepatocellular carcinoma and gallstones in animal models. In humans, however, the association of H. hepaticus infection with biliary and pancreatic diseases has not been elucidated. The aim of this study was to serologically examine the prevalence of H. hepaticus infection in patients with biliary and pancreatic diseases., Methods: Serum samples obtained from 55 patients with cholelithiasis, 18 with bile duct or gallbladder cancer and 19 with pancreatic cancer were studied. Sera were obtained from 34 control subjects who underwent endoscopy and were diagnosed as not having peptic ulcers or cancers. Seropositivity of H. hepaticus was examined by western blot analysis using a H. hepaticus-specific antigen. To validate the specificity, positive sera were also tested after absorption with H. hepaticus whole-cell sonicate. Serum samples were also tested for the presence of anti-Helicobacter pylori antibody., Results: Prevalence of antibody to H. hepaticus-specific antigen in patients with bile tract cancer was 38.8% and was significantly higher than in control subjects (13.1%, P < 0.05). Prevalence of antibody to H. hepaticus-specific antigen was 18.2% and 10.5% in patients with cholelithiasis and pancreatic cancer, respectively. Seropositivity for H. pylori was similar in all groups. Detection of the H. hepaticus-specific band was significantly decreased after the sera were absorbed with H. hepaticus whole-cell sonicate., Conclusion: Infection with H. hepaticus might be associated with bile duct cancer. Results obtained from absorbed sera suggested high specificity of the western blot analysis.

The key role of interleukin (IL)-23 in the pathogenesis of autoimmune and chronic inflammatory disorders is supported by the identification of IL-23 receptor (IL-23R) susceptibility alleles associated with inflammatory bowel disease, psoriasis and ankylosing spondylitis. IL-23-driven inflammation has primarily been linked to the actions of T-helper type 17 (TH17) cells. Somewhat overlooked, IL-23 also has inflammatory effects on innate immune cells and can drive T-cell-independent colitis. However, the downstream cellular and molecular pathways involved in this innate intestinal inflammatory response are poorly characterized. Here we show that bacteria-driven innate colitis is associated with an increased production of IL-17 and interferon-gamma in the colon. Stimulation of colonic leukocytes with IL-23 induced the production of IL-17 and interferon-gamma exclusively by innate lymphoid cells expressing Thy1, stem cell antigen 1 (SCA-1), retinoic-acid-related orphan receptor (ROR)-gammat and IL-23R, and these cells markedly accumulated in the inflamed colon. IL-23-responsive innate intestinal cells are also a feature of T-cell-dependent models of colitis. The transcription factor ROR-gammat, which controls IL-23R expression, has a functional role, because Rag-/-Rorc-/- mice failed to develop innate colitis. Last, depletion of Thy1+ innate lymphoid cells completely abrogated acute and chronic innate colitis. These results identify a previously unrecognized IL-23-responsive innate lymphoid population that mediates intestinal immune pathology and may therefore represent a target in inflammatory bowel disease.

The gastrointestinal tract harbors a diverse microbiota that has coevolved with mammalian hosts. Though most associations are symbiotic or commensal, some resident bacteria (termed pathobionts) have the potential to cause disease. Bacterial type VI secretion systems (T6SSs) are one mechanism for forging host-microbial interactions. Here we reveal a protective role for the T6SS of Helicobacter hepaticus, a Gram-negative bacterium of the intestinal microbiota. H. hepaticus mutants with a defective T6SS display increased numbers within intestinal epithelial cells (IECs) and during intestinal colonization. Remarkably, the T6SS directs an anti-inflammatory gene expression profile in IECs, and CD4+ T cells from mice colonized with T6SS mutants produce increased interleukin-17 in response to IECs presenting H. hepaticus antigens. Thus, the H. hepaticus T6SS limits colonization and intestinal inflammation, promoting a balanced relationship with the host. We propose that disruption of such balances contributes to human disorders such as inflammatory bowel disease and colon cancer., (Copyright 2010 Elsevier Inc. All rights reserved.)

Catalase is reported to be one of the target antigens for autoantibodies in various pathologies. To understand the mechanism of autoantibody production, we compared the several properties of autoantigenic epitopes (AE)-1 and -2 of mouse catalase, which reported to react with antibodies from sera of Helicobacter hepaticus-infected mice; AE-3 and -4 of rat catalase, which we found to be susceptible to autoimmunity; and antigenic epitope (E)-1 of H. pylori catalase, which is recognized by monoclonal antibodies produced by immunized mice. Amino acid sequences of AE-1 and -2 were similar among both mammalian and pathogenic microorganism catalases, whereas that of E-1 differed. Amino acid sequences of AE-3 and -4 were similar among mammalian catalases but differed from pathogenic microorganism catalases. Based on local relative rates of evolution, these vertebrate catalases were divided into 5 segments. E-1 included a faster evolving region, whereas AE-1 and -2 included a slowly evolving region; AE-3 and -4 comprised a slowly evolving patch within a faster evolving region. In conclusion, although AE-1 and -2 of catalase have been reported to contribute to autoimmune responses in animals infected with catalase-producing pathogens, AE-3 and -4 appear to have a different mechanism for autoantibody production.

Background: Since the discovery of Helicobacter pylori, various enterohepatic Helicobacter spices have been detected in the guts of humans and animals. Some enterohepatic Helicobacters have been associated with inflammatory bowel disease or liver disease in mice. However the association of these bacteria with human diseases remains unknown., Materials and Methods: We collected 126 bile samples from patients with cholelithiasis, cholecystitis, gallbladder polyp, and other nonbiliary diseases. Samples were screened for the presence of enterohepatic Helicobacter spp. using cultures, nested PCR, or in situ hybridization. We tested for antibodies to H. pylori and H. hepaticus by Western blot analysis., Results: Attempts at cultivation were unsuccessful. However, H. hepaticus was detected in bile samples with nested PCR whereas H. bilis was not. Helicobacter hepaticus in the bile was confirmed by in situ hybridization, but H. hepaticus from bile samples was coccoid in appearance. We detected immunoglobulin G antibodies to H. hepaticus in bile samples by Western blotting. Helicobacter hepaticus was detected in 40 (32%) of total 126 samples as H. hepaticus positive if at least one of the three methods with nested PCR, in situ, or Western blotting. Patients with cholelithiasis (41%) and cholecystitis with gastric cancer (36%) had significantly higher (p = .029) prevalence of H. hepaticus infection than samples from patients with other diseases., Conclusion: Helicobacter hepaticus may closely associate with diseases of the liver and biliary tract in humans.

Background & Aims: The commensal microbiota is believed to have an important role in regulating immune responsiveness and preventing intestinal inflammation. Intestinal microbes produce signals that regulate inflammation via Toll-like receptor (TLR) signaling, but the mechanisms of this process are poorly understood. We investigated the role of the anti-inflammatory cytokine interleukin (IL)-10 in this signaling pathway using a mouse model of colitis., Methods: Clinical, histopathologic, and functional parameters of intestinal inflammation were evaluated in TLR4(-/-), IL-10(-/-), and TLR4(-/-) x IL-10(-/-) mice that were free of specific pathogens and in TLR4(-/-) x IL-10(-/-) mice following eradication and reintroduction of Helicobacter hepaticus. Regulatory T-cell (Treg) function was evaluated by crossing each of the lines with transgenic mice that express green fluorescent protein under control of the endogenous regulatory elements of Foxp3. Apoptotic cells in the colonic lamina propria were detected by a TUNEL assay., Results: TLR4-mediated signals have 2 interrelated roles in promoting inflammation in TLR4(-/-) x IL-10(-/-) mice. In the absence of TLR4-mediated signals, secretion of proinflammatory and immunoregulatory cytokines is dysregulated. Tregs (Foxp3(+)) that secrete interferon-gamma and IL-17 accumulate in the colonic lamina propria of TLR4(-/-) x IL-10(-/-) mice and do not prevent inflammation. Aberrant control of epithelial cell turnover results in the persistence of antigen-presenting cells that contain apoptotic epithelial fragments in the colonic lamina propria of Helicobacter-infected TLR4(-/-) mice., Conclusions: In mice that lack both IL-10- and TLR4-mediated signals, aberrant regulatory T-cell function and dysregulated control of epithelial homeostasis combine to exacerbate intestinal inflammation.

Background and Aims: Infection with Helicobacter hepaticus is suggested to play a role in the pathogenesis of chronic liver disease in humans. However, reactive antigens among Helicobacter species make the development of an H. hepaticus ELISA test with high specificity difficult. A new monoclonal antibody from a hybridoma clone (HRII-51) showed high specificity to H. hepaticus without cross-reaction to other gastrointestinal bacteria., Methods: The molecular weight of HRII-51 immunoreactive antigen was examined by Western blot of H. hepaticus probed with the monoclonal antibody HRII-51. A HRII-51-immunoreactive antigen capture ELISA was prepared in which the specific antigen was anchored by HRII-51-immobilized ELISA plate. Accuracy of HRII-51 antigen capture ELISA was examined using sera obtained from mice inoculated with Helicobacter species. Specificity of HRII-51 antigen capture ELISA was compared to that of H. hepaticus antigen-based ELISA using human sera with absorption by H. pylori cell lysate., Results: HRII-51 immunoreactive antigen had a molecular weight of 15 kDa. Sensitivity and specificity of HRII-51 antigen capture ELISA were 87.0% and 97.6% in mice inoculated with Helicobacter species. In human sera, modification of the results by absorption with H. pylori lysate was smaller in HRII-51 antigen capture ELISA comparing with H. hepaticus-antigen-based ELISA., Conclusion: Use of the HRII-51 antigen capture ELISA would be a useful approach for the serodiagnosis of H. hepaticus infection in both experimental animals and humans.

Recombinase-activating gene-2-deficient (Rag2(-/-)) mice lacking functional lymphocytes provide a useful model of chronic inflammatory bowel disease-emulating events in human colon cancer. Infection of Rag2(-/-) mice with Helicobacter hepaticus led to accumulation of macrophages and neutrophils in the colon, a process temporally related to up-regulation of tissue inducible nitric oxide synthase (iNOS) expression at the site of infection and increased nitric oxide (NO) production, as evidenced by urinary excretion of nitrate. Progressive development of increasingly severe inflammation, hyperplasia, dysplasia, and cancer accompanied these changes. Concurrent administration of an iNOS inhibitor prevented NO production and abrogated epithelial pathology and inhibited the onset of cancer. The presence of Gr-1(+) neutrophils and elevated tumor necrosis factor-alpha (TNF-alpha) expression in colon were required for increased iNOS expression and cancer, whereas interleukin-10 (IL-10) down-regulated TNF-alpha and iNOS expression and suppressed cancer. Anti-inflammatory CD4(+) regulatory lymphocytes also down-regulated iNOS and reduced cancer formation. Collectively, these results confirm essential roles for inflammation, increased TNF-alpha expression, and elevated NO production in colon carcinogenesis.

Urease activity contributes to bacterial survival in the acidic environment of the stomach and is essential for persistent infection by known gastric helicobacters such as the human pathogen Helicobacter pylori. Several enterohepatic Helicobacter species (EHS) that primarily infect the less acidic intestine also have very active urease enzymes. The importance of urease and its contribution to pathogenesis for these EHS are poorly understood. In this study, we generated a urease-deficient, isogenic mutant (HhureNT9) of Helicobacter hepaticus 3B1 (Hh 3B1), an EHS that possesses a urease gene cluster similar to that of H. pylori. Lack of urease activity did not affect the level of cecal colonization by HhureNT9 compared to Hh 3B1 in male A/JCr mice (P=0.48) at 4 months post-inoculation (MPI). In contrast, there was no HhureNT9 detected in the livers of any infected mice, whereas all livers from the Hh 3B1-infected mice were PCR-positive for Hh 3B1. The mice infected with HhureNT9 developed significantly less severe hepatitis (P=0.017) and also produced significantly lower hepatic mRNA levels of proinflammatory cytokines IFN-gamma (P=0.0007) and TNF-alpha (P<0.0001) compared to the Hh 3B1-infected mice. The Hh 3B1-infected mice developed significantly higher total IgG, Th1-associated IgG2a and Th2-associated IgG1 responses to infection. These results indicate that H. hepaticus urease activity plays a crucial role in hepatic disease but is not required for cecal colonization by H. hepaticus.

Inflammatory bowel disease is a chronic inflammatory response of the gastrointestinal tract mediated in part by an aberrant response to intestinal microflora. Expression of IL-23 subunits p40 and p19 within cells of the innate immune system plays a central role in the development of lower bowel inflammation in response inflammatory challenge. The NF-kappaB subunit c-Rel can regulate expression of IL-12/23 subunits suggesting that it could have a critical role in mediating the development of chronic inflammation within the lower bowel. In this study, we have analyzed the role of c-Rel within the innate immune system in the development of lower bowel inflammation, in two well-studied models of murine colitis. We have found that the absence of c-Rel significantly impaired the ability of Helicobacter hepaticus to induce colitis upon infection of RAG-2-deficient mice, and ameliorated the ability of CD4(+)CD45RB(high) T cells to induce disease upon adoptive transfer into RAG-deficient mice. The absence of c-Rel interfered with the expression of IL-12/23 subunits both in cultured primary macrophages and within the colon. Thus, c-Rel plays a critical role in regulating the innate inflammatory response to microflora within the lower bowel, likely through its ability to modulate expression of IL-12/23 family members.

The astonishing density of microbes in the mammalian gut has raised numerous questions, including how such colonization is tolerated in an immunocompetent location. Clearly the organisms perform a beneficial role, but until now the mechanisms have been less than clear. In a recent study in Nature, Mazmanian et al. (2008) reveal the ability of specific moieties on the surface of Bacteroides fragilis to direct the host's immune response.

Enterohepatic Helicobacter species infect the intestinal tracts and biliary trees of various mammals, including mice and humans, and are associated with chronic inflammatory diseases of the intestine, gallstone formation, and malignant transformation. The recent analysis of the whole genome sequence of the mouse enterohepatic species Helicobacter hepaticus allowed us to perform a functional analysis of bacterial factors that may play a role in these diseases. We tested the hypothesis that H. hepaticus suppresses or evades innate immune responses of mouse intestinal epithelial cells, which allows this pathogen to induce or contribute to chronic inflammatory disease. We demonstrated in the present study that the innate immune responses of intestinal epithelial cells to lipopolysaccharide (LPS) via Toll-like receptor 4 (TLR4) and to flagellin-mediated activation via TLR5 are reduced by H. hepaticus infection through soluble bacterial factors. In particular, H. hepaticus lysate and the soluble component LPS antagonized TLR4- and TLR5-mediated immune responses of intestinal epithelial cells. H. hepaticus lysate and LPS inhibited development of endotoxin tolerance to Escherichia coli LPS. Suppression of innate immune responses by H. hepaticus LPS thus may affect intestinal responses to the resident microbial flora, epithelial homeostasis, and intestinal inflammatory conditions.

Defects within the innate immune system sensitize NF-kappaB-deficient (p50(-/-); p65(+/-)) mice to Helicobacter hepaticus (Hh)-induced colitis. Because IL-10 plays a central role in the inhibition of Hh-induced colitis, we hypothesized that the ability of IL-10 to inhibit the innate inflammatory response to Hh may be compromised in NF-kappaB-deficient mice. To test this hypothesis, we evaluated the ability of an IL-10-Ig fusion protein with IL-10-like properties to inhibit Hh-induced colitis in RAG-2(-/-) (RAG) and p50(-/-); p65(+/-); RAG-2(-/-) (3X/RAG) mice. As expected, IL-10-Ig efficiently inhibited the development of colitis in RAG mice. In contrast, the ability of IL-10-Ig to inhibit colitis was compromised in 3X/RAG mice. The defect in response to IL-10-Ig appeared to be primarily the result of the absence of the p50/p105 subunit, because the ability of IL-10-Ig to inhibit colitis was also compromised in p50(-/-); RAG-2(-/-) (p50/RAG) mice. Radiation chimeras demonstrated that the presence of p50/p105 within hemopoietic cells of the innate immune system was necessary for efficient inhibition of colitis by IL-10-Ig. Consistent with a defect in the suppressive effects of IL-10 in the absence of p50/p105, we found that the ability of IL-10 to control LPS-induced expression of IL-12 p40 was significantly compromised in macrophages lacking p50/p105. These results suggest that the absence of the p50/p105 subunit of NF-kappaB within hemopoietic cells of the innate immune system interferes with the ability of IL-10 to suppress inflammatory gene expression and Hh-induced colitis.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract that is caused in part by a dysregulated immune response to the intestinal flora. The common interleukin (IL)-12/IL-23p40 subunit is thought to be critical for the pathogenesis of IBD. We have analyzed the role of IL-12 versus IL-23 in two models of Helicobacter hepaticus-triggered T cell-dependent colitis, one involving anti-IL-10R monoclonal antibody treatment of infected T cell-sufficient hosts, and the other involving CD4+ T cell transfer into infected Rag-/- recipients. Our data demonstrate that IL-23 and not IL-12 is essential for the development of maximal intestinal disease. Although IL-23 has been implicated in the differentiation of IL-17-producing CD4+ T cells that alone are sufficient to induce autoimmune tissue reactivity, our results instead support a model in which IL-23 drives both interferon gamma and IL-17 responses that together synergize to trigger severe intestinal inflammation.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract involving aberrant activation of innate and adaptive immune responses. We have used two complementary models of IBD to examine the roles of interleukin (IL)-12 family cytokines in bacterially induced intestinal inflammation. Our results clearly show that IL-23, but not IL-12, is essential for the induction of chronic intestinal inflammation mediated by innate or adaptive immune mechanisms. Depletion of IL-23 was associated with decreased proinflammatory responses in the intestine but had little impact on systemic T cell inflammatory responses. These results newly identify IL-23 as a driver of innate immune pathology in the intestine and suggest that selective targeting of IL-23 represents an attractive therapeutic approach in human IBD.

Inflammation associated with bacterial infections is a risk factor for cancers in humans, yet its role in breast cancer remains poorly understood. We have previously shown that innate immune inflammatory response against intestinal bacteria is sufficient to induce colon cancer. Here we report that infecting Rag2-deficient C57BL/6 Apc(Min/+) mice with an intestinal bacterial pathogen, Helicobacter hepaticus, significantly promotes mammary carcinoma in females and enhances intestinal adenoma multiplicity by a tumor necrosis factor alpha (TNFalpha)-dependent mechanism. The mammary and intestinal tumor development as well as the increase in proinflammatory mediators is suppressed by adoptive transfer of interleukin 10-competent CD4+CD45RB(lo)CD25+ regulatory (T(R)) cells. Furthermore, prior exposure of donor mice to H. hepaticus significantly enhances antitumor potency of their T(R) cells. Interestingly, these microbially experienced T(R) cells suppress tumorigenesis more effectively in recipient mice irrespective of their tumor etiology. These data suggest that infections with enteric pathogens enhance T(R)-cell potency and protect against epithelial cancers later in life, potentially explaining paradoxical increases in cancer risk in developed countries having more stringent hygiene practices. The possibility that dysregulated gut microbial infections in humans may lead to cancer in anatomically distant organs, such as breast, highlights the need for novel immune-based strategies in cancer prevention and treatment.

Helicobacter hepaticus is an enterohepatic Helicobacter species that induces lower bowel inflammation in susceptible mouse strains, including those lacking the p50/p105 subunit of NF-kappaB. H. hepaticus-induced colitis is associated with elevated levels of IL-12 p40 expression, and p50/p105-deficient macrophages express higher levels of IL-12 p40 than wild-type macrophages after challenge with H. hepaticus. However, the molecular mechanisms by which the p50/p105 subunit of NF-kappaB suppresses IL-12 p40 expression have not yet been elucidated. In this study we have demonstrated that H. hepaticus challenge of macrophages induces ERK activation, and this event plays a critical role in inhibiting the ability of H. hepaticus to induce IL-12 p40. Activation of ERK requires both p50/p105 and the MAPK kinase kinase, Tpl-2. Inhibition of the induction of IL-12 p40 by ERK was independent of c-Rel, a known positive regulator of IL-12 p40. Instead, it was linked to the induction of c-Fos, a known inhibitor of IL-12 p40 expression. These results suggest that H. hepaticus induces ERK activation by a pathway dependent upon Tpl-2 and p105, and that activation of ERK inhibits the expression of IL-12 p40 by inducing c-Fos. Thus, a defect in ERK activation could play a pivotal role in the superinduction of IL-12 p40 observed after challenge of macrophages lacking the p50/p105 subunit of NF-kappaB with H. hepaticus.

Lactic acid bacteria or their secretion products can modulate immune responses differently in normal and inflammatory conditions. This comparative study analyzes the effect of oral administration of living lactic acid bacteria, or their conditioned media, on the epithelial and immune functions of colitis-prone C57BL/6 IL-10-deficient mice. Mice were untreated (control) or infected with Helicobacter hepaticus with or without oral treatment with living bacteria, Bifidobacterium breve C50 and Streptococcus thermophilus 065 (LB), or their culture-conditioned media (CM). Histology, cytokine mRNA, electrical resistance, and barrier capacity of colonic samples as well as cytokine secretion by mesenteric lymph node (MLN) cells were studied. Helicobacter hepaticus mice developed only mild colitis, which was not modified in LB or CM groups. In the CM (but not the LB) group, the colonic barrier was reinforced as compared to the other groups, as evidenced by decreased horseradish peroxidase (HRP) transcytosis and mannitol fluxes and increased electrical resistance. In MLN, the percentage of CD4+ and CD8+ T cells secreting IFNgamma was significantly higher in CM (2.06% and 1.98%, respectively) mice than in H. hepaticus (1.1% and 0.47%, P < 0.05) or control mice. In addition, the nonspecific stimulation of IFNgamma, TNFalpha, and IL-12 secretion by MLN cells was significantly higher in the CM group as compared to the other groups. In the absence of severe colitis, Bifidobacterium breve C50- and Streptococcus thermophilus 065-conditioned media can reinforce intestinal barrier capacity and stimulate Th1 immune response, highlighting the involvement of lactic acid bacteria-derived components in host defense.