Role of natural killer T cells in the mouse colitis-associated colon cancer model

Invariant natural killer T (iNKT) cells are considered innate-like lymphocytes, and regulate the immunity against inflammation and tumorigenesis. However, the impact of iNKT cells in inflammation-associated tumorigenesis remains unclear. In this study, we examined the physiological role of iNKT cells in a mouse colitis-associated colorectal cancer model. C57BL/6 (B6) and Jα18 NKT cell-deficient KO (KO) mice were used. Colitis-associated colorectal cancer was induced by azoxymethane (AOM) and dextran sodium sulfate (DSS). The resulting inflammation and tumours were examined. The surface markers of mononuclear cells from the liver and the colon were assessed by FACS. The levels of IL-13 from the colon were measured by ELISA. α-galactosylceramide (GC), or its close analog OCH, was administered intraperitoneally on the first day of each cycle of DSS-administration. In the AOM/DSS model, hepatic iNKT cells were significantly decreased. In KO mice there were significantly greater numbers of colon tumours and more severe inflammation than in B6 mice. FACS analysis revealed that the population of NK1.1 (+) T cells (non-invariant NKT cells) in the colon was increased when compared to B6 mice. The secretion of IL-13 was increased in the colon of KO mice after AOM/DSS. The number of colon tumours was significantly decreased in the GC-treated group compared to the control group. GC-treatment significantly inhibited IL-13 secretion from the colonic mononuclear cells and the number of colonic NK1.1 (+) T cells was significantly decreased. These results suggest that iNKT cells may play a critical role in the prevention of tumour progression and inflammation in the AOM/DSS model.     

SIGIRR,a negative regulator of Toll-like receptor-interleukin 1 receptor signaling

The Toll-like receptor-interleukin 1 receptor signaling (TLR-IL-1R) receptor superfamily is important in differentially recognizing pathogen products and eliciting appropriate immune responses. These receptors alter gene expression, mainly through the activation of nuclear factor-kappaB and activating protein 1. SIGIRR (single immunoglobulin IL-1R-related molecule), a member of this family that does not activate these factors, instead negatively modulates immune responses. Inflammation is enhanced in SIGIRR-deficient mice, as shown by their enhanced chemokine induction after IL-1 injection and reduced threshold for lethal endotoxin challenge. Cells from SIGIRR-deficient mice showed enhanced activation in response to either IL-1 or certain Toll ligands. Finally, biochemical analysis indicated that SIGIRR binds to the TLR-IL-1R signaling components in a ligand-dependent way. Our data show that SIGIRR functions as a biologically important modulator of TLR-IL-1R signaling.     

NLRP6 function in inflammatory monocytes reduces susceptibility to chemically induced intestinal injury

NLRP6 is a member of the Nod-like receptor family, whose members are involved in the recognition of microbes and/or tissue injury. NLRP6 was previously demonstrated to regulate the production of interleukin (IL)-18 and is important for protecting mice against chemically induced intestinal injury and colitis-associated colon cancer. However, the cellular mechanisms by which NLRP6 reduces susceptibility to colonic inflammation remain unclear. Here, we determined that NLRP6 expression is specifically upregulated in Ly6C^hi inflammatory monocytes that infiltrate into the colon during dextran sulfate sodium (DSS)-induced inflammation. Adoptive transfer of wild-type (WT) Ly6C^hi inflammatory monocytes into Nlrp6^−/− mice was sufficient to protect them from mortality, significantly reducing intestinal permeability and damage. NLRP6-deficient inflammatory monocytes were defective in tumor necrosis factor α (TNFα) production, which was important for reducing DSS-induced mortality and was dependent on autocrine IL-18 signaling by inflammatory monocytes. Our data reveal a previously unappreciated role for NLRP6 in inflammatory monocytes, which are recruited after DSS-induced intestinal injury to promote barrier function and limit bacteria-driven inflammation. This study highlights the importance of early cytokine responses, particularly NLRP6-dependent and IL-18-dependent TNFα production, in preventing chronic dysregulated inflammation.     

Commensal Gram-positive bacteria initiates colitis by inducing monocyte/macrophage mobilization

Breakdown of the intestinal epithelial layer's barrier function results in the inflow of commensal flora and improper immune responses against the commensal flora, leading to inflammatory bowel disease (IBD) development. Using a mouse dextran sodium sulfate (DSS)-induced colitis model, we show here that commensal Gram-positive bacteria trigger the mobilization of inflammatory monocytes and macrophages into the colon. Monocytes/macrophages are major producers of tumor necrosis factor-α (TNF-α), a representative cytokine that aggravates colitis. Notably, pretreating mice with vancomycin, which eliminated Gram-positive bacteria, particularly the Lachnospiraceae family, significantly reduced the severity of the colitis by selectively blocking the recruitment of monocytes/macrophages, but not of other cells. Importantly, vancomycin treatment specifically downregulated the colonic epithelial cell (cEC) expression of C–C chemokine receptor type-2 (CCR2) ligands, which are critical chemokines for monocyte/macrophage mobilization into the inflamed colon. Collectively, these results provide previously undiscovered evidence that Gram-positive commensal bacteria induce colitis by recruiting colitogenic monocytes and macrophages. Our findings may lead to new avenues of treatment for IBD.     

Microbial metabolites,short‐chain fatty acids,restrain tissue bacterial load,chronic inflammation,and associated cancer in the colon of mice

The intestinal immune system is regulated by microbes and their metabolites. The roles of gut microbial metabolites in regulating intestinal inflammation and tumorigenesis are incompletely understood. We systematically studied the roles of short‐chain fatty acids (SCFAs) and their receptors (GPR43 or GPR41) in regulating tissue bacterial load, acute versus chronic inflammatory responses, and intestinal cancer development. SCFA receptor‐, particularly GPR43‐, deficient mice were defective in mounting appropriate acute immune responses to promote barrier immunity, and developed uncontrolled chronic inflammatory responses following epithelial damage. Further, intestinal carcinogenesis was increased in GPR43‐deficient mice. Dietary fiber and SCFA administration suppressed intestinal inflammation and cancer in both GPR43‐dependent and independent manners. The beneficial effect of GPR43 was not mediated by altered microbiota but by host tissue cells and hematopoietic cells to a lesser degree. We found that inability to suppress commensal bacterial invasion into the colonic tissue is associated with the increased chronic Th17‐driven inflammation and carcinogenesis in the intestine of GPR43‐deficient mice. In sum, our results reveal the beneficial function of the SCFA‐GPR43 axis in suppressing bacterial invasion and associated chronic inflammation and carcinogenesis in the colon.     

B cell-intrinsic MyD88 signaling prevents the lethal dissemination of commensal bacteria during colonic damage

The Toll-like receptor adaptor protein MyD88 is essential for the regulation of intestinal homeostasis in mammals. In this study, we determined that Myd88-deficient mice are susceptible to colonic damage that is induced by dextran sulfate sodium (DSS) administration resulting from uncontrolled dissemination of intestinal commensal bacteria. The DSS-induced mortality of Myd88-deficient mice was completely prevented by antibiotic treatment to deplete commensal bacteria. By using cell type-specific Myd88-deficient mice, we established that B cell-intrinsic MyD88 signaling plays a central role in the resistance to DSS-induced colonic damage via the production of IgM and complement-mediated control of intestinal bacteria. Our results indicate that the lack of intact MyD88 signaling in B cells, coupled with impaired epithelial integrity, enables commensal bacteria to function as highly pathogenic organisms, causing rapid host death.     

Regulatory T cells with superior immunosuppressive capacity emigrate from the inflamed colon to draining lymph nodes

Foxp3⁺ Regulatory T cells (Tregs) play a critical role in the maintenance of colon homeostasis. Here we utilized photoconvertible KikGR mice to track immune cells from the caecum and ascending (proximal) colon in the steady state and DSS-induced colitis. We found that Tregs from the proximal colon (colonic migratory Tregs) migrated exclusively to the distal part of mesenteric lymph nodes (dMLN) in an S1PR1-dependent process. In the steady state, colonic migratory CD25⁺ Tregs expressed higher levels of CD103, ICOS, LAG3 and CTLA-4 in comparison with pre-existing LN Tregs. Intestinal inflammation led to accelerated Treg replacement in the colon, bidirectional Treg migration from the colon to dMLN and vice versa, as well as increases in Treg number, proliferation and expression of immunosuppressive molecules. This was especially apparent for CD25 very high Tregs induced in colitis. Furthermore, colonic migratory Tregs from the inflamed colon included more interleukin (IL)-10 producing cells, and demonstrated greater inhibition of T-cell proliferation in comparison with pre-existing LN Tregs. Thus, our results suggest that Tregs with superior immunosuppressive capacity are increased both in the colon and dMLN upon inflammation. These Tregs recirculate between the colon and dMLN, and are likely to contribute to the downregulation of intestinal inflammation.     

Quantitative assessment of azoxymethane-induced aberrant crypt foci in inbred mice

Heritable differences in tumor susceptibility are observed in mice after repetitive exposures to the organotropic colon carcinogen azoxymethane (AOM). The following study was undertaken to determine whether early morphological alterations within the colonic epithelium correlate with subsequent cancer risk. A/J and SWR/J (susceptible) and AKR/J (resistant) mice were injected once a week with AOM at a dose of 10 mg/kg, i.p., for a total of 6 weeks. Four weeks after the last injection, methylene blue-stained whole-mount colons were examined for the presence of colonic epithelial lesions referred to as aberrant crypt foci (ACF). Putative lesions identified under low magnification were further characterized by H&E staining of corresponding sections. AOM produced a treatment-related increase in ACFs in each of the mouse lines examined. The tumor-susceptible SWR/J and A/J mice developed on average between three- and sixfold more ACFs in the distal colon (32 and 15/cm of colon, respectively) than the resistant AKR/J mice (5/cm colon). The size distribution of ACFs was further analyzed in each of the strains. In SWR/J and A/J, 20-35% of lesions were classified as large ACFs, consisting of 5 or more aberrant crypts per focus. This is in striking contrast to the size distribution of lesions identified in the AKR/J colons, where fewer than 5% of grossly identified lesions were classified as large. In fact, the majority (> 80%) of ACFs in AKR/J mice consisted of only 1-2 aberrant crypts@focus. In addition, there was no evidence of dysplasia in any of the AKR/J lesions examined, whereas the lesions in susceptible mice were dysplastic (adenomas). Our data indicate that tumorigenic response is associated with the extent and multiplicity of ACFs that form within the colonic epithelium at an early time point after carcinogen exposure. These studies further support the use of this morphological biomarker as a short-term endpoint of colon tumorigenesis.     

IL-36α expression is elevated in ulcerative colitis and promotes colonic inflammation

A role for the IL-36 family of cytokines has been identified in the pathogenesis of psoriasis. Although significant mechanistic overlap can exist between psoriasis and inflammatory bowel disease (IBD), to date there have been no reports investigating the IL-36 family in gastrointestinal inflammation. Here we demonstrate that expression levels of IL-36α are specifically elevated in the colonic mucosa of ulcerative colitis patients. This elevated expression is mirrored in the inflamed colonic mucosa of mice, wherein IL-36 receptor deficiency confirmed this pathway as a mediator of mucosal inflammation. Il36r−/− mice exhibited reduced disease severity in an acute DSS-induced model of colitis in association with decreased innate inflammatory cell infiltration to the colon lamina propria. Consistent with these data, infection with the enteropathogenic bacteria Citrobacter rodentium, resulted in reduced innate inflammatory cell recruitment and increased bacterial colonization in the colons of il36r−/− mice. Il36r−/− mice also exhibited altered T helper cell responses in this model, with enhanced Th17 and reduced Th1 responses, demonstrating that IL-36R signaling also regulates intestinal mucosal T-cell responses. These data identify a novel role for IL-36 signaling in colonic inflammation and indicate that the IL-36R pathway may represent a novel target for therapeutic intervention in IBD.     

The germ-free mice monocolonization with Bacteroides fragilis improves azoxymethane/dextran sulfate sodium induced colitis-associated colorectal cancer

Objective: Inflammatory bowel disease (IBD) is generally considered as a major risk factor in the progression of colitis-associated colorectal cancer (CAC). Previous studies have indicated that the composition of gut microflora may be involved in CAC induction and progress. Bacteroides fragilis (BF) is a Gram-negative anaerobe belonging to colonic symbiotic bacteria of the host. This study was aimed to investigate the protective role of BF in a colorectal cancer (CRC) model induced by azoxymethane (AOM) and dextran sulfate sodium (DSS) in germ-free (GF) mice.

Materials and methods: Total 22 GF mice were divided into two groups: GF and BF group. Half of the GF mice were colonized with BF for 28?days before CRC induction by AOM/DSS.

Results: BF colonization increased animal survival (100%). Cecum weight and cecum/body weight ratio significantly decreased in BF/AOM/DSS group. Interestingly, there was a significant decrease in tumor number and tumor incidence in the BF/AOM/DSS group as compared to the GF/AOM/DSS group. The adenocarcinoma/adenoma incidence and histologic score were also decreased in the BF/AOM/DSS group. In addition, immunohistochemistry staining found decreased numbers of cell proliferation (PCNA) and inflammatory cell (granulocytes) infiltration in the colon mucosa of the BF group. The β-catenin staining in the BF/AOM/DSS group had fewer and weaker positive signal expressions. Taking together, the BF colonization significantly ameliorated AOM/DSS-induced CRC by suppressing the activity of cell proliferation-related molecules and reducing the number of inflammatory cells.

Conclusions: Symbiotic BF may play a pivotal role in maintaining the gastrointestinal immunophysiologic balance and regulating anti-tumorigenesis responses.     

Non-hematopoietic STAT6 induces epithelial tight junction dysfunction and promotes intestinal inflammation and tumorigenesis

Enhanced gut permeability due to dysregulated epithelial tight junction is often associated with inflammatory bowel diseases (IBD), which have a greater risk for developing colorectal cancer. STAT6 activation was detected in inflamed colonic epithelium of active IBD patients, suggesting a role of epithelial STAT6 in colitis development. Here, we demonstrated that non-hematopoietic STAT6, but not hematopoietic STAT6, triggered DSS-induced colitis and subsequent tumorigenesis. This could be due to the enhancing-effect of STAT6 on gut permeability and microbiota translocation via interruption of epithelial tight junction integrity. Mechanistically, long-myosin light-chain kinase (MLCK1) was identified as a target of STAT6, leading to epithelial tight junction dysfunction and microbiota-driven colitis. Furthermore, neutralization of IL-13, which was primarily derived from type 2 innate lymphoid cells (ILC2) in a microbiota-dependent way, inhibited epithelial STAT6 activation and improved gut permeability and DSS-induced colitis. Importantly, pharmacological inhibition of STAT6 reduces murine intestinal tumor formation, and tumoral p-STAT6 levels positively correlated to the clinical stage and poor prognosis of human colorectal cancer. Thus, our study reveals a direct role of STAT6 in the disruption of epithelial tight junction integrity and colitis development, and suggests STAT6 as a potential therapeutic and prophylactic target for IBD and colitis-associated cancer.     

Upregulation of PD-1 follows tumour development in the AOM/DSS model of inflammation-induced colorectal cancer in mice

Chronic inflammation may drive development of cancer as observed in inflammation-induced colorectal cancer (CRC). Though immune cells can infiltrate the tumour microenvironment, cancer cells seem to evade anti-tumour responses, which is one of the established hallmarks of cancer. Targeting the programmed cell death protein-1 (PD-1)/PD-L1 signalling pathway is currently at the forefront in the development of anti-tumour immunity-based therapies for multiple malignancies. By blocking the immune-checkpoint of activated T-cells, it is possible to rewire the adaptive resistance induced by the PD-1 ligands expressed in the tumour microenvironment. However, adverse immunotherapy-modulated events could complicate the treatment of individuals with preexisting chronic inflammatory conditions. In this study, we investigated the expression of different systemic and mucosal T-cell subsets during the course of azoxymethane (AOM)/dextran sulphate sodium (DSS)-induced colitis and colitis-associated CRC. In addition, we examined the expression of PD-1 and its ligands PD-L1 and PD-L2 as well as other molecular targets related to T-cell exhaustion. We found a significant increase in PD-1 expression on all examined mucosal T-cell subsets of the colon and the ileum, which correlated with disease progression. We also observed an upregulation of PD-L1 and PD-L2 mRNA expression throughout the AOM/DSS regime. Blocking PD-1 signalling with an anti-PD1 antibody did not affect the tumour burden in the AOM/DSS-treated mice, but did potentiate the weight loss in the third DSS cycle, indicating possible immune-mediated toxicity. This raises a concern for patients with colitis-associated CRCs and should be further investigated.     

Negative regulation of Toll-like receptor signaling plays an essential role in homeostasis of the intestine

A healthy intestinal tract is characterized by controlled homeostasis due to the balanced interaction between commensal bacteria and the host mucosal immune system. Human and animal model studies have supported the hypothesis that breakdown of this homeostasis may underlie the pathogenesis of inflammatory bowel diseases. However, it is not well understood how intestinal microflora stimulate the intestinal mucosal immune system and how such activation is regulated. Using a spontaneous, commensal bacteria-dependent colitis model in IL-10-deficient mice, we investigated the role of TLR and their negative regulation in intestinal homeostasis. In addition to IL-10(-/-) MyD88(-/-) mice, IL-10(-/-) TLR4(-/-) mice exhibited reduced colitis compared to IL-10(-/-) mice, indicating that TLR4 signaling plays an important role in inducing colitis. Interestingly, the expression of IRAK-M, a negative regulator of TLR signaling, is dependent on intestinal commensal flora, as IRAK-M expression was reduced in mice re-derived into a germ-free environment, and introduction of commensal bacteria into germ-free mice induced IRAK-M expression. IL-10(-/-) IRAK-M(-/-) mice exhibited exacerbated colitis with increased inflammatory cytokine gene expression. Therefore, this study indicates that intestinal microflora stimulate the colitogenic immune system through TLR and negative regulation of TLR signaling is essential in maintaining intestinal homeostasis.     

The myeloid immune signature of enterotoxigenic Bacteroides fragilis-induced murine colon tumorigenesis

Enterotoxigenic Bacteroides fragilis (ETBF), a human commensal and candidate pathogen in colorectal cancer (CRC), is a potent initiator of interleukin-17 (IL-17)-dependent colon tumorigenesis in Min^Apc+/− mice. We examined the role of IL-17 and ETBF on the differentiation of myeloid cells into myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages, which are known to promote tumorigenesis. The myeloid compartment associated with ETBF-induced colon tumorigenesis in Min mice was defined using flow cytometry and gene expression profiling. Cell-sorted immature myeloid cells were functionally assayed for inhibition of T-cell proliferation and inducible nitric oxide synthase expression to delineate MDSC populations. A comparison of ETBF infection with that of other oncogenic bacteria (Fusobacterium nucleatum or pks⁺Escherichia coli) revealed a specific, ETBF-associated colonic immune infiltrate. ETBF-triggered colon tumorigenesis is associated with an IL-17-driven myeloid signature characterized by subversion of steady-state myelopoiesis in favor of the generation of protumoral monocytic-MDSCs (MO-MDSCs). Combined action of the B. fragilis enterotoxin BFT and IL-17 on colonic epithelial cells promoted the differentiation of MO-MDSCs, which selectively upregulated Arg1 and Nos2, produced NO, and suppressed T-cell proliferation. Evidence of a pathogenic inflammatory signature in humans colonized with ETBF may allow for the identification of populations at risk for developing colon cancer.     

Interleukin-1beta mediates the extra-intestinal thrombosis associated with experimental colitis

Inflammatory bowel diseases (IBDs) are associated with an increased risk for thromboembolism, which is often manifested as deep vein thrombosis or pulmonary embolism, at extra-intestinal sites. Although some of the cytokines that contribute to IBD pathogenesis are also known to alter the coagulation pathway, it remains unclear whether these mediators also contribute to the extra-intestinal thrombosis often associated with IBD. The objective of this study is to evaluate the role of interleukin (IL)-1β in enhanced extra-intestinal thrombosis observed in mice with dextran sodium sulfate (DSS)-induced colitis. IL-1β concentrations were measured in plasma, colon, and skeletal muscle of wild-type (WT) control and colitic mice. Microvascular thrombosis was induced in cremaster muscle microvessels by using a light/dye injury model. The effects of exogenous IL-1β on thrombus formation were determined in control WT mice. DSS-induced thrombogenesis was evaluated in WT mice treated with an IL-1β antibody and in IL-1 receptor-deficient (IL-1r(-/-)) mice. DSS-induced colonic inflammation in WT mice was associated with enhanced thrombus formation in arterioles. IL-1β concentrations were elevated in inflamed colon and skeletal muscle. Exogenous IL-1β enhanced thrombosis in control mice in a dose-dependent manner. DSS colitic mice treated with the IL-1β antibody as well as IL-1r(-/-) mice exhibited significantly blunted thrombogenic responses. These findings implicate IL-1β as a mediator of enhanced microvascular thromboses that occur in extra-intestinal tissues during colonic inflammation.     

Neutrophil infiltration favors colitis-associated tumorigenesis by activating the interleukin-1 (IL-1)/IL-6 axis

Neutrophil infiltration is a key event in chronic intestinal inflammation and associated colorectal cancer, but how these cells support cancer development is poorly understood. In this study, using a mouse model of colitis-associated cancer (CAC), we have demonstrated that infiltrated neutrophils produce large amounts of interleukin-1 (IL)-1β that is critical for the development of CAC. Depletion of neutrophil or blockade of IL-1β activity significantly reduced mucosal damage and tumor formation. This protumorigenic function of IL-1β was mainly attributed to increased IL-6 secretion by intestine-resident mononuclear phagocytes (MPs). Furthermore, commensal flora-derived lipopolysaccharide (LPS) was identified to trigger IL-1β expression in neutrophils. Importantly, accumulation of IL-1β-expressing neutrophils was seen in lesions of patients suffering from ulceratic CAC and these infiltrated neutrophils induced IL-6 production by intestinal MPs in an IL-1β-dependent manner. Overall, these findings reveal that in CAC milieu, infiltrating neutrophils secrete IL-1β that promotes tumorigenesis by inducing IL-6 production by intestinal MPs.     

Oral administration of a recombinant cholera toxin B subunit promotes mucosal healing in the colon

Cholera toxin B subunit (CTB) is a component of a licensed oral cholera vaccine. However, CTB has pleiotropic immunomodulatory effects whose impacts on the gut are not fully understood. Here, we found that oral administration in mice of a plant-made recombinant CTB (CTBp) significantly increased several immune cell populations in the colon lamina propria. Global gene expression analysis revealed that CTBp had more pronounced impacts on the colon than the small intestine, with significant activation of TGFβ-mediated pathways in the colon epithelium. The clinical relevance of CTBp-induced impacts on colonic mucosa was examined. In a human colon epithelial model using Caco2 cells, CTBp, but not the non-GM1-binding mutant G33D-CTBp, induced TGFβ-mediated wound healing. In a dextran sodium sulfate (DSS) acute colitis mouse model, oral administration of CTBp protected against colon mucosal damage as manifested by mitigated body weight loss, decreased histopathological scores, and blunted escalation of inflammatory cytokine levels while inducing wound healing-related genes. Furthermore, biweekly oral administration of CTBp significantly reduced disease severity and tumorigenesis in the azoxymethane/DSS model of ulcerative colitis and colon cancer. Altogether, these results demonstrate CTBp's ability to enhance mucosal healing in the colon, highlighting its potential application in ulcerative colitis therapy besides cholera vaccination.