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

Background & AimsTight junction dysregulation and epithelial damage contribute to barrier loss in patients with inflammatory bowel disease. However, the mechanisms that regulate these processes and their relative contributions to disease pathogenesis are not completely understood. We investigated these processes using colitis models in mice.MethodsWe induced colitis by adoptive transfer of CD4+CD45RBhi cells or administration of dextran sulfate sodium 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, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction assays.ResultsInduction 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. Long MLCK upregulation, myosin II light chain phosphorylation, barrier loss, and weight loss were attenuated in TNFR2−/− , but not TNFR1−/− , recipients of wild-type CD4+CD45RBhi cells. Similarly, long MLCK−/− mice had limited increases in myosin II light chain phosphorylation, claudin-2 expression, and intestinal permeability and delayed onset of adoptive transfer−induced colitis. However, coincident with onset of epithelial apoptosis, long MLCK−/− mice ultimately developed colitis. 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 dextran sulfate sodium colitis.ConclusionsIn immune-mediated inflammatory bowel disease 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 result 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 can lead to improved inflammatory bowel disease therapies. Tight junction dysregulation and epithelial damage contribute to barrier loss in patients with inflammatory bowel disease. However, the mechanisms that regulate these processes and their relative contributions to disease pathogenesis are not completely understood. We investigated these processes using colitis models in mice. We induced colitis by adoptive transfer of CD4+CD45RBhi cells or administration of dextran sulfate sodium 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, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction assays. 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. Long MLCK upregulation, myosin II light chain phosphorylation, barrier loss, and weight loss were attenuated in TNFR2−/− , but not TNFR1−/− , recipients of wild-type CD4+CD45RBhi cells. Similarly, long MLCK−/− mice had limited increases in myosin II light chain phosphorylation, claudin-2 expression, and intestinal permeability and delayed onset of adoptive transfer−induced colitis. However, coincident with onset of epithelial apoptosis, long MLCK−/− mice ultimately developed colitis. 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 dextran sulfate sodium colitis. In immune-mediated inflammatory bowel disease 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 result 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 can lead to improved inflammatory bowel disease therapies.