17 Increased Expression of LRRK2, a Susceptibility Gene of IBD Results in Enhanced Pro-Inflammatory Response and Severe Experimental Colitis

Abstract

Background Many genetic risk loci of inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis have been identified by genome wide association studies in recent years. LRRK2/MUC19 locus has the third strongest association in IBD. LRRK2 gene has been intensively studied as a causative and susceptibility gene of Parkinson's disease. However the immunological function and the mechanisms about how this locus is involved in the pathogenesis of IBD remain largely unknown. The aim of this study is to investigate the immunological role of LRRK2 in the pathogenesis of IBD. Methods We used Lrrk2BAC transgenic mice (Lrrk2 Tg) that contains entire LRRK2 gene region including endogenous promoter and enhancer. Dextran sulfate sodium (DSS)-induced colitis was performed and bone marrow derived dendritic cells (BMDC) from Lrrk2 Tg and littermate control (CTL) were used to examine the immunological function. Immunoprecipitation and western blotting were performed to find the binding partners of LRRK2. Results Lrrk2 Tg exhibited more severe DSS induced colitis than CTL. BMDC from Lrrk2 Tg and CTL were stimulated with various TLR ligands including zymosan, a yeast wall extract. We found that TLR2 and TLR4 ligands did not show an increased response in Lrrk2 Tg compared to CTL. However zymosan induced significant increase in TNF-a synthesis in Lrrk2 Tg. Zymosan can stimulate Dectin1 as well. So we next stimulated BMDC with Dectin-1 agonists such as zymosan depleted (ZymD), a more specific ligand for Dectin-1, heat-killed Saccharomyces cerevisiae (HK-SC) and heat-killed Candida albicans (HK-CA). These stimulations significantly increased TNFa production in Lrrk2 Tg compared to CTL. To investigate the role of LRRK2 in Dectin-1 signaling, BMDC were stimulated with ZymD and the phosphorylation of NF-kB and MAP kinase-related molecules were examined by western blotting. BMDC from Lrrk2 Tg showed increased NF-kB signaling and slight increase in MAP kinase signaling compared to CTL. To identify the binding partners of LRRK2, HEK293T cells were transfected with TAK1, NEMO, TRAF6 and TAB2 expressing plasmids and the immunoprecipitants were subjected to western blotting. We found that these signaling molecules interacted with LRRK2. Conclusions The result of DSS colitis indicates that increased expression of LRRK2 leads to an increased inflammatory response in experimental colitis. Our data suggest that LRRK2 is involved in Dectin-1 signaling and activates NF-kB signaling through TAK1 complex. It has been shown that patients with Crohn's disease had increased LRRK2 expression in colonic mucosa and LRRK2 was phosphorylated during the course of DSS colitis. The activation and increased expression of LRRK2 cause more proinflammatory cytokines production in response to gut microbiota-derived innate immune stimuli and may lead to intestinal inflammation.