TNFR2 Activates MLCK-Dependent Tight Junction Dysregulation to Cause Apoptosis-Mediated Barrier Loss and Experimental Colitis

Abstract

BACKGROUND & AIMS Tight junction dysregulation and epithelial damage contribute to barrier loss in patients with inflammatory bowel disease (IBD). However, the mechanisms that regulate these processes and their relative contributions to disease pathogenesis are incompletely understood. We investigated these processes using colitis models in mice. METHODS We induced colitis by adoptive transfer of CD4+CD45RBhi cells or administration of dextran sulfate sodium (DSS) to mice, including those deficient in tumor necrosis factor receptor (TNFR) 1, TNFR2, or the long isoform of myosin light chain kinase (MLCK). Intestinal tissues and isolated epithelial cells were analyzed by immunoblot, immunofluorescence, ELISA, and real-time PCR assays. RESULTS Induction of immune-mediated colitis by CD4+CD45RBhi adoptive transfer increased intestinal permeability; epithelial expression of claudin-2, the long isoform of MLCK, and TNFR2 (but not TNFR1); and phosphorylation of the myosin II light chain (MLC). Long MLCK upregulation, MLC phosphorylation, barrier loss, and weight loss were attenuated in TNFR2−/−, but not TNFR1−/−, recipients of wildtype CD4+CD45RBhi cells. Similarly, long MLCK−/− mice had limited increases in MLC phosphorylation, claudin-2 expression, and intestinal permeability and delayed onset of cell transfer-induced colitis. However, coincident with onset of epithelial apoptosis, colitis ultimately developed. This indicates that disease progresses via apoptosis in the absence of MLCK-dependent tight junction regulation. In support of this conclusion, long MLCK−/− mice were not protected from epithelial apoptosis-mediated, damage-dependent DSS colitis. CONCLUSIONS In immune-mediated IBD models, TNFR2 signaling increases long MLCK expression, resulting in tight junction dysregulation, barrier loss and induction of colitis. At advanced stages, colitis progresses by apoptosis and mucosal damage that results in tight junction- and MLCK-independent barrier loss. Therefore, barrier loss in immune-mediated colitis occurs via two temporally and morphologically distinct mechanisms. Differential targeting of these mechanisms may lead to improved IBD therapies.