Background and Aims Matrix Metalloproteinases (MMPs) play an important role in extracellular matrix regulation during cell growth and wound healing. Increased expression of MMP-12 (human macrophage elastase) has been reported in inflammatory bowel disease (IBD) which is characterized by the loss of epithelial tight junction (TJ) barrier function and an excessive inflammatory response. The aim of this study was to investigate the role of MMP-12 in intestinal TJ barrier function and inflammation. Methods Wild type (WT) and MMP-12 -/- mice were subjected to experimental acute or chronic dextran sodium sulfate (DSS) colitis. The mouse colonic permeability was measured in vivo by recycling perfusion of the entire colon and ex vivo by Ussing chamber studies. Results DSS administration increased colonic permeability through modulation of TJ proteins and also increased MMP-12 expression in the colonic mucosa of WT mice. The acute as well as chronic DSS-induced increase in colonic TJ permeability and the severity of DSS colitis was found to be markedly attenuated in MMP-12 -/- mice. The resistance of MMP-12 -/- mice to DSS colitis was characterized by reduced macrophage infiltration and transmigration, and reduced basement membrane laminin degradation. Further in vitro and in vivo studies show that macrophage transmigration across epithelial layer is MMP-12 dependent and the epithelial TJ barrier is compromised during macrophage transmigration. Together, this data demonstrates that MMP-12-mediated degradation of basement membrane laminin, macrophage transmigration, and associated loss of intestinal TJ barrier are key pathogenic factors for intestinal inflammation.

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Background and aim Functional loss of the gut epithelium's paracellular tight junction (TJ) barrier and defective autophagy are factors potentiating inflammatory bowel disease (IBD). Previously, we showed the role of autophagy in enhancing the intestinal TJ barrier via pore-forming claudin-2 degradation. How autophagy regulates the TJ barrier forming proteins remain unknown. Here, we investigated the role of autophagy in the regulation of occludin, a principal TJ component involved in TJ barrier enhancement. Results Autophagy induction using pharmacological activators and nutrient starvation increased total occludin levels in intestinal epithelial cells, mouse colonocytes, and human colonoids. Autophagy induction enriched membrane occludin levels and reduced paracellular permeability of macromolecules. Autophagy-mediated TJ barrier enhancement was contingent on the presence of occludin as OCLN-/- nullified its TJ barrier enhancing effect against macromolecular flux. Autophagy inhibited the constitutive degradation of occludin by preventing its caveolar endocytosis from the membrane and protected against inflammation-induced TJ barrier loss. Autophagy enhanced the phosphorylation of ERK-1/2 and inhibition of these kinases in Caco-2 cells and human colonic mucosa prevented the macromolecular barrier-enhancing effects of autophagy. In-vivo, autophagy induction by rapamycin enhanced occludin levels in WT mouse intestines and protected against LPS and TNF-α-induced TJ barrier loss. Disruption of autophagy with acute Atg7 knockout in adult mice decreased intestinal occludin levels, increasing baseline colonic TJ permeability and exacerbating the effect of experimental colitis. Conclusion Our data suggest a novel role of autophagy in promoting the intestinal TJ barrier by increasing occludin levels in an ERK1/2 MAPK-dependent mechanism.

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Background Proton pump inhibitors (PPIs) are widely used to treat a number of gastroesophageal disorders. PPI-induced elevation in intra-gastric pH may alter gastrointestinal physiology. The tight junctions (TJs) residing at the apical intercellular contacts act as a paracellular barrier. The TJ barrier dysfunction is an important pathogenic factor in Inflammatory Bowel Disease (IBD). Recent studies suggest that PPI may promote disease flares in IBD patients. The role of PPI in intestinal permeability is not clear. Aim The aim of the present study was to study the effect of PPI on the intestinal TJ barrier function. Methods Human intestinal epithelial cell culture and organoid models and mouse IBD models of Dextran sodium sulfate (DSS) and spontaneous enterocolitis in IL-10 -/- mice were used to study the role of PPI in intestinal permeability. Results PPI increased TJ barrier permeability via an increase in a principal TJ regulator, myosin light chain kinase (MLCK) activity and expression, in a p38MAPK dependent manner. PPI-induced increase in extracellular pH caused MLCK activation via p38MAPK. Long term PPI administration in mice exaggerated increase in intestinal TJ permeability and disease severity in two independent models of DSS colitis and IL-10 -/- enterocolitis. The TJ barrier disruption by PPI was prevented in MLCK -/- mice. Human database studies revealed increased hospitalizations associated with PPI use in IBD patients. Conclusions Our results suggest that long term use of PPIs increases intestinal TJ permeability and exaggerates experimental colitis via increase in MLCK expression and activity.

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Background and Aim: Functional loss of paracellular tight junction (TJ) barrier of the gut epithelium and mutations in autophagy genes are factors potentiating inflammatory bowel disease (IBD). Previously we showed the role of autophagy in enhancing the TJ barrier via claudin-2 degradation, however, its role in the regulation of the barrier-forming protein occludin remains unknown. Here, we investigate the role of autophagy in the regulation of occludin and its role in inflammation-mediated TJ barrier loss. Methods: Pharmacological and genetic tools were used to study the effect of autophagy on occludin levels and localization, and the role of the MAPK pathway. Results: Autophagy induction using pharmacological activators and nutrient starvation increased total occludin levels in different epithelial cells. Starvation enriched membrane occludin levels and reduced paracellular inulin flux in Caco-2 cells. Starvation-induced TJ barrier enhancement was contingent on the presence of occludin as OCLN-/- nullified its TJ barrier enhancing effect. Autophagy inhibited the constitutive degradation of occludin and protected against inflammation-induced TJ barrier loss. Starvation-induced TJ barrier enhancement was prevented by inhibition of autophagy. Autophagy enhanced the phosphorylation of ERK-1/2. Inhibition of these kinases in Caco-2 cells and human intestinal mucosa inhibited the protective effects of autophagy. In-vivo, autophagy induction by rapamycin increased occludin levels in mouse intestines and protected against LPS and TNF-α-induced TJ barrier loss. Additionally, acute Atg7 knockout in adult mice decreased intestinal occludin levels, increasing baseline colonic TJ-permeability and exacerbating the effect of DSS-induced colitis. Conclusion: Our data suggest a novel role of autophagy in promoting the intestinal TJ barrier by increasing occludin levels in an ERK1/2 MAPK-dependent mechanism.

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