Abstract
Inflammatory bowel disease (IBD) is characterized by abnormal host-microbe interactions. Proinflammatory cytokine IFNγ and a novel TNF superfamily member, TL1A, have been implicated in epithelial barrier dysfunction. The divergent regulatory mechanisms of transcellular versus paracellular hyperpermeability remain poorly understood. Intestinal epithelia express two splice variants of long myosin light chain kinase (MLCK), of which the full-length MLCK1 differ from the shorter isoform MLCK2 by a Src kinase phosphorylation site. The aim was to investigate the roles of MLCK splice variants in gut barrier defects under proinflammatory stress. Upregulated expression of TL1A, IFNγ, and two MLCK variants was observed in human IBD biopsy specimens. The presence of intraepithelial bacteria preceded tight junction (TJ) damage in dextran sodium sulfate-treated and TL1A-transgenic mouse models. Lack of barrier defects was observed in long MLCK(-/-) mice. TL1A induced MLCK-dependent terminal web (TW) contraction, brush border fanning, and transepithelial bacterial internalization. The bacterial taxa identified in the inflamed colonocytes included Escherichia, Enterococcus, Staphylococcus,and Lactobacillus. Recombinant TL1A and IFNγ at low doses induced PI3K/Akt/MLCK2-dependent bacterial endocytosis, whereas high-dose IFNγ caused TJ opening via the iNOS/Src/MLCK1 axis. Bacterial internalization was recapitulated in MLCK-knockout cells individually expressing MLCK2 but not MLCK1. Immunostaining showed different subcellular sites of phosphorylated MLC localized to the TJ and TW in the MLCK1- and MLCK2-expressing cells, respectively. In conclusion, proinflammatory cytokines induced bacterial influx through transcellular and paracellular routes via divergent pathways orchestrated by distinct MLCK isoforms. Bacterial transcytosis induced by TL1A may be an alternative route causing symptom flares in IBD.
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