Gastroenterology

Gastroenterology

Volume 145, Issue 2, August 2013, Pages 407-415
Gastroenterology

Original Research
Full Report: Basic and Translational—Alimentary Tract
TNFR2 Activates MLCK-Dependent Tight Junction Dysregulation to Cause Apoptosis-Mediated Barrier Loss and Experimental Colitis

https://doi.org/10.1053/j.gastro.2013.04.011Get rights and content

Background & Aims

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.

Methods

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.

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. 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.

Conclusions

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.

Section snippets

Mice and Colitis Models

All experiments used C57BL/6 mice bred and maintained at the University of Chicago in accordance with Institutional Animal Care and Use Committee regulations. CD4+CD45RBhi and dextran sulfate sodium (DSS) colitis was induced in 6- to 8-week-old mice as described previously.1 For adoptive transfer colitis, CD4+ splenocytes were isolated using magnetic-activated cell sorting beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and enriched for CD45RBhi lymphocytes using a MoFlo Cell Sorter

CD4+CD45RBhi Adoptive Transfer Colitis Induces Changes in Tight Junction Protein Expression and Organization That Are Similar to Human IBD

Recent studies have shown that ileal and colonic epithelial long MLCK expression and activity as well as claudin-2 expression are increased in human ulcerative colitis and Crohn’s disease.12, 13 Immunofluorescence analysis confirmed increased long MLCK expression and activity, measured as increased myosin light chain (MLC) phosphorylation, increased claudin-2 expression, and occludin endocytosis in ileal enterocytes during CD4+CD45RBhi-induced disease (Figure 1A). As expected, similar changes

Discussion

Increased intestinal permeability has been linked to a variety of autoimmune and inflammatory disorders. The case is strongest in Crohn’s disease, where reduced barrier function is a marker of impending disease reactivation.22 However, increased intestinal permeability is also increased in ulcerative colitis.23 Despite this association, intestinal barrier loss alone is insufficient to cause disease in either animal models or human subjects.1, 2, 3, 24 Nevertheless, intestinal barrier defects do

Acknowledgments

The authors thank Drs Cathryn Nagler and Warren Strober for insightful discussions and advice.

References (40)

Cited by (299)

View all citing articles on Scopus

Author names in bold designate shared co-first authorship.

Conflicts of interest The authors disclose no conflicts.

Funding Supported by the National Institutes of Health (R01DK61931, R01DK68271, P01D067887, P30DK042086, P30CA14599, UL1RR024999, T32HL007237, K01DK09238, R01DK77905), Department of Defense (W81XWH-09-1-0341), the Broad Medical Research Foundation (IBD-022), the Crohn’s and Colitis Foundation of America, the Chicago Biomedical Consortium (with support from The Searle Funds at The Chicago Community Trust), and the National Natural Science Foundation of China (30900670, 81272749).

Authors share co-first authorship.

View full text