Modulation of the Intestinal Microbiota Alters Colitis-Associated Colorectal Cancer Susceptibility

It is well established that the intestinal microbiota plays a key role in the pathogenesis of Crohn''s disease (CD) and ulcerative colitis (UC) collectively referred to as inflammatory bowel disease (IBD). Epidemiological studies have provided strong evidence that IBD patients bear increased risk for the development of colorectal cancer (CRC). However, the impact of the microbiota on the development of colitis-associated cancer (CAC) remains largely unknown. In this study, we established a new model of CAC using azoxymethane (AOM)-exposed, conventionalized-Il10^−/− mice and have explored the contribution of the host intestinal microbiota and MyD88 signaling to the development of CAC. We show that 8/13 (62%) of AOM-Il10^−/− mice developed colon tumors compared to only 3/15 (20%) of AOM- wild-type (WT) mice. Conventionalized AOM-Il10^−/− mice developed spontaneous colitis and colorectal carcinomas while AOM-WT mice were colitis-free and developed only rare adenomas. Importantly, tumor multiplicity directly correlated with the presence of colitis. Il10^−/− mice mono-associated with the mildly colitogenic bacterium Bacteroides vulgatus displayed significantly reduced colitis and colorectal tumor multiplicity compared to Il10^−/− mice. Germ-free AOM-treated Il10^−/− mice showed normal colon histology and were devoid of tumors. Il10^−/−; Myd88^−/− mice treated with AOM displayed reduced expression of Il12p40 and Tnfα mRNA and showed no signs of tumor development. We present the first direct demonstration that manipulation of the intestinal microbiota alters the development of CAC. The TLR/MyD88 pathway is essential for microbiota-induced development of CAC. Unlike findings obtained using the AOM/DSS model, we demonstrate that the severity of chronic colitis directly correlates to colorectal tumor development and that bacterial-induced inflammation drives progression from adenoma to invasive carcinoma.     

Probiotic Bifidobacterium breve Induces IL-10-Producing Tr1 Cells in the Colon

Specific intestinal microbiota has been shown to induce Foxp3⁺ regulatory T cell development. However, it remains unclear how development of another regulatory T cell subset, Tr1 cells, is regulated in the intestine. Here, we analyzed the role of two probiotic strains of intestinal bacteria, Lactobacillus casei and Bifidobacterium breve in T cell development in the intestine. B. breve, but not L. casei, induced development of IL-10-producing Tr1 cells that express cMaf, IL-21, and Ahr in the large intestine. Intestinal CD103⁺ dendritic cells (DCs) mediated B. breve-induced development of IL-10-producing T cells. CD103⁺ DCs from Il10 ^−/−, Tlr2 ^−/−, and Myd88 ^−/− mice showed defective B. breve-induced Tr1 cell development. B. breve-treated CD103⁺ DCs failed to induce IL-10 production from co-cultured Il27ra ^−/− T cells. B. breve treatment of Tlr2 ^−/− mice did not increase IL-10-producing T cells in the colonic lamina propria. Thus, B. breve activates intestinal CD103⁺ DCs to produce IL-10 and IL-27 via the TLR2/MyD88 pathway thereby inducing IL-10-producing Tr1 cells in the large intestine. Oral B. breve administration ameliorated colitis in immunocompromised mice given naïve CD4⁺ T cells from wild-type mice, but not Il10 ^−/− mice. These findings demonstrate that B. breve prevents intestinal inflammation through the induction of intestinal IL-10-producing Tr1 cells.     

Rag2-deficient IL-1 Receptor Antagonist-deficient Mice Are a Novel Colitis Model in Which Innate Lymphoid Cell-derived IL-17 Is Involved in the Pathogenesis

Il1rn^−/− mice spontaneously develop arthritis and aortitis by an autoimmune mechanism and also develop dermatitis by an autoinflammatory mechanism. Here, we show that Rag2^−/−Il1rn^−/− mice develop spontaneous colitis with high mortality, making a contrast to the suppression of arthritis in these mice. Enhanced IL-17A expression in group 3 innate lymphoid cells (ILC3s) was observed in the colon of Rag2^−/−Il1rn^−/− mice. IL-17A-deficiency prolonged the survival of Rag2^−/−Il1rn^−/− mice, suggesting a pathogenic role of this cytokine in the development of intestinal inflammation. Although IL-17A-producing T cells were increased in Il1rn^−/− mice, these mice did not develop colitis, because CD4⁺Foxp3⁺ regulatory T cell population was also expanded. Thus, excess IL-1 signaling and IL-1-induced IL-17A from ILC3s cause colitis in Rag2^−/−Il1rn^−/− mice in which Treg cells are absent. These observations suggest that the balance between IL-17A-producing cells and Treg cells is important to keep the immune homeostasis of the colon.     

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.     

Chronic Proliferative Dermatitis in Sharpin Null Mice: Development of an Autoinflammatory Disease in the Absence of B and T Lymphocytes and IL4/IL13 Signaling

SHARPIN is a key regulator of NFKB and integrin signaling. Mice lacking Sharpin develop a phenotype known as chronic proliferative dermatitis (CPDM), typified by progressive epidermal hyperplasia, apoptosis of keratinocytes, cutaneous and systemic eosinophilic inflammation, and hypoplasia of secondary lymphoid organs. Rag1^−/− mice, which lack mature B and T cells, were crossed with Sharpin^−/− mice to examine the role of lymphocytes in CDPM. Although inflammation in the lungs, liver, and joints was reduced in these double mutant mice, dermatitis was not reduced in the absence of functional lymphocytes, suggesting that lymphocytes are not primary drivers of the inflammation in the skin. Type 2 cytokine expression is increased in CPDM. In an attempt to reduce this aspect of the phenotype, Il4ra^−/− mice, unresponsive to both IL4 and IL13, were crossed with Sharpin^−/− mice. Double homozygous Sharpin^−/−, Il4ra^−/− mice developed an exacerbated granulocytic dermatitis, acute system inflammation, as well as hepatic necrosis and mineralization. High expression of CHI3L4, normally seen in CPDM skin, was abolished in Sharpin^−/−, Il4ra^−/− double mutant mice indicating the crucial role of IL4 and IL13 in the expression of this protein. Cutaneous eosinophilia persisted in Sharpin^−/−, Il4ra^−/− mice, although expression of Il5 mRNA was reduced and the expression of Ccl11 and Ccl24 was completely abolished. TSLP and IL33 were both increased in the skin of Sharpin^−/− mice and this was maintained in Sharpin^−/−, Il4ra^−/− mice suggesting a role for TSLP and IL33 in the eosinophilic dermatitis in SHARPIN-deficient mice. These studies indicate that cutaneous inflammation in SHARPIN-deficient mice is autoinflammatory in nature developing independently of B and T lymphocytes, while the systemic inflammation seen in CPDM has a strong lymphocyte-dependent component. Both the cutaneous and systemic inflammation is enhanced by loss of IL4 and IL13 signaling indicating that these cytokines normally play an anti-inflammatory role in SHARPIN-deficient mice.     

Pathogenicity of Helicobacter ganmani in Mice Susceptible and Resistant to Infection with H. hepaticus

Helicobacter spp. are some of the most prevalent bacterial contaminants of laboratory mice. Although abundant data regarding the diseases associated with H. hepaticus infection are available, little is known about the pathogenicity of H. ganmani, which was first isolated in 2001 from the intestines of laboratory mice. The objective of this study was to evaluate the host response to H. ganmani colonization in H. hepaticus disease-resistant C57BL/6 and disease-susceptible A/J and IL10-deficient mice. Mice were inoculated with H. ganmani, H. hepaticus, or Brucella broth. Cecal lesion scores, cecal gene expression, and Helicobacter load were measured at 4 and 90 d after inoculation. At both time points, mice inoculated with H. ganmani had similar or significantly more copies of cecum-associated Helicobacter DNA than did mice inoculated with H. hepaticus. When compared with those of sham-inoculated control mice, cecal lesion scores at 4 and 90 d after inoculation were not significantly greater in H. ganmani-inoculated A/J, C57BL/6, or IL10-deficient mice. Analysis of cecal gene expression demonstrated that H. ganmani infection failed to cause significant elevations of IFNγ in A/J, C57BL/6, or IL10-deficient mice. However, in IL10-deficient mice, H. ganmani infection was associated with a significant increase in the expression of the proinflammatory cytokine IL12/23p40. Although H. ganmani infection in this study failed to induce the typhlitis that is the hallmark of H. hepaticus infection, infection with H. ganmani was associated with alterations in inflammatory cytokines in IL10-deficient mice.Abbreviations: B6, C57BL/6NCr; HPRT, hypoxanthine guanine phosphoribosyl transferase; IL10 KO, B6129P2-IL10^tm1Cgn/JSince the discovery of the link between Helicobacter pylori and chronic gastritis in 1982,¹⁷ Helicobacter spp. in humans and animals have become a field of extensive study. Due to improved detection methods, there has been a rapid expansion in our understanding and ability to detect native Helicobacter spp. in mouse models. Several reports investigating their prevalence in mice housed in research institutions have found Helicobacter spp. to be some of the most common bacterial contaminants of laboratory rodents.^2,3,12,16,23 Helicobacter hepaticus is perhaps the most notorious of the murine helicobacters, by virtue of the early realization of its pathogenicity in adult mice.^8,24 The hallmarks of infection by H. hepaticus are typhlitis, colitis, and hepatitis.¹⁰ In addition, H. hepaticus is commonly used as a microbial trigger in susceptible mouse strains used as models of inflammatory bowel disease.^5,9,19,21,28 In 2001, less than 10 y after H. hepaticus was discovered, H. ganmani was isolated from the intestines of laboratory mice.²⁶ During its initial characterization, 16S rDNA sequence analysis placed H. ganmani phylogenetically closest to H. rodentium, a urease-negative helicobacter that had been previously isolated from mouse intestines.²⁶Despite the reported endemic presence of H. ganmani in many research colonies,^2,3,12 only a few reports to date have attempted to address H. ganmani’s potential pathogenicity.^22,30 One report describes an outbreak of inflammatory bowel-like disease associated with H. ganmani infection in an otherwise Helicobacter-free conventional colony of IL10-deficient mice.²² The findings from another report describe the effect of natural colonization of IL10-deficient mice with H. ganmani, H. hepaticus, or both.³⁰ In that study, 8- to 20-wk-old mice monoinfected with H. ganmani had significantly lower lesion scores than did mice monoinfected with H. hepaticus, suggesting that infection with H. ganmani alone was not sufficient to cause severe typhlocolitis.³⁰ However, by 34 wk of age, clinical typhlocolitis (diarrhea) and grossly enlarged ceca were observed at necropsy in 2 of the 6 mice monoinfected with H. ganmani.³⁰Although these reports of naturally occurring infections have provided a glimpse into H. ganmani’s potential to produce intestinal disease in immunodeficient mice, a controlled study in immunocompetent and immunodeficient mice had not been conducted previously. The objectives of the current study were to evaluate the effect of H. ganmani infection on intestinal disease and to characterize alterations of inflammatory gene expression associated with infection. To this end, we selected A/J and IL10-deficient mice for this study because of their known susceptibility to H. hepaticus-induced typhlocolitis.^{9,13,14,19,21,28} In contrast, although C57BL/6 mice show an initial spike in inflammatory cytokines after H. hepaticus infection, they do not typically develop chronic disease.¹⁹ We did not expect C57BL/6 mice to develop H. hepaticus-induced disease, but we deemed it prudent to characterize the possible effects—through unknown mechanisms—of H. ganmani on this common strain.Previous studies characterizing cecal gene expression during H. hepaticus induced typhlocolitis demonstrated that IFNγ and IL12/23p40 (IL12/23) are key proinflammatory cytokines that drive typhlitis.¹⁹ Expression of these cytokines was increased in H. hepaticus-inoculated A/J mice but not in H. hepaticus-inoculated C57BL/6 mice.¹⁹ In addition, treatment with neutralizing monoclonal antibodies against these cytokines significantly decreased cecal lesion severity, implicating the roles of IFNγ and IL12/23 in modulating the pathogenesis of typhlitis.¹⁹ We hypothesized that characterizing the effect of H. ganmani infection on expression of IFNγ and IL12/23 would uncover aspects of the host response that are not readily apparent by histologic evaluation of cecal tissue alone.To date, our understanding of the potential for H. ganmani to cause intestinal disease has been limited to reports that focused on the evaluation of histologic disease in naturally infected IL10-deficient mice. Despite the reported endemic presence of H. ganmani in many research colonies,^2,3,12 there are no published reports of disease associated with H. ganmani infection in immunocompetent mice. In addition, H. ganmani shares close sequence homology with H. rodentium, which has been found to be nonpathogenic in monoinfected immunodeficient and immunocompetent mice.²⁰ Therefore, we hypothesized that experimental infection with H. ganmani would not produce disease in H. hepaticus-susceptible or -resistant mice.     

Salmonella enterica serovar typhimurium exploits inflammation to compete with the intestinal microbiota

Most mucosal surfaces of the mammalian body are colonized by microbial communities (“microbiota”). A high density of commensal microbiota inhabits the intestine and shields from infection (“colonization resistance”). The virulence strategies allowing enteropathogenic bacteria to successfully compete with the microbiota and overcome colonization resistance are poorly understood. Here, we investigated manipulation of the intestinal microbiota by the enteropathogenic bacterium Salmonella enterica subspecies 1 serovar Typhimurium (S. Tm) in a mouse colitis model: we found that inflammatory host responses induced by S. Tm changed microbiota composition and suppressed its growth. In contrast to wild-type S. Tm, an avirulent invGsseD mutant failing to trigger colitis was outcompeted by the microbiota. This competitive defect was reverted if inflammation was provided concomitantly by mixed infection with wild-type S. Tm or in mice (IL10^−/−, VILLIN-HA^CL4-CD8) with inflammatory bowel disease. Thus, inflammation is necessary and sufficient for overcoming colonization resistance. This reveals a new concept in infectious disease: in contrast to current thinking, inflammation is not always detrimental for the pathogen. Triggering the host's immune defence can shift the balance between the protective microbiota and the pathogen in favour of the pathogen.     

The Microbiota Mediates Pathogen Clearance from the Gut Lumen after Non-Typhoidal Salmonella Diarrhea

Many enteropathogenic bacteria target the mammalian gut. The mechanisms protecting the host from infection are poorly understood. We have studied the protective functions of secretory antibodies (sIgA) and the microbiota, using a mouse model for S. typhimurium diarrhea. This pathogen is a common cause of diarrhea in humans world-wide. S. typhimurium (S. tm ^att, sseD) causes a self-limiting gut infection in streptomycin-treated mice. After 40 days, all animals had overcome the disease, developed a sIgA response, and most had cleared the pathogen from the gut lumen. sIgA limited pathogen access to the mucosal surface and protected from gut inflammation in challenge infections. This protection was O-antigen specific, as demonstrated with pathogens lacking the S. typhimurium O-antigen (wbaP, S. enteritidis) and sIgA-deficient mice (TCRβ^−/−δ^−/−, J_H ^−/−, IgA^−/−, pIgR^−/−). Surprisingly, sIgA-deficiency did not affect the kinetics of pathogen clearance from the gut lumen. Instead, this was mediated by the microbiota. This was confirmed using ‘L-mice’ which harbor a low complexity gut flora, lack colonization resistance and develop a normal sIgA response, but fail to clear S. tm ^att from the gut lumen. In these mice, pathogen clearance was achieved by transferring a normal complex microbiota. Thus, besides colonization resistance ( = pathogen blockage by an intact microbiota), the microbiota mediates a second, novel protective function, i.e. pathogen clearance. Here, the normal microbiota re-grows from a state of depletion and disturbed composition and gradually clears even very high pathogen loads from the gut lumen, a site inaccessible to most “classical” immune effector mechanisms. In conclusion, sIgA and microbiota serve complementary protective functions. The microbiota confers colonization resistance and mediates pathogen clearance in primary infections, while sIgA protects from disease if the host re-encounters the same pathogen. This has implications for curing S. typhimurium diarrhea and for preventing transmission.     

NADPH Oxidase Deficient Mice Develop Colitis and Bacteremia upon Infection with Normally Avirulent,TTSS-1- and TTSS-2-Deficient Salmonella Typhimurium

Infections, microbe sampling and occasional leakage of commensal microbiota and their products across the intestinal epithelial cell layer represent a permanent challenge to the intestinal immune system. The production of reactive oxygen species by NADPH oxidase is thought to be a key element of defense. Patients suffering from chronic granulomatous disease are deficient in one of the subunits of NADPH oxidase. They display a high incidence of Crohn’s disease-like intestinal inflammation and are hyper-susceptible to infection with fungi and bacteria, including a 10-fold increased risk of Salmonellosis. It is not completely understood which steps of the infection process are affected by the NADPH oxidase deficiency. We employed a mouse model for Salmonella diarrhea to study how NADPH oxidase deficiency (Cybb ^−/−) affects microbe handling by the large intestinal mucosa. In this animal model, wild type S. Typhimurium causes pronounced enteropathy in wild type mice. In contrast, an avirulent S. Typhimurium mutant (S.Tm^avir; invGsseD), which lacks virulence factors boosting trans-epithelial penetration and growth in the lamina propria, cannot cause enteropathy in wild type mice. We found that Cybb ^−/− mice are efficiently infected by S.Tm^avir and develop enteropathy by day 4 post infection. Cell depletion experiments and infections in Cybb ^−/− Myd88 ^−/− mice indicated that the S.Tm^avir-inflicted disease in Cybb ^−/− mice hinges on CD11c⁺CX₃CR1⁺ monocytic phagocytes mediating colonization of the cecal lamina propria and on Myd88-dependent proinflammatory immune responses. Interestingly, in mixed bone marrow chimeras a partial reconstitution of Cybb-proficiency in the bone marrow derived compartment was sufficient to ameliorate disease severity. Our data indicate that NADPH oxidase expression is of key importance for restricting the growth of S.Tm^avir in the mucosal lamina propria. This provides important insights into microbe handling by the large intestinal mucosa and the role of NADPH oxidase in maintaining microbe-host mutualism at this exposed body surface.     

Quantitative trait loci in a bacterially induced model of inflammatory bowel disease

Inflammatory bowel diseases (IBDs) are complex disorders caused by a combination of environmental, microbial, and genetic factors. Genome-wide association studies in humans have successfully identified multiple genes and loci associated with disease susceptibility, but the mechanisms by which these loci interact with each other and/or with environmental factors (i.e., intestinal microbiota) to cause disease are poorly understood. Helicobacter hepaticus-induced intestinal inflammation in mice is an ideal model system for elucidating the genetic basis of IBD susceptibility in a bacterially induced system, as there are significant differences in H. hepaticus-induced disease susceptibility among inbred mouse strains. Infected A/J mice develop acute overexpression of proinflammatory cytokines followed 2?C3?months later by chronic cecal inflammation, whereas infected C57BL/6 mice fail to develop cecal inflammation or increased cytokine expression. The goal of this project was to use quantitative trait locus (QTL) mapping to evaluate genetic factors that contribute to the differential disease susceptibility between these two mouse strains. Using acute cecal IL-12/23p40 expression as a biomarker for disease susceptibility, QTL analysis of H. hepaticus-infected F₂ mice revealed involvement of multiple loci. The loci with the strongest association were located on Chromosome 3 and Chromosome 17, with logarithm of odds (LOD) scores of 6.89 and 3.09, respectively. Cecal expression of IL-12/23p40 in H. hepaticus-infected C57BL/6J-Chr3^A/J/NaJ chromosome substitution mice had an intermediate phenotype, significantly higher than in resistant C57BL/6 but lower than in susceptible A/J mice, confirming the importance of this locus to the immune response to H. hepaticus infection.     

Hcp and VgrG1 are secreted components of the Helicobacter hepaticus type VI secretion system and VgrG1 increases the bacterial colitogenic potential

The enterohepatic Epsilonproteobacterium Helicobacter hepaticus persistently colonizes the intestine of mice and causes chronic inflammatory symptoms in susceptible mouse strains. The bacterial factors causing intestinal inflammation are poorly characterized. A large genomic pathogenicity island, HHGI1, which encodes components of a type VI secretion system (T6SS), was previously shown to contribute to the colitogenic potential of H. hepaticus. We have now characterized the T6SS components Hcp, VgrG1, VgrG2 and VgrG3, encoded on HHGI1, including the potential impact of the T6SS on intestinal inflammation in a mouse T‐cell transfer model. The H. hepaticus T6SS components were expressed during the infection and secreted in a T6SS‐dependent manner, when the bacteria were cultured either in the presence or in the absence of mouse intestinal epithelial cells. Mutants deficient in VgrG1 displayed a significantly lower colitogenic potential in T‐cell‐transferred C57BL/6 Rag2^?/? mice, despite an unaltered ability to colonize mice persistently. Intestinal microbiota analyses demonstrated only minor changes in mice infected with wild‐typeH. hepaticus as compared with mice infected with VgrG1‐deficient isogenic bacteria. In addition, competitive assays between both wild‐type and T6SS‐deficient H. hepaticus, and between wild‐type H. hepaticus and Campylobacter jejuni or Enterobacteriaceae species did not show an effect of the T6SS on interbacterial competitiveness. Therefore, we suggest that microbiota alterations did not play a major role in the changes of pro‐inflammatory potential mediated by the T6SS. Cellular innate pro‐inflammatory responses were increased by the secreted T6SS proteins VgrG1 and VgrG2. We therefore concluded that the type VI secretion component VgrG1 can modulate and specifically exacerbate the innate pro‐inflammatory effect of the chronic H. hepaticus infection.     

Host adaptive immunity alters gut microbiota

It has long been recognized that the mammalian gut microbiota has a role in the development and activation of the host immune system. Much less is known on how host immunity regulates the gut microbiota. Here we investigated the role of adaptive immunity on the mouse distal gut microbial composition by sequencing 16 S rRNA genes from microbiota of immunodeficient Rag1^−/− mice, versus wild-type mice, under the same housing environment. To detect possible interactions among immunological status, age and variability from anatomical sites, we analyzed samples from the cecum, colon, colonic mucus and feces before and after weaning. High-throughput sequencing showed that Firmicutes, Bacteroidetes and Verrucomicrobia dominated mouse gut bacterial communities. Rag1⁻ mice had a distinct microbiota that was phylogenetically different from wild-type mice. In particular, the bacterium Akkermansia muciniphila was highly enriched in Rag1^−/− mice compared with the wild type. This enrichment was suppressed when Rag1^−/− mice received bone marrows from wild-type mice. The microbial community diversity increased with age, albeit the magnitude depended on Rag1 status. In addition, Rag1^−/− mice had a higher gain in microbiota richness and evenness with increase in age compared with wild-type mice, possibly due to the lack of pressure from the adaptive immune system. Our results suggest that adaptive immunity has a pervasive role in regulating gut microbiota''s composition and diversity.     

Secretory Antibodies Do Not Affect the Composition of the Bacterial Microbiota in the Terminal Ileum of 10-Week-Old Mice

Terminal restriction fragment length polymorphism (T-RFLP) analysis was conducted on the 16S rRNA genes of the bacterial communities colonizing the epithelial surfaces of the terminal ilea of open conventionally housed mice in an institutional small-animal facility. Polymeric-immunoglobulin-receptor-deficient (pIgR^−/−) mice that were unable to secrete antibodies across mucosal surfaces were cohoused with normal and otherwise genetically identical wild-type (C57BL/6) mice for 4 weeks. If secretory antibodies played a role in modeling the gastrointestinal microbiota, C57BL/6 mice would have had a more distinct and uniform microbiota than their pIgR^−/− cage mates. The T-RFLP profiles of the bacterial communities were compared by using Sorensen's pairwise similarity coefficient, a newly developed weighted pairwise similarity coefficient, and on the basis of Shannon's and Simpson's diversity indices. No systematic differences were observed between the dominant components of the mucosa-associated bacterial communities of the terminal ileal walls of the two types of mice, indicating that secretory antibodies do not control the composition of this microbiota. Similar analyses of experiments conducted at two different times, between which the bacterial community composition of the mouse colony in the small-animal facility appeared to have changed, showed that differences could have been detected, had they existed.     

Role of Mast Cells in Inflammatory Bowel Disease and Inflammation-Associated Colorectal Neoplasia in IL-10-Deficient Mice

Background

Inflammatory bowel disease (IBD) is hypothesized to result from stimulation of immune responses against resident intestinal bacteria within a genetically susceptible host. Mast cells may play a critical role in IBD pathogenesis, since they are typically located just beneath the intestinal mucosal barrier and can be activated by bacterial antigens.

Methodology/Principal Findings

This study investigated effects of mast cells on inflammation and associated neoplasia in IBD-susceptible interleukin (IL)-10-deficient mice with and without mast cells. IL-10-deficient mast cells produced more pro-inflammatory cytokines in vitro both constitutively and when triggered, compared with wild type mast cells. However despite this enhanced in vitro response, mast cell-sufficient Il10 ^−/− mice actually had decreased cecal expression of tumor necrosis factor (TNF) and interferon (IFN)-γ mRNA, suggesting that mast cells regulate inflammation in vivo. Mast cell deficiency predisposed Il10 ^−/− mice to the development of spontaneous colitis and resulted in increased intestinal permeability in vivo that preceded the development of colon inflammation. However, mast cell deficiency did not affect the severity of IBD triggered by non-steroidal anti-inflammatory agents (NSAID) exposure or helicobacter infection that also affect intestinal permeability.

Conclusions/Significance

Mast cells thus appear to have a primarily protective role within the colonic microenvironment by enhancing the efficacy of the mucosal barrier. In addition, although mast cells were previously implicated in progression of sporadic colon cancers, mast cells did not affect the incidence or severity of colonic neoplasia in this inflammation-associated model.     

Comparison of Effects of Trichuris muris and Spontaneous Colitis on the Proximal Colon Microbiota in C3H/HeJ and C3Bir IL10–/– Mice

The nematode Trichuris muris has been shown to interact with specific enteric bacteria, but its effects on the composition of its host''s microbial community are not fully understood. We hypothesized that Trichuris muris-infected mice would have altered colon microbiota as compared with uninfected mice. Colon histopathology and microbial community structure and composition were examined in mouse models of colitis (C3BirTLR4^−/− IL10^−/− and C3H/HeJ TLR4^−/− IL10^+/+ mice) with and without T. muris infection, in uninfected C3BirIL10^−/− mice with and without spontaneous colitis, and in normal C3H/HeJ mice. T. muris-infected mice developed colon lesions that were more severe than those seen in IL10-deficient mice. Approximately 80% of infected IL10^−/− mice had colon neutrophilic exudates, and some had extraintestinal worms and bacteria. The composition and structure of proximal colon microbiota were assessed by using terminal restriction fragment length polymorphism analysis targeting the bacterial 16S rRNA gene. Colon microbiota in C3BirIL10^−/− and C3H/HeJ mice differed both qualitatively and quantitatively. Trichuris infection significantly altered the relative abundance of individual operational taxonomic units [OTU] but not the composition (presence or absence of OTU) of colon microbiota in the 2 mouse genotypes. When C3BirIL10^−/− and C3H/HeJ mouse OTU were considered separately, Trichuris was found to affect the microbiota of C3BirIL10^−/− mice but not of C3H/HeJ mice. Even though 34 of the 75 (45%) C3BirIL10^−/− mice had spontaneous colitis, neither qualitative nor quantitative differences were detected in microbiota between colitic or noncolitic C3BirIL10^−/− mice or noncolitic C3H/HeJ mice. Therefore, Trichuris-infected mice developed distinct microbial communities that were influenced by host background genes; these alterations cannot be attributed solely to colonic inflammation.roup method with arithmetic averaging; OTU, operational taxonomic unit; qPCR, quantitative real-time PCR; SIMPER, similarity percentage; T-RFLP, terminal restriction fragment length polymorphism

Trichuris spp. are gastrointestinal nematodes that dwell in close association with a complex bacterial community in the host''s colon. After ingestion, embryonated eggs hatch in the cecum or colon releasing first-stage larvae that penetrate the epithelium and undergo 4 molts before becoming sexually mature. Both larval and adult Trichuris form syncytial tunnels in the colonic epithelium^21,30 that anchor the organisms in the proximal colon, where females produce eggs that pass in feces and embryonate in the environment.T. suis excretory secretory products (ESP) condition the colonic environment for enhanced worm survival, including effects on intestinal bacteria. Previous work demonstrated that T. suis ESP had dose-dependent effects on the tight junctions of epithelial cells.¹ The ESP fraction below a molecular weight of 10,000 kDa was mainly composed of an antimicrobial moiety² with bactericidal activity against gram-negative (Campylobacter jejuni, C. coli, and Escherichia coli) and gram-positive (Staphylococcus aureus) bacteria. In addition, due to several enzymatic activities, T. suis ESP have been demonstrated to aid the worms in burrowing into the host''s colonic epithelium and in feeding.^1,10,12 In addition to a 20-kDa diagnostic antigen,^10,11 higher molecular-weight fractions of ESP harbored a 42-kDa zinc metalloprotease that likely functions to provide nutrition for the worms through collagenase and elastase activities.¹⁰ Furthermore, a serine protease inhibitor (TsCEI) was purified from adult-stage T. suis by using acid precipitation, affinity chromatography, and reverse-phase HPLC.³³ This 6.43-kDa TsCEI inhibited chymotrypsin, pancreatic elastase, neutrophil elastase, and cathepsin G and was suggested to function as a parasite defense mechanism by modulating host immune responses. Indeed, exposure of cultured epithelial cells to T. suis ESP elicited IL6 and IL10 cytokine responses.³¹Trichuris has also been reported to interact with bacteria in vivo. Early studies demonstrated development of diarrhea in weaning age pigs concurrently harboring T. suis and various bacteria.³⁵ A mixed inoculum of T. suis and cecal scrapings containing Brachyspira, Campylobacter spp., or Salmonella spp. were implicated in this diarrhea by means of passive transfer to SPF pigs.³⁵ Interactions between this helminth and enteric bacteria were also explored by antibiotic treatment of T. suis-infected pigs.^20,27 Results of both passive transfer and antibiotic treatment experiments in pigs showed that Trichuris and various bacterial strains were necessary to produce the type of diarrhea and colonic lesions seen in weaning aged pigs in production, but did not implicate a single bacterial agent. In 2003, synergism between T. suis and C. jejuni was proven to cause mucohemorrhagic colitis in that germ-free piglets inoculated with both agents developed disease, whereas those infected with a single agent did not.²⁵ Recent studies in T. suis-infected pigs show changes in the microbial community of the colon with some accompanying metabolic changes.^22,45 Similar interactions have been found in extensive studies of captive rhesus monkeys with chronic enterocolitis. In these monkeys, severe disease was associated with presence of Trichuris trichiura and several enteric pathogens including C. coli, C. jejuni, Shigella flexneri, Yersinia enterocolitica, adenovirus, and Strongyloides fulleborni.³⁸ Therefore, Trichuris interacts with and may demonstrate synergy in disease production with the host''s colonic microflora.Interactions between Trichuris and bacteria have also been studied in mice.^9,20,36 One study found 100% morbidity in C57BL/6 IL10^−/− and congenic IL10^−/− IL4^−/− mice after challenge with T. muris.³⁶ The authors hypothesized that this high morbidity was due to an overgrowth of opportunistic invasive bacteria that use the mechanical damage caused by T. muris larvae to breach the intestinal tract. Adding the broad-spectrum antibiotic neomycin sulfate to the drinking water of IL10^−/− IL4^−/− mice and then infecting them with T. muris resulted in a statistically significant increase in the percentage of mice that survived infection.³⁶ The authors concluded that growth of opportunistic bacteria may have contributed to the previously observed morbidity and mortality. Most recently, another group⁹ found that increased levels of colonic microflora favor increased numbers of T. muris and chronic infections. The group also demonstrated that T. muris eggs hatched more efficiently in vitro when incubated with explants of mouse cecum containing 5 isolates of bacteria (E. coli, Staphylococcus aureus, Salmonella typhimurium, or Pseudomonas aeruginosa) and the yeast Saccharomyces cerevisiae, with the greatest effects seen at 37 °C. Similarly, work from our laboratory²⁰ demonstrated that treatment of T. muris-infected C57BL/6 IL10^−/− mice with metronidazole but not prednisolone increased survival.²⁰ Most recently, chronic infections with T. muris in C57BL/6 mice have been shown to decrease the diversity of intestinal microbiota,¹³ increase the abundance of Lactobacillus spp., and alter the metabolome.¹⁴Taken together, these data suggest an important microbial component to the pathogenesis of Trichuris infections in a variety of species. Given that Trichuris suis has been administered to patients with inflammatory bowel disease (IBD), and in some studies appeared to diminish IBD symptoms^42,43 we sought to understand the community-wide interactions of this worm with enteric bacteria in a mouse model of colitis. We hypothesized that the microbiota of the proximal colon would differ significantly in mice infected with T. muris as compared with uninfected mice. We theorized that these effects would occur due to the worm''s immunomodulatory properties in the host and may contribute to the successful outcomes of Trichuris treatment in patients with IBD.     

Characterization of the Fecal Microbiota of Pigs before and after Inoculation with “Brachyspira hampsonii”

“Brachyspira hampsonii” causes disease indistinguishable from swine dysentery, and the structure of the intestinal microbiome likely plays a role in determining susceptibility of individual pigs to infection and development of clinical disease. The objectives of the current study were to determine if the pre-inoculation fecal microbiota differed between inoculated pigs that did (INOC MH) or did not (INOC non-MH) develop mucohaemorrhagic diarrhea following challenge with “B. hampsonii”, and to quantify changes in the structure of the microbiome following development of clinical disease. Fecal microbiota profiles were generated based on amplification and sequencing of the cpn60 universal target sequence from 89 samples from 18 pigs collected at −8, −5, −3 and 0 days post-inoculation, and at termination. No significant differences in richness, diversity or taxonomic composition distinguished the pre-inoculation microbiomes of INOC MH and INOC non-MH pigs. However, the development of bloody diarrhea in inoculated pigs was associated with perturbation of the microbiota relative to INOC non-MH or sham-inoculated control pigs. Specifically, the fecal microbiota of INOC MH pigs was less dense (fewer total 16S rRNA copies per gram of feces), and had a lower Bacteroidetes:Firmicutes ratio. Further investigation of the potential long-term effects of Brachyspira disease on intestinal health and performance is warranted.     

Role of the Helicobacter hepaticus flagellar sigma factor FliA in gene regulation and murine colonization

The enterohepatic Helicobacter species Helicobacter hepaticus colonizes the murine intestinal and hepatobiliary tract and is associated with chronic intestinal inflammation, gall stone formation, hepatitis, and hepatocellular carcinoma. Thus far, the role of H. hepaticus motility and flagella in intestinal colonization is unknown. In other, closely related bacteria, late flagellar genes are mainly regulated by the sigma factor FliA (σ²⁸). We investigated the function of the H. hepaticus FliA in gene regulation, flagellar biosynthesis, motility, and murine colonization. Competitive microarray analysis of the wild type versus an isogenic fliA mutant revealed that 11 genes were significantly more highly expressed in wild-type bacteria and 2 genes were significantly more highly expressed in the fliA mutant. Most of these were flagellar genes, but four novel FliA-regulated genes of unknown function were identified. H. hepaticus possesses two identical copies of the gene encoding the FliA-dependent major flagellin subunit FlaA (open reading frames HH1364 and HH1653). We characterized the phenotypes of mutants in which fliA or one or both copies of the flaA gene were knocked out. flaA_1 flaA_2 double mutants and fliA mutants did not synthesize detectable amounts of FlaA and possessed severely truncated flagella. Also, both mutants were nonmotile and unable to colonize mice. Mutants with either flaA gene knocked out produced flagella morphologically similar to those of wild-type bacteria and expressed FlaA and FlaB. flaA_1 mutants which had flagella but displayed reduced motility did not colonize mice, indicating that motility is required for intestinal colonization by H. hepaticus and that the presence of flagella alone is not sufficient.     

Helicobacter typhlonius and Helicobacter rodentium Differentially Affect the Severity of Colon Inflammation and Inflammation-Associated Neoplasia in IL10-Deficient Mice

Infection with Helicobacter species is endemic in many animal facilities and may alter the penetrance of inflammatory bowel disease (IBD) phenotypes. However, little is known about the relative pathogenicity of H. typhlonius, H. rodentium, and combined infection in IBD models. We infected adult and neonatal IL10^−/− mice with H. typhlonius, H. rodentium, or both bacteria. The severity of IBD and incidence of inflammation-associated colonic neoplasia were assessed in the presence and absence of antiHelicobacter therapy. Infected IL10^−/− mice developed IBD with severity of noninfected (minimal to no inflammation) < H. rodentium < H. typhlonius < mixed H. rodentium + H. typhlonius (severe inflammation). Inflammation-associated colonic neoplasia was common in infected mice and its incidence correlated with IBD severity. Combined treatment with amoxicillin, clarithromycin, metronidazole, and omeprazole eradicated Helicobacter in infected mice and ameliorated established IBD in both infected and noninfected mice. Infection of IL10^−/− mice with H. rodentium, H. typhlonius, or both organisms can trigger development of severe IBD that eventually leads to colonic neoplasia. The high incidence and multiplicity of neoplastic lesions in infected mice make this model well-suited for future research related to the development and chemoprevention of inflammation-associated colon cancer. The similar antiinflammatory effect of antibiotic therapy in Helicobacter-infected and -noninfected IL10^−/− mice with colitis indicates that unidentified microbiota in addition to Helicobacter drive the inflammatory process in this model. This finding suggests a complex role for both Helicobacter and other intestinal microbiota in the onset and perpetuation of IBD in these susceptible hosts.Abbreviations: IBD, Inflammatory bowel diseaseInflammatory bowel disease (IBD) is hypothesized to develop due to aberrant immune responses induced by gut microbes.⁵ IBD does not occur in germ-free IL10^−/− mice,^2,15 indicating the importance of microorganisms as environmental triggers of intestinal inflammation. However, conventionally colonized or specific pathogen-free IL10^−/− mice may develop colitis spontaneously^2,32 or in response to specific triggers such as nonsteroidal antiinflammatory drugs^3,14 or infections with certain bacteria.^6,16,18 The normal lack of ongoing immune responses against bacteria in subjects without IBD has been attributed to the immunologic tolerance that specifically downregulates immune responses against antigens derived from these bacteria. Nevertheless, despite a large number of studies, no single bacterial type has fulfilled Koch postulates and been confirmed as a cause of IBD in animals or humans.Previous studies used fluorescence in situ hybridization with probes specific for bacterial 16S rRNA combined with conventional histologic techniques to study the relationships between various species of intestinal bacteria and the mucosa in mice and humans with IBD.^33,34 Those studies showed that in normal mice, most bacterial groups are separated from the mucosal surface by either a mucus layer that excludes bacteria or, in the cecum and proximal colon, by an ‘interlaced’ layer that is composed of tightly packed bacteria. The interlaced or mucus layer thus limits the contact of the bulk of the enteric bacteria with the mucosal epithelium. In contrast, complex biofilms composed of multiple species of bacteria that were firmly adherent to the mucosal surface were identified in the majority of colon tissue samples collected from humans and mice with IBD.^33,34 The presence of a biofilm abrogates the protective effects of the normal layer of mucus and can allow luminal bacterial antigens and toxins to reach the unshielded epithelial surface, where they can trigger cascades of host inflammatory responses. Situations that cause defects in the epithelial surface or degrade the protective qualities of mucus or the interlaced layer (or both) may allow contact of bacterial antigens and adjuvants with immune cells located in the lamina propria and lead to the generation of immune responses that result in IBD.³⁴Helicobacter species are used frequently to model microbial triggers of colon inflammation, because they have previously been linked to the development of both IBD- and inflammation-associated neoplasia.^11,21,29 Most studies have been performed by using Helicobacter hepaticus or H. bilis.²⁰ However, H. typhlonius, H. rodentium, H. muridarum, H. ganmani, H. trogontum and other species^{8,12,17,29,35} can also be endemic in research animal facilities. The pathophysiologic effects of these less-common Helicobacter species are, for the most part, poorly investigated.Most rodent Helicobacter species are urease-negative and therefore preferentially colonize the intestine, but some species produce urease enzyme and can translocate to the liver or colonize the biliary system.¹³ H. typhlonius was shown to cause an enteric disease characterized by mucosal hyperplasia and associated inflammation in the cecum and colon in immunodeficient mice^11,23 and IL10^−/− mice.¹⁸ H. typhlonius is genetically related most closely to H. hepaticus, having only 2.36% difference in the 16S rRNA gene sequence, but H. typhlonius has a unique intervening sequence in this gene that makes it easily recognizable by PCR.^9,12 Molecular detection of this pathogen with PCR is rapid, sensitive and allows the detection of the early phases of infection; further enhanced sensitivity is achieved with nested primers.²² One of the most important features of PCR is that it can be performed noninvasively on fecal pellets. Data regarding the pathogenetic mechanisms of H. rodentium are scarce.^35,36 H. rodentium alone apparently does not cause hepatitis or enteritis in A/JCr or C.B-17/IcrCrl-scidBr mice; however, coinfection with H. hepaticus and H. rodentium was associated with augmented cecal gene expression and clinical diarrheal disease in immunodeficient mice compared with mice infected with H. hepaticus alone.²³Previous reports demonstrated that H. typhlonius was capable of initiating colitis in adult IL10^−/− mice.^10,11 In those studies, colitis was relatively mild, with no development of inflammation-associated neoplasia. H. rodentium has been described to be nonpathogenic in adult wild-type mice but did enhance cytokine production in the cecum of mice also infected with H. hepaticus.²³ We recently observed rapid onset of severe IBD and a high incidence of inflammation-associated neoplasia in IL10^−/− mice that were coinfected with both H. typhlonius and H. rodentium as pups.¹⁶ The current study was undertaken to determine the relative roles of H. rodentium and H. typhlonius, individually and in combination, and age at infection in the development of colon inflammation and inflammation-associated neoplasia in IL10^−/− mice. Novel features of our model include controlled infection of the combination of H. typhlonius and H. rodentium⁹ and infection of IL10^−/− mice during the neonatal period.