Rishu Dheer

Cancer in Inflammatory Bowel Disease: lessons from animal models

Purpose of review Human colitis-associated cancers (CAC) represent a heterogeneous group of conditions in which multiple oncogenic pathways are involved. In this article, we review the latest studies using genetic, chemical, bacterial and innate immune-mediated experimental models of CAC. Recent findings Using the azoxymethane-dextran sodium sulfate model, wound healing pathways seem to be required in the development of CAC. There is also an emerging understanding that commensal and/or pathogenic bacteria can promote tumorigenesis, through T cell and TLR-mediated inflammation. Using specific transgenic mice (villin-CD98, T cell SMAD7, villin-TLR4) or specific knockout mice, investigators have determined that derangements in epithelial or innate and adaptive immune pathways can result in CAC. Subtle perturbations in epithelial repair – both too little or too exuberant – can render mice susceptible to tumorigenesis. Summary With the aid of animal models, we have witnessed a rapid expansion of our knowledge of the molecular and immunologic mechanisms underlying inflammatory cancers. Though animal models have contributed a discrete amount of information to our understanding of tumorigenesis in the setting of intestinal inflammation, it is clear that no single animal model will be able to adequately recapitulate the pathogenesis of complex colorectal cancers, but each model gets us one step closer to comprehending the nature of CAC.

Intestinal Epithelial Toll-Like Receptor 4 Signaling Affects Epithelial Function and Colonic Microbiota and Promotes a Risk for Transmissible Colitis

ABSTRACT Evidence obtained from gene knockout studies supports the role of Toll-like receptor 4 (TLR4) in intestinal inflammation and microbiota recognition. Increased epithelial TLR4 expression is observed in patients with inflammatory bowel disease. However, little is known of the effect of increased TLR4 signaling on intestinal homeostasis. Here, we examined the effect of increased TLR4 signaling on epithelial function and microbiota by using transgenic villin-TLR4 mice that overexpress TLR4 in the intestinal epithelium. Our results revealed that villin-TLR4 mice are characterized by increases in the density of mucosa-associated bacteria and bacterial translocation. Furthermore, increased epithelial TLR4 signaling was associated with an impaired epithelial barrier, altered expression of antimicrobial peptide genes, and altered epithelial cell differentiation. The composition of the colonic luminal and mucosa-associated microbiota differed between villin-TLR4 and wild-type (WT) littermates. Interestingly, WT mice cohoused with villin-TLR4 mice displayed greater susceptibility to acute colitis than singly housed WT mice did. The results of this study suggest that epithelial TLR4 expression shapes the microbiota and affects the functional properties of the epithelium. The changes in the microbiota induced by increased epithelial TLR4 signaling are transmissible and exacerbate dextran sodium sulfate-induced colitis. Together, our findings imply that host innate immune signaling can modulate intestinal bacteria and ultimately the hosts susceptibility to colitis.

Stool Phospholipid Signature is Altered by Diet and Tumors

Intake of saturated fat is a risk factor for ulcerative colitis (UC) and colon cancer. Changes in the microbiota have been implicated in the development of UC and colon cancer. The host and the microbiota generate metabolites that may contribute to or reflect disease pathogenesis. We used lipid class specific quantitative mass spectrometry to assess the phospholipid (PL) profile (phosphatidylcholine [PC], phosphatidylethanolamine [PE], phosphatidylinositol [PI], phosphatidylserine [PS]) of stool from mice fed a high fat (HFD) or control diet with or without induction of colitis-associated tumors using azoxymethane and dextran sodium sulfate. The microbiota was assessed using qPCR for several bacterial groups. Colitis-associated tumors were associated with reduced bulk PI and PE levels in control diet fed mice compared to untreated mice. Significant decreases in the relative quantities of several PC species were found in colitis-associated tumor bearing mice fed either diet. Statistical analysis of the PL profile revealed distinct clustering by treatment group. Partial least squares regression analysis found that the relative quantities of the PS class profile best predicted bacterial abundance of Clostridium leptum and Prevotella groups. Abundance of selected PL species correlated with bacterial group quantities. Thus, we have described that a HFD and colitis-associated tumors are associated with changes in phospholipids and may reflect host-microbial interactions and disease states.

Radiation induces proinflammatory dysbiosis: transmission of inflammatory susceptibility by host cytokine induction

Objective Radiation proctitis (RP) is a complication of pelvic radiotherapy which affects both the host and microbiota. Herein we assessed the radiation effect on microbiota and its relationship to tissue damage using a rectal radiation mouse model. Design We evaluated luminal and mucosa-associated dysbiosis in irradiated and control mice at two postradiation time points and correlated it with clinical and immunological parameters. Epithelial cytokine response was evaluated using bacterial–epithelial co-cultures. Subsequently, germ-free (GF) mice were colonised with postradiation microbiota and controls and exposed to radiation, or dextran sulfate-sodium (DSS). Interleukin (IL)-1β correlated with tissue damage and was induced by dysbiosis. Therefore, we tested its direct role in radiation-induced damage by IL-1 receptor antagonist administration to irradiated mice. Results A postradiation shift in microbiota was observed. A unique microbial signature correlated with histopathology. Increased colonic tumor necrosis factor (TNF)α, IL-1β and IL-6 expression was observed at two different time points. Adherent microbiota from RP differed from those in uninvolved segments and was associated with tissue damage. Using bacterial–epithelial co-cultures, postradiation microbiota enhanced IL-1β and TNFα expression compared with naïve microbiota. GF mice colonisation by irradiated microbiota versus controls predisposed mice to both radiation injury and DSS-induced colitis. IL-1 receptor antagonist administration ameliorated intestinal radiation injury. Conclusions The results demonstrate that rectal radiation induces dysbiosis, which transmits radiation and inflammatory susceptibility and provide evidence that microbial-induced radiation tissue damage is at least in part mediated by IL-1β. Environmental factors may affect the host via modifications of the microbiome and potentially allow for novel interventional approaches via its manipulation.

Intestinal Exposure to PCB 153 Induces Inflammation via the ATM/NEMO Pathway

Background: Polychlorinated biphenyls (PCBs) are persistent organic pollutants that adversely affect human health. PCBs bio‐accumulate in organisms important for human consumption. PCBs accumulation in the body leads to activation of the transcription factor NF‐&kgr;B, a major driver of inflammation. Despite dietary exposure being one of the main routes of exposure to PCBs, the gut has been widely ignored when studying the effects of PCBs. Objectives: We investigated the effects of PCB 153 on the intestine and addressed whether PCB 153 affected intestinal permeability or inflammation and the mechanism by which this occurred. Methods: Mice were orally exposed to PCB 153 and gut permeability was assessed. Intestinal epithelial cells (IECs) were collected and evaluated for evidence of genotoxicity and inflammation. A human IEC line (SW480) was used to examine the direct effects of PCB 153 on epithelial function. NF‐&kgr;B activation was measured using a reporter assay, DNA damage was assessed, and cytokine expression was ascertained with real‐time PCR. Results: Mice orally exposed to PCB 153 had an increase in intestinal permeability and inflammatory cytokine expression in their IECs; inhibition of NF‐&kgr;B ameliorated both these effects. This inflammation was associated with genotoxic damage and NF‐&kgr;B activation. Exposure of SW480 cells to PCB 153 led to similar effects as seen in vivo. We found that activation of the ATM/NEMO pathway by genotoxic stress was upstream of NF‐kB activation. Conclusions: These results demonstrate that oral exposure to PCB 153 is genotoxic to IECs and induces downstream inflammation and barrier dysfunction in the intestinal epithelium. HighlightsPCB 153 causes an increase in inflammatory cytokines in intestinal epithelial cells.PCB 153 increases intestinal permeability.PCB 153 activates the transcription factor NF‐&kgr;B to cause these effects.PCB 153 is genotoxic to intestinal epithelial cells.PCB 153 activates NF‐ &kgr;B through the ATM/NEMO pathway.

Inflammation and Colorectal Cancer

Chronic intestinal Inflammation occurs in response to environmental factors, infection and genetics; and plays a critical role in initiation, promotion, progression and metastasis of colon cancer. Colitis associated colon cancer (CAC) is a classic example of multifactorial, multi-step colorectal cancer associated with inflammatory bowel diseases. In recent years, the generation of animal models of CAC and recognition of the importance of the gut microbiota, altered immune system, and other environmental factors in CAC, has expanded the basic understanding of inflammation associated colon cancer. In this chapter, we discuss the cellular alterations and mechanisms by which inflammation contributes towards the development of colon cancer using CAC as a model system. We have also explored some of the promising strategies for preventing progression of inflammation to colon cancer. The emerging role of dietary factors, obesity and gut microbiota in colon cancer is also reviewed.

688 Obesity Induces Colonic and Mesenteric Fat Inflammation and Promotes Tumorigenesis in a Model of Murine Colon Cancer

Background: Obesity is defined as a chronic low-level inflammatory condition characterized by high levels of circulating IL-6 and LPS and increases the risk of colon cancer development. Aim: We aimed to investigate the mechanism of obesity enhanced colon tumorigenesis in a murine model of diet-induced obesity. Method: Mice were fed either a control diet (10% calories from fat) or a high fat (HF) diet (60% calories from fat) and colonic tumors were induced by intraperitoneal injection of the genotoxic compound azoxymethane (AOM) followed by 2 rounds of oral dextran-sodium sulfate (DSS). Proximal and distal colon tissue was assessed for gene expression of inflammatory markers by qRT-PCR. The mesenteric fat was similarly assessed. The macrophage infiltrate in mesenteric fat was measured by immunohistochemistry staining for CD68. Bacterial translocation of live bacteria to the mesenteric lymph nodes (MLN) and spleen was determined using culture-dependent techniques. Results: Mice fed a HF diet for 10 weeks gained significantly more weight during the course of the experiment compared to mice fed a lean diet. They also had increased proximal colon gene expression of inflammatory mediators including tnfa, nos2 and il1b which was not observed distally. However, increased proximal gene expression did not lead to overt inflammation in the colon as determined by histological grading. Feeding a HF diet also resulted in mesenteric adipose tissue gene expression of tnfa and il1b and increased infiltration of CD68+ macrophages. Mice fed a HF diet and given AOM-DSS had an increased number of colonic tumors compared to control diet fed mice. However, there were few differences in inflammatory gene expression between the control diet or HF diet fed mice given AOM-DSS, and this observation extended to cytokine protein secretion from distal colon explant supernatants. The highest levels of live bacteria were recovered from the MLN of mice fed a HF diet and given AOM-DSS. Macrophages isolated from the mesenteric fat of mice fed a HF diet did not robustly respond to LPS stimulation, while those isolated from mice fed a HF diet and given AOM-DSS had increased baseline cytokine secretion that was further enhanced by LPS stimulation. Conclusion: Obesity increases tumor formation in a model of inflammation-induced colon cancer. Obesity itself induces inflammation of the colon and mesenteric fat. Mesenteric fat-associated macrophages from obese tumor bearing mice are primed to respond to LPS or translocated bacteria leading to an increase in local and systemic inflammation. We believe our results highlight the yet unknown role of mesenteric fat as a rheostat for intestinal and systemic inflammatory states.

Distinct Inflammatory Pathways Induce Altered Intracellular Bacterial Community in Murine Colonic Lamina Propria Macrophages: P-264

of cecal tissues and severe inflammation. Although intestinal mucus is the first line of defense in the mouse GI tract, its role in providing host defense against Salmonella is still unclear. The mucus barrier is made up of the highly glycosylated mucin Muc2, which is secreted by goblet cells. Muc2 glycosylation occurs within the goblet cell and likely has significant implications for the function and effectiveness of the mucus barrier. Glycosylation involves the actions of several enzymes, for example, Core 3O derived glycans are synthesized by Core 3 b1,3N-acetylglucosaminyltransferase (C3GnT). Mice lacking these glycans still produce the Muc2 protein, but display a thinner mucus barrier, and show increased susceptibility to chemical induced colitis. METHODS: We began our investigations by comparing Salmonella induced colitis and mucus dynamics in Muc2 deficient (-/-) mice, C3GnT-/mice and wildtype C57BL/6 mice. RESULTS: We observed that mucus secretion increased in response to Salmonella infection in C3GnT-/and C57BL/6, with Salmonella found within the mucus layer. In contrast, Muc2-/mice showed dramatic susceptibility to Salmonella infection, carrying 100 fold heavier cecal pathogen burdens and developing significantly increased barrier disruption compared with C57BL/6 mice. As a result, Muc2 -/mice displayed high rates of morbidity and mortality. We also tested the susceptibility of C3GnT -/mice, finding they carry WT pathogen burdens but developed exaggerated barrier disruption like Muc2 -/mice. CONCLUSION(S): These data suggest that the intestinal mucus layer plays a critical role in controlling Salmonella intestinal burdens, whereas core-3 glycosylation plays an important role in controlling intestinal epithelial barrier function.