Methotrexate modulates tight junctions through NF-κB, MEK, and JNK pathways.

OBJECTIVES: Chemotherapy often induces intestinal mucositis, which is associated with an increase in intestinal permeability; however, underlying mechanisms remain incompletely understood. Thus, we aimed to study the regulation of 3 tight junction (TJ) proteins, claudin-1, occludin, and zonula occludens-1, after anticancer treatment. METHODS: Methotrexate (MTX) was subcutaneously injected for 3 consecutive days in Sprague-Dawley rats to induce intestinal mucositis and was applied on Caco-2 cell monolayers. TJ protein expression and cellular distribution were studied by Western blot and microscopy, respectively. In Caco-2 cells, the paracellular permeability was evaluated by both transepithelial electrical resistance and flux of fluorescein isothiocyanate-dextran marker. Cytokine production and signaling pathways were also assessed. RESULTS: In MTX-treated rats, the cellular distribution of the 3 TJ proteins was altered and claudin-1 and occludin expression was reduced during the acute phase of mucositis compared with controls. During the recovery phase, these parameters were restored. In vitro, MTX treatment led to an increase in proinflammatory cytokine production at the apical side but did not affect Caco-2 cell apoptosis and necrosis. Increase in paracellular permeability was associated with altered occludin and zonula occludens-1 expression and cellular distribution. All of these alterations were prevented by MEK1 and 2, JNK, and NF-κB inhibitors. CONCLUSIONS: MTX treatment induced an increase in intestinal permeability partially related to alteration of TJs protein expression and cellular distribution that may be mediated by MAPK and NF-κB pathways. These are potential targets to limit the adverse effects of chemotherapy.

Role of toll like receptors in irritable bowel syndrome: differential mucosal immune activation according to the disease subtype.

BACKGROUND: The irritable bowel syndrome (IBS) is a functional gastrointestinal disorder whose pathogenesis is not completely understood. Its high prevalence and the considerable effects on quality of life make IBS a disease with high social cost. Recent studies suggest that low grade mucosal immune activation, increased intestinal permeability and the altered host-microbiota interactions that modulate innate immune response, contribute to the pathophysiology of IBS. However, the understanding of the precise molecular pathophysiology remains largely unknown. METHODOLOGY AND FINDINGS: In this study our objective was to evaluate the TLR expression as a key player in the innate immune response, in the colonic mucosa of IBS patients classified into the three main subtypes (with constipation, with diarrhea or mixed). TLR2 and TLR4 mRNA expression was assessed by real time RT-PCR while TLRs protein expression in intestinal epithelial cells was specifically assessed by flow cytometry and immunofluorescence. Mucosal inflammatory cytokine production was investigated by the multiplex technology. Here we report that the IBS-Mixed subgroup displayed a significant up-regulation of TLR2 and TLR4 in the colonic mucosa. Furthermore, these expressions were localized in the epithelial cells, opening new perspectives for a potential role of epithelial cells in host-immune interactions in IBS. In addition, the increased TLR expression in IBS-M patients elicited intracellular signaling pathways resulting in increased expression of the mucosal proinflammatory cytokines IL-8 and IL1ß. CONCLUSIONS: Our results provide the first evidence of differential expression of TLR in IBS patients according to the disease subtype. These results offer further support that microflora plays a central role in the complex pathophysiology of IBS providing novel pharmacological targets for this chronic gastrointestinal disorder according to bowel habits.