Mo1736 Knockdown of STE20-Like Proline/Alanine-Rich Kinase (SPAK) Attenuates Intestinal Inflammation in Mouse

Abstract

Background: Inflammatory bowel diseases (IBDs), principally Crohn's disease and ulcerative colitis, are characterized by epithelial barrier disruption and alterations in immune regulation. Ste20-like proline/alanine-rich kinase (SPAK) plays a role in intestinal inflammation, but its underlying mechanisms need to be defined. Methods: SPAK knockout mice (KO) colon mucosa was subjected to transepithelial resistance (TER) assay and dilution potential assay with ussing chamber, and transepithelial permeability assay with fluorescein isothiocyanatedextran (FITC-dextran). SPAK KO mice were treated with dextran sulfate sodium (DSS) or trinitrobenzene sulfuric acid (TNBS) to evaluate the effect of SPAK on the experimental colitis in terms of clinical and histological characteristics, mouse endoscopic features, enzymatic activities, and production of pro-inflammatory cytokines and translocation of luminal bacteria. The expressions of junction proteins were characterized using real time PCR, western blots and immunohistochemistry. Results: SPAK KO mice exhibited significant increases of intestinal barrier function. Knock-down of SPAK significantly decreased paracellular intestinal permeability to FITC-dextran, and also lowered significantly the sodium ion selectivity of tight junctions in intestinal epithelial cells. Further, the expression of junction proteins βcatenin, claudin-2 decreased. In contrast, expression of ZO-1, Occludin, E-cadeherin increased significantly, whereas the change of expressions of Claudin-4 and Claudin-1 was not noticeable. In Vivo studies using mouse model of colitis induced by DSS and TNBS showed that KO mice were more tolerant to DSS or TNBS treatment than were wild-type animals, as demonstrated by clinical and histological characteristics and MPO enzymatic activities. KO mice also demonstrated ameliorated colon inflammation by mouse endoscopy compared to wild type mice. Consistent with this notion, we found that SPAK knockdown attenuated the increases of inflammatory cytokines production In Vivo and also ameliorated bacterial translocation under DSS treatment, which together likely reduce intestinal epithelial permeability. Conclusion: Knockdown of SPAK increases intestinal innate immune homeostasis, showing that SPAK is important in control or attenuation of pathological responses in IBD.