Cecilia Espana

Constitutive activation of epithelial TLR4 augments inflammatory responses to mucosal injury and drives colitis-associated tumorigenesis

Background: Chronic intestinal inflammation culminates in cancer and a link to Toll‐like receptor‐4 (TLR4) has been suggested by our observation that TLR4 deficiency prevents colitis‐associated neoplasia. In the current study we address the effect of the aberrant activation of epithelial TLR4 on induction of colitis and colitis‐associated tumor development. We take a translational approach to address the consequences of increased TLR signaling in the intestinal mucosa. Methods: Mice transgenic for a constitutively active TLR4 under the intestine‐specific villin promoter (villin‐TLR4 mice) were treated with dextran sodium sulfate (DSS) for acute colitis and azoxymethane (AOM)‐DSS TLR4 expression was analyzed by immunohistochemistry in colonic tissue from patients with ulcerative colitis (UC) and UC‐associated cancer. The effect of an antagonist TLR4 antibody (Ab) was tested in prevention of colitis‐associated neoplasia in the AOM‐DSS model. Results: Villin‐TLR4 mice were highly susceptible to both acute colitis and colitis‐associated neoplasia. Villin‐TLR4 mice had increased epithelial expression of COX‐2 and mucosal PGE2 production at baseline. Increased severity of colitis in villin‐TLR4 mice was characterized by enhanced expression of inflammatory mediators and increased neutrophilic infiltration. In human UC samples, TLR4 expression was upregulated in almost all colitis‐associated cancer and progressively increased with grade of dysplasia. As a proof of principle, a TLR4/MD‐2 antagonist antibody inhibited colitis‐associated neoplasia in the mouse model. Conclusions: Our results show that regulation of TLRs can affect the outcome of both acute colitis and its consequences, cancer. Targeting TLR4 and other TLRs may ultimately play a role in prevention or treatment of colitis‐associated cancer. (Inflamm Bowel Dis 2010;)

TLR4 Activates the β-catenin Pathway to Cause Intestinal Neoplasia

Colonic bacteria have been implicated in the development of colon cancer. We have previously demonstrated that toll-like receptor 4 (TLR4), the receptor for bacterial lipopolysaccharide (LPS), is over-expressed in humans with colitis-associated cancer. Genetic epidemiologic data support a role for TLR4 in sporadic colorectal cancer (CRC) as well, with over-expression favoring more aggressive disease. The goal of our study was to determine whether TLR4 played a role as a tumor promoter in sporadic colon cancer. Using immunofluorescence directed to TLR4, we found that a third of sporadic human colorectal cancers over-express this marker. To mechanistically investigate this observation, we used a mouse model that over-expresses TLR4 in the intestinal epithelium (villin-TLR4 mice). We found that these transgenic mice had increased epithelial proliferation as measured by BrdU labeling, longer colonic crypts and an expansion of Lgr5+ crypt cells at baseline. In addition, villin-TLR4 mice developed spontaneous duodenal dysplasia with age, a feature that is not seen in any wild-type (WT) mice. To model human sporadic CRC, we administered the genotoxic agent azoxymethane (AOM) to villin-TLR4 and WT mice. We found that villin-TLR4 mice showed an increased number of colonic tumors compared to WT mice as well as increased β-catenin activation in non-dysplastic areas. Biochemical studies in colonic epithelial cell lines revealed that TLR4 activates β-catenin in a PI3K-dependent manner, increasing phosphorylation of β-cateninSer552, a phenomenon associated with activation of the canonical Wnt pathway. Our results suggest that TLR4 can trigger a neoplastic program through activation of the Wnt/β-catenin pathway. Our studies highlight a previously unexplored link between innate immune signaling and activation of oncogenic pathways, which may be targeted to prevent or treat CRC.

Host innate recognition of an intestinal bacterial pathogen induces TRIF-dependent protective immunity

TRIF signaling triggers the amplification of macrophage bactericidal activity sufficient to eliminate invading intestinal pathogens through the sequential induction of IFN-β and IFN-γ from macrophages and NK cells, respectively.

TLR4 Activates the B-Catenin Pathway to Cause Intestinal Neoplasia: P-247

tem are critical for establishing a proper balance between immune host defense mechanisms and tissue health. Changes in the composition of gut bacterial communities have been associated with intestinal inflammation and obesity. Recent studies have begun to note that a fraction of mucosa-associated microorganisms are not bacterial. Mucosal fungal infections are relatively common in Crohn’s Disease patients, and antibodies against fungal antigens (ASCA) are a widely accepted clinical marker for disease severity. What fungi populate the intestine and how immunity to them might play a role in inflammatory disease is currently unknown. Fungi are sensed by number of innate immune receptors among which Dectin-1 has emerged as the main innate immune receptor for recognition, phagocytosis, and killing of fungi by myeloid phagocytes. METHODS: Commensal fungi were visualized and quantified by staining with Dectin-1 probe followed by microscopy and FACS analysis, and additionally detected in fecal samples by qPCR for fungal 18S rDNA. To define the mouse intestinal fungal microbiome (the mycobiome), we isolated DNA from murine feces, amplified the internal transcribed spacer region (ITS1-2) of fungal rDNA, and performed high-throughput sequencing. To induce colitis wild type and Dectin-1-/mice were treated with DSS. In some cases mice were supplemented with C. tropicalis a common commensal fungus we found in murine gut and subjected to DSS. To determine whether the altered fungal burden during colitis contributes to disease severity, we suppressed fungal growth with fluconazole, a specific antifungal drug. RESULTS: We found that mice lacking Dectin-1, recognizing fungal cell wall b-glucan, are more susceptible to experimental colitis characterized by increased infiltration of Th17 and Th1 cells in the colon. Interestingly this pathology was driven by intestinal fungus, and antifungal therapy ameliorated colitis severity in knockout mice. Deep sequencing analysis of the fungal mycobiome revealed fungal species that are overrepresented in the gut during experimental colitis. When Dectin-1-/mice were supplemented with Candida tropicalis, a specific commensal fungus found in the intestine during colitis, they experienced more severe intestinal inflammation and augmented Th17 mucosal responses in absence of Dectin-1. Consistently, intestinal dendritic cells (DCs) from Dectin-1-/mice, but not WT DCs, showed reduced ability to kill fungi. Therefore the data suggest that an inability of Dectin-1-/mice to mount effective immune responses to specific intestinal fungi creates conditions that promote inflammation. Since the mouse model suggested that Dectin-1 is involved in contributing to the severity of colonic disease, we focused on the severity of ulcerative colitis (UC), the form of IBD that always affects the colon. In particular we focused on severe UC, termed medically refractory UC (MRUC), consisting of patients requiring colectomy as a result of lack of response to medication. We found that a specific variant of the gene for Dectin-1 is strongly associated with a severe form of ulcerative colitis requiring colectomy. CONCLUSION(S): Together our findings reveal a novel eukaryotic fungal community in the gut and show that altered interactions between the fungal microflora and the host intestinal phagocytes can profoundly influence intestinal pathology.

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.

M1776 Toll-Like Receptor 4 (TLR4) is a Target of Wnt/β-Catenin Signaling in Intestinal Epithelial Cells (IEC): Implications for Sporadic Colon Cancer

whether colonic tissue-specific factors are responsible for re-educating DC into a gut-like phenotype. To address this issue we cultured human colonic biopsies from healthy controls and collected supernatants (SN) to condition human blood DC for 24 hours. RESULTS: Conditioning DC with SN generated In Vitro a human gut-like DC phenotype, characterized by a higher proportion of cells expressing the CCR9 (p=0.0017, n=11), β7 (p=0.0328, n=13) and CD103 (p=0.0354, n=10) gut-homingmarkers compared with their basal counterparts. A tolerogenic phenotype was also induced, characterized by reduced surface expression of the co-stimulatory markers CD40 (p=0.0297, n=9) and CD83 (p=0.0315, n=8), and also of TLR2 (p=0.0029, n=13) and TLR4 (p=0.0372, n=11). In addition, a regulatory intracellular DC cytokine profile was induced with reduced IL-12 and increased IL-10 production (n= 3). Furthermore, SN-conditioned DC demonstrated enhanced phagocytic properties (n=3), compared with those in basal conditions. These data demonstrate that human blood DC can be modulated to express the phenotype and function of human gut DC. Moreover, SNconditioned DC were less effective in stimulating proliferation of allogeneic T-cells, which subsequently expressed an increased gut/skin homing profile (n=3). CONCLUSION: We have demonstrated the relevance of the local tissue microenvironment in modulating DC since conditioning blood DC with colonic biopsy SN -through a mechanism in which vitamin A was revealed as essential but not sufficienthave the capacity to condition DC into gutlike DC with unique properties rendering them crucial for oral tolerance. Identifying all the molecules related to the generation of the tolerogenic gut-like DC would provide us with new targets for immunomodulation in patients suffering from IBD, where the functionality of gut-DC is altered leading to a lack of oral tolerance against the commensal microbiota.