IL-35 inhibits gut microbiota-produced uremic toxin-accelerated endothelial cell activation Chronic kidney disease (CKD) affects 15% of the adult population worldwide. CKD promotes cardiovascular morbidity and mortality. Increased endothelial cell (EC) activation/dysfunction and vascular inflammation play a critical role the development of CKD-accelerated cardiovascular disease (CVD), which begins in the early stages of CKD. New therapies are urgently needed to inhibit EC activation/dysfunction accelerated by CKD. EC dysfunction is associated with reduced nitric oxide (NO) production whereas our new JBC paper showed EC activation features include: 1) increased secretion of cytokines and chemokines; 2) upregulation of EC adhesion molecules; 3) upregulation of additional DAMP receptors; and 4) upregulation of T cell co-stimulation/co-inhibition receptors. Novel strategies targeted at reducing EC activation/dysfunction and vascular inflammation may provide effective treatment in the early stages of CKD. Trimethylamine-N-Oxide (TMAO) is a gut microbiota generated, choline-derived metabolite. TMAO is a newly identified as a uremic toxin, which is strongly elevated in CKD and associated with atherosclerotic CVDs. TMAO induces EC dysfunction and increased vascular inflammation via binding to G-protein coupled receptor, thereby activating mitogen?activated protein kinase and NF??B, and increasing circulating cytokines. We and others reported that interleukin-35 (IL-35) is a new and powerful anti-inflammatory cytokine that inhibits various inflammation. Plasma IL-35 levels are increased in CKD patients. Therefore, the central hypothesis of this proposal is that IL-35 suppresses uremic toxin TMAO-induced EC activation/dysfunction and CKD-accelerated vascular inflammation. Therefore, we will examine this hypothesis via following three aims: 1) To determine expression and suppressive function of IL-35/IL-35R subunits in TMAO-induced human aortic ECs (HAECs) and mouse aortic ECs (MRECs) from CKD mice; 2) To determine the molecular mechanisms, by which IL-35 inhibits EC activation via inhibiting mitochondrial reactive oxygen species (mtROS) generation, suppressing caspase-1(casp1) canonical/casp11 non-canonical inflammasome signaling, and inhibiting histone 3 lysine 14 acetylation (H3K14ac) induced EC activation gene expression; and 3) To determine the suppressing roles of two IL-35 subunits (p35, and EBI3) and an IL-35 receptor (IL-35R) subunit (IL12R?2) in CKD mouse model. The aim 3 will be explored through the use of a novel IL-35 therapy (gain of function) in CKD mouse model, and four loss of function CKD models including p35-/- CKD mice, EBI-3-/- CKD mice, global IL-12R?2-/- CKD mice and EC-specific IL12Rb2-/- CKD mice to determine the suppressive roles of IL-35 signaling in EC dysfunction and vascular inflammation in CKD. The significance of this proposal is that the success of these studies should have a major impact in the field, and provide novel mechanistic insights into IL-35 in suppressing TMAO-induced EC activation and CKD-accelerated vascular inflammation, which could lead to the potential development of novel therapeutics for CKD-accelerated CVDs.