Toll-like receptors (TLRs) and Nod-like receptors (NLRs) in inflammatory disorders.

Toll-like receptors (TLRs) and Nod-like receptors (NLRs) are two major forms of innate immune sensors, which provide immediate responses against pathogenic invasion or tissue injury. Activation of these sensors induces the recruitment of innate immune cells such as macrophages and neutrophils, initiates tissue repair processes, and results in adaptive immune activation. Abnormalities in any of these innate sensor-mediated processes may cause excessive inflammation due to either hyper responsive innate immune signaling or sustained compensatory adaptive immune activation. Recent gene association studies appear to reveal strong associations of NLR gene mutations and development of several idiopathic inflammatory disorders. In contrast, TLR polymorphisms are less often associated with inflammatory disorders. Nevertheless, TLRs are up-regulated in the affected tissue of most inflammatory disorders, suggesting TLR signaling is involved in the pathogenesis of chronic and/or idiopathic inflammatory disorders. NLR signaling results in the formation of a molecular scaffold complex (termed an inflammasome) and orchestrates with TLRs to induce IL-1beta and IL-18, both of which are important mediators in the majority of inflammatory disorders. Therefore, understanding the roles of TLRs and NLRs in the pathogenesis of chronic and idiopathic inflammatory disorders may provide novel targets for the prevention and/or treatment of many common and uncommon diseases involving inflammation.

Toll-like receptor-4 promotes the development of colitis-associated colorectal tumors.

BACKGROUND & AIMS: Chronic inflammation is a risk factor for colon cancer in patients with ulcerative colitis (UC). The molecular mechanisms linking inflammation and colon carcinogenesis are incompletely understood. We tested the hypothesis that Toll-like receptor 4 (TLR4) is involved in tumorigenesis in the setting of chronic inflammation. METHODS: Tissues from UC patients with cancer were examined for TLR4 expression. Colitis-associated neoplasia was induced using azoxymethane injection followed by dextran sodium sulfate treatment in TLR4-deficient or wild-type mice. Inflammation, polyps, and microscopic dysplasia were scored. Cyclooxygenase (Cox)-2 and prostaglandin E(2) production were analyzed by real-time polymerase chain reaction, immunohistochemistry, or enzyme immunoassay. Epidermal growth factor receptor (EGFR) phosphorylation and amphiregulin production were examined by Western blot analysis and enzyme-linked immunosorbent assay, respectively. RESULTS: We show that TLR4 is overexpressed in human and murine inflammation-associated colorectal neoplasia. TLR4-deficient mice were protected markedly from colon carcinogenesis. Mechanistically, we show that TLR4 is responsible for induction of Cox-2, increased prostaglandin E(2) production, and activation of EGFR signaling in chronic colitis. Amphiregulin, an EGFR ligand, was induced in a TLR4, Cox-2-dependent fashion and contributes to activation of EGFR phosphorylation in colonic epithelial cells. CONCLUSIONS: TLR4 signaling is critical for colon carcinogenesis in chronic colitis. TLR4 activation appears to promote the development of colitis-associated cancer by mechanisms including enhanced Cox-2 expression and increased EGFR signaling. Inhibiting TLR4 signaling may be useful in the prevention or treatment of colitis-associated cancer.

Performing forward-viewing endoscopy at time of pancreaticobiliary EUS and ERCP may detect additional upper gastrointestinal lesions.

BACKGROUND AND STUDY AIMS: It is unknown whether significant incidental upper gastrointestinal lesions are missed when using non-forward-viewing endoscopes without completing a forward-viewing exam in linear endoscopic ultrasound (EUS) or endoscopic retrograde cholangiopancreatography (ERCP) exams. We evaluated whether significant upper GI lesions are missed during EUS and ERCP when upper endoscopy is not performed routinely with a gastroscope. PATIENTS AND METHODS: A retrospective analysis was performed in which an EGD with a forward-viewing gastroscope was performed after using a non-forward-viewing endoscope (linear echoendoscope, duodenoscope, or both) during a single procedure. Upper gastrointestinal tract findings were recorded separately for each procedure. Significant lesions found with a forward-viewing gastroscope were defined as findings that led to a change in the patient's medication regimen, additional endoscopic surveillance/interventions, or the need for other imaging studies. RESULTS: A total of 168 patients were evaluated. In 83 patients, a linear echoendoscope was used, in 52 patients a duodenoscope was used, and in 33 patients both devices were used. Clinically significant additional lesions diagnosed with a gastroscope but missed by a non-forward-viewing endoscope were found in 30 /168 patients (18 %). EGD after linear EUS resulted in additional lesion findings in 17 /83 patients (20.5 %, χ(2) = 13.385, P = 0.00025). EGD after use of a duodenoscope resulted in additional lesions findings in 10 /52 patients (19.2 %, χ(2) = 9.987, P = 0.00157). EGD after the use of both a linear echoendoscope and a duodenoscope resulted in additional lesions findings in 3/33 patients (9 %, χ(2) = 3.219, P = 0.07). CONCLUSION: Non forward-viewing endoscopes miss a significant amount of incidental upper gastrointestinal lesions during pancreaticobiliary endoscopy. Performing an EGD with a gastroscope at the time of linear EUS or ERCP can lead to increased yield of upper gastrointestinal lesions.

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.

Regulation of Toll-like receptor 4-associated MD-2 in intestinal epithelial cells: a comprehensive analysis.

The intestinal epithelium maintains a state of controlled inflammation despite continuous contact with Gram-negative commensal bacteria and lipopolysaccharide (LPS) on its luminal surface. Recognition of LPS by the Toll-like receptor (TLR) 4/MD-2 complex results in pro-inflammatory gene expression and cytokine secretion in intestinal epithelial cells (IECs). We have shown that IECs express low levels of MD-2 and TLR4 and are poorly responsive to LPS. In this study, we did a comprehensive analysis to understand the immune-mediated and epigenetic mechanisms by which IECs down-regulate MD-2 expression. Expression of MD-2 and TLR4 mRNA was examined in human inflammatory bowel disease and intestinal epithelial cell lines (T84, HT-29, Caco-2). Nuclear factor-kappaB transcriptional activation was used as a measure of LPS responsiveness. Intestinal epithelial cells in patients with inflammatory bowel disease exhibited increased expression of MD-2 and TLR4 mRNA. Lipopolysaccharide responsiveness in IECs was polarized to the basolateral membrane. Bisulfite sequencing of the MD-2 promoter demonstrated methylation of CpG dinucleotides. Inhibition of methylation by 5-azacytidine and histone de-actylation by trichostatin A, two forms of epigenetic silencing, resulted in increased mRNA expression of MD-2 in IECs. These results demonstrate various molecular mechanisms by which IECs down-regulate MD-2 and, thereby, protect against dysregulated inflammation to commensal bacteria in the intestinal lumen.

The myeloid differentiation factor 88 (MyD88) is required for CD4+ T cell effector function in a murine model of inflammatory bowel disease.

Abnormal T cell responses to commensal bacteria are involved in the pathogenesis of inflammatory bowel disease. MyD88 is an essential signal transducer for TLRs in response to the microflora. We hypothesized that TLR signaling via MyD88 was important for effector T cell responses in the intestine. TLR expression on murine T cells was examined by flow cytometry. CD4(+)CD45Rb(high) T cells and/or CD4(+)CD45Rb(low)CD25(+) regulatory T cells were isolated and adoptively transferred to RAG1(-/-) mice. Colitis was assessed by changes in body weight and histology score. Cytokine production was assessed by ELISA. In vitro proliferation of T cells was assessed by [(3)H]thymidine assay. In vivo proliferation of T cells was assessed by BrdU and CFSE labeling. CD4(+)CD45Rb(high) T cells expressed TLR2, TLR4, TLR9, and TLR3, and TLR ligands could act as costimulatory molecules. MyD88(-/-) CD4(+) T cells showed decreased proliferation compared with WT CD4(+) T cells both in vivo and in vitro. CD4(+)CD45Rb(high) T cells from MyD88(-/-) mice did not induce wasting disease when transferred into RAG1(-/-) recipients. Lamina propria CD4(+) T cell expression of IL-2 and IL-17 and colonic expression of IL-6 and IL-23 were significantly lower in mice receiving MyD88(-/-) cells than mice receiving WT cells. In vitro, MyD88(-/-) T cells were blunted in their ability to secrete IL-17 but not IFN-gamma. Absence of MyD88 in CD4(+)CD45Rb(high) cells results in defective T cell function, especially Th17 differentiation. These results suggest a role for TLR signaling by T cells in the development of inflammatory bowel disease.

Cox-2 is regulated by Toll-like receptor-4 (TLR4) signaling: Role in proliferation and apoptosis in the intestine.

BACKGROUND & AIMS: We recently showed that mice deficient in Toll-like receptor 4 (TLR4) or its adapter molecule MyD88 have increased signs of colitis compared with wild-type (WT) mice after dextran sodium sulfate (DSS)-induced injury. We wished to test the hypothesis that cyclooxygenase 2 (Cox-2)-derived prostaglandin E2 (PGE2) is important in TLR4-related mucosal repair. METHODS: Cox-2 expression was analyzed by real-time polymerase chain reaction, immunohistochemistry, Western blotting, and luciferase reporter constructs. Small interfering RNA was used to inhibit expression of MyD88. TLR4-/- or WT mice were given 2.5% DSS for 7 days. Proliferation and apoptosis were assessed using bromodeoxyuridine staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays, respectively. PGE2 was given orally to DSS-treated mice. RESULTS: Intestinal epithelial cell lines up-regulated Cox-2 expression in a TLR4- and MyD88-dependent fashion. Lipopolysaccharide-mediated stimulation of PGE2 production was blocked by a selective Cox-2 inhibitor or small interfering RNA against MyD88. After DSS injury, Cox-2 expression increased only in WT mice. TLR4-/- mice have significantly reduced proliferation and increased apoptosis after DSS injury compared with WT mice. PGE2 supplementation of TLR4-/- mice resulted in improvement in clinical signs of colitis and restoration of proliferation and apoptosis to WT values. The mechanism for improved epithelial repair may be through PGE2-dependent activation of the epidermal growth factor receptor. CONCLUSIONS: We describe an important link between TLR4 signaling and Cox-2 expression in the gut. TLR4 and MyD88 signaling are required for optimal proliferation and protection against apoptosis in the injured intestine. Although TLR4 signaling is beneficial in the short term, chronic signaling through TLR4 may lower the threshold for colitis-associated cancer.