Background & aims Goblet cells (GC) facilitate mucosal protection and epithelial barrier repair, yet the innate immune mechanisms that selectively drive GC functions have not been defined. The aim of this study was to determine whether TLR2 and modulation of GC-derived TFF3 are functionally linked in the intestine. Methods GC modulation was assessed using qRT-PCR, western blotting and confocal microscopy. DSS colitis was induced in wild-type, TFF3−/− and TLR2−/− mice. Recombinant TLR2 ligand or TFF3 peptide were orally administered after DSS termination. Caco-2 overexpressing full-length TLR2 or mutant TLR2-R753Q were tested for TFF3 synthesis and functional-related effects in a wounding-assay. Results Data from in-vitro (Ls174T) and ex-vivo models of murine and human GC reveal that TLR2 activation selectively induces synthesis of TFF3. In-vivo studies using TFF3−/− or TLR2−/− mice demonstrate the ability for oral treatment with a TLR2 agonist to confer anti-apoptotic protection of the intestinal mucosa against inflammatory stress-induced damage through TFF3. Recombinant TFF3 rescues TLR2-deficient mice from increased morbidity and mortality during acute colonic injury. Severe ulcerative colitis has recently been found to be associated with the R753Q polymorphism of the TLR2 gene. The relevance of the observed functional effect of TLR2 in regulating GC is confirmed by the finding that the UC-associated TLR2-R753Q variant is functionally deficient in the ability to induce TFF3 synthesis, thus leading to impaired wound healing. Conclusions These data demonstrate a novel function of TLR2 in intestinal GC that links products of commensal bacteria to innate immune protection of the host via TFF3.

Gap junctional intercellular communication (GJIC) coordinates cellular functions essential for sustaining tissue homeostasis; yet its regulation in the intestine is not well understood. Here, we identify a novel physiological link between Toll-like receptor (TLR) 2 and GJIC through modulation of Connexin-43 (Cx43) during acute and chronic inflammatory injury of the intestinal epithelial cell (IEC) barrier. Data from in vitro studies reveal that TLR2 activation modulates Cx43 synthesis and increases GJIC via Cx43 during IEC injury. The ulcerative colitis-associated TLR2-R753Q mutant targets Cx43 for increased proteasomal degradation, impairing TLR2-mediated GJIC during intestinal epithelial wounding. In vivo studies using mucosal RNA interference show that TLR2-mediated mucosal healing depends functionally on intestinal epithelial Cx43 during acute inflammatory stress-induced damage. Mice deficient in TLR2 exhibit IEC-specific alterations in Cx43, whereas administration of a TLR2 agonist protects GJIC by blocking accumulation of Cx43 and its hyperphosphorylation at Ser 368 to prevent spontaneous chronic colitis in MDR1␣-deficient mice. Finally, adding the TLR2 agonist to three-dimensional intestinal mucosalike cultures of human biopsies preserves intestinal epithelial Cx43 integrity and polarization ex vivo. In conclusion, Cx43 plays an important role in innate immune control of commensal-mediated intestinal epithelial wound repair.

Intestinal mucositis represents the most common complication of intensive chemotherapy, which has a severe adverse impact on quality of life of cancer patients. However, the precise pathophysiology remains to be clarified and there is so far no successful therapeutic intervention. Here, we investigated the role of innate immunity through TLR signaling in modulating genotoxic chemotherapy-induced small intestinal injury in vitro and in vivo. Genetic deletion of TLR2, but not MD-2, in mice resulted in severe chemotherapy-induced intestinal mucositis in the proximal jejunum with villous atrophy, accumulation of damaged DNA, CD11b+-myeloid cell infiltration and significant gene alterations in xenobiotic metabolism, including a decrease in ABCB1/MDR1 p-glycoprotein (p-gp) expression. Functionally, stimulation of TLR2 induced synthesis and drug efflux activity of ABCB1/MDR1 p-gp in murine and human CD11b+-myeloid cells, thus inhibiting chemotherapy-mediated cytotoxicity. Conversely, TLR2 activation failed to protect small intestinal tissues genetically deficient in MDR1A against DNA-damaging drug-induced apoptosis. Gut microbiota depletion by antibiotics led to increased susceptibility to chemotherapy-induced mucosal injury in wildtype mice, which was suppressed by administration of a TLR2 ligand, preserving ABCB1/MDR1 p-gp expression. Findings were confirmed in a preclinical model of human chemotherapy-induced intestinal mucositis using duodenal biopsies, by demonstrating that TLR2 activation limited the toxic-inflammatory reaction and maintained assembly of the drug transporter p-gp. In conclusion, this study identifies a novel molecular link between innate immunity and xenobiotic metabolism. TLR2 acts as a central regulator of xenobiotic defense via the multidrug transporter ABCB1/MDR1 p-gp. Targeting TLR2 may represent a novel therapeutic approach in chemotherapy-induced intestinal mucositis.

Background & Aims The Toll-like receptor (TLR) 4 mediates homeostasis of the intestinal epithelial cell (IEC) barrier. We investigated the effects of TLR4-D299G on IEC functions. Methods We engineered IECs (Caco-2) to stably overexpress hemagglutinin-tagged wild-type TLR4, TLR4-D299G, or TLR4-T399I. We performed gene expression profiling using DNA microarray analysis. Findings were confirmed by real-time, quantitative, reverse-transcriptase polymerase chain reaction, immunoblot, enzyme-linked immunosorbent assay, confocal immunofluorescence, and functional analyses. Tumorigenicity was tested using the CD1 nu/nu mice xenograft model. Human colon cancer specimens (N = 214) were genotyped and assessed for disease stage. Results Caco-2 cells that expressed TLR4-D299G underwent the epithelial-mesenchymal transition and morphologic changes associated with tumor progression, whereas cells that expressed wild-type TLR4 or TLR4-T399I did not. Caco-2 cells that expressed TLR4-D299G had significant increases in expression levels of genes and proteins associated with inflammation and/or tumorigenesis compared with cells that expressed other forms of TLR4. The invasive activity of TLR4-D299G Caco-2 cells required Wnt-dependent activation of STAT3. In mice, intestinal xenograft tumors grew from Caco-2 cells that expressed TLR4-D299G, but not cells that expressed other forms of TLR4; tumor growth was blocked by a specific inhibitor of STAT3. Human colon adenocarcinomas from patients with TLR4-D299G were more frequently of an advanced stage (International Union Against Cancer [UICC] ≥III, 70% vs 46%; P = .0142) with metastasis (UICC IV, 42% vs 19%; P = .0065) than those with wild-type TLR4. Expression of STAT3 messenger RNA was higher among colonic adenocarcinomas with TLR4-D299G than those with wild-type TLR4. Conclusions TLR4-D299G induces features of neoplastic progression in intestinal epithelial Caco-2 cells and associates with aggressive colon cancer in humans, implying a novel link between aberrant innate immunity and colonic cancerogenesis.