IL-35 suppression of endothelial cell activation and atherosclerosis

动脉粥样硬化及其并发症，如心肌梗塞、中风和外周动脉 疾病是美国发病率和死亡率的主要原因。因此，迫切需要新的治疗方法 需要抑制动脉粥样硬化的进展。CD4Foxp3调节性T细胞(Treg)抑制 人类和小鼠的血管炎症和动脉粥样硬化。然而，潜在的机制 Treg对动脉粥样硬化的抑制作用仍不明确。白介素35(IL-35)是一种新的特征 抗炎细胞因子，主要由Treg分泌，它抑制由各种原因引起的炎症 病情包括自身免疫性糖尿病和关节炎等，与其他特点比较好 抗炎细胞因子IL-10和转化生长因子-β，IL-35作用更强，IL-35显著转化 效应T细胞和B细胞转化为可诱导的Treg和调节性B细胞。 IL-35是由p35和EB病毒诱导基因3(EBI3)亚基组成的异源二聚体。 IL-35亚基在血管内皮细胞(EC)和巨噬细胞(M)中均有较强的表达。 动脉粥样硬化斑块，提示IL-35，而不是IL-27，在动脉粥样硬化病变中上调。 与健康对照组比较，冠心病患者血浆IL-35水平降低。 冠心病患者相关细胞因子IL-12、IL-27水平升高。然而，IL-2的作用- 35在抑制EC激活和动脉粥样硬化形成中的作用尚未见研究。 目的是确定抑制IL-35的作用和机制。 内皮细胞活化与动脉粥样硬化。我们有很长的研究EC激活的出版记录，EC 血管加速中的功能障碍、单核细胞(MC)募集、动脉粥样硬化和Treg降低 发炎。我们已经获得了强有力的初步数据，并发表了两篇论文(J.B.C.；PLOS One) 表明1)IL-35是一种反应性细胞因子，在大多数细胞类型和 在脂多糖(LPS)诱导的炎症和致动脉粥样硬化的载脂蛋白中显著诱导 E(ApoE-/-)小鼠；2)IL-35与其相关细胞因子IL-27共享EBI3亚基。与IL-27相比，IL-35 抑制促动脉粥样硬化的Toll样受体4(TLR4)配体脂多糖诱导的内皮细胞活化 血管细胞黏附分子-1(VCAM-1)通过丝裂原活化蛋白激酶(MAPK)的表达 人主动脉内皮细胞AP-1依赖通路；3)IL-35抑制溶血磷脂酰胆碱。 衍生的致动脉粥样硬化脂质刺激)诱导内皮细胞活化；4)机制上，IL-35抑制溶血PC-1。 人主动脉内皮细胞线粒体活性氧的诱导性产生 建立载脂蛋白E-/-小鼠IL-35治疗模型，发现IL-35治疗可抑制动脉粥样硬化； 最后，我们已经培育出EBI3-/-/ApoE-/-双基因敲除(KO)小鼠 额外产生两只双KO小鼠，包括IL-12Rβ2(IL-35受体(IL35R)亚单位)-/- /ApoE-/-Double KO小鼠和p35(IL-35亚单位)-/-/ApoE-/-Double KO小鼠。这些强劲的初步数据 研究表明，IL-35可以抑制EC的活化和动脉粥样硬化。 F H T是项目 中环 假设 需要检查的是，IL-35抑制EC的激活和促炎作用 Ly6促进MC募集，从而有助于抑制动脉粥样硬化。我们将检验这一假设 使用下面简要描述的三个相互关联的具体目标。目标1将确定表达式和 致动脉内皮细胞激活的IL-35/IL-35R亚基对动脉内皮细胞的抑制作用 载脂蛋白E-/-小鼠的主动脉(相关研究)。目标2将研究IL-35的潜在机制 在动脉粥样硬化期间抑制EC激活和单核细胞/M募集(机制研究)。 AIM 3将确定IL-35亚基p35、EBI3和IL-35R亚基IL-12Rβ2的调节作用 载脂蛋白E-/-小鼠的动脉粥样硬化形成(治疗研究)。这项提议的成功将导致确定 IL-35抑制EC活化和MC/M募集的作用和机制 动脉粥样硬化的形成。这一结果将为基于IL-35的治疗学的未来发展带来巨大的希望 用于抑制血管炎症和动脉粥样硬化。

Atherosclerosis and its complications, such as myocardial infarction, stroke, and peripheral artery disease, are the leading cause of morbidity and mortality in the U.S. Therefore, novel therapies are urgently needed to inhibit the progression of atherosclerosis. CD4+Foxp3+ regulatory T cells (Treg) suppress vascular inflammation and atherosclerosis in both humans and mice. However, the mechanisms underlying Treg suppression of atherosclerosis remain poorly defined. Interleukin-35 (IL-35) is a newly characterized anti-inflammatory cytokine, mainly secreted from Treg, which inhibits inflammation resulting from various conditions, including autoimmune diabetes and arthritis, etc. Compared with the other well-characterized anti-inflammatory cytokines, IL-10 and TGF-β, IL-35 is more powerful, and IL-35 significantly converts effector T cells and B cells to inducible Treg and regulatory B cells. IL-35 is a heterodimer composed of p35 and Epstein-Barr virus induced gene 3 (EBI3) subunits. Both IL-35 subunits are strongly expressed in endothelial cells (EC) and macrophages (M) in patients' atherosclerotic plaques, suggesting that IL-35, but not IL-27, is upregulated in atherosclerotic lesions. Compared with healthy controls, IL-35 is decreased in the plasma of patients with coronary artery disease (CAD) while related cytokines IL-12 and IL-27 are increased in patients with CAD. However, the roles of IL- 35 in suppressing EC activation and atherogenesis have not been studied. The goal o is to determine the roles and mechanisms underlying IL-35 suppression of EC activation and atherosclerosis. We have a long publication record of studying EC activation, EC dysfunction, monocyte (MC) recruitment, atherosclerosis, and decreased Treg in acceleration of vascular inflammation. We have obtained strong preliminary data and published two papers (J.B.C.; PLOS ONE) showing that 1) IL-35 is a responsive cytokine, which is not constitutively expressed in most cell types and is significantly induced in lipopolysaccharide (LPS)-induced inflammation and in atherogenic apolipoprotein E (ApoE-/-) mice; 2) IL-35 shares EBI3 subunit with its relative cytokine, IL-27. In contrast to IL-27, IL-35 inhibits EC activation induced by proatherogenic Toll-like receptor 4 (TLR4) ligand, LPS, by suppressing the expression of vascular cell adhesion molecule 1 (VCAM-1) via mitogen-activated protein kinase (MAPK)- AP-1 dependent pathway in human aortic EC; 3) IL-35 inhibits lysophosphatidylcholine (lysoPC, an oxLDL- derived proatherogenic lipid stimulus)-induced EC activation; 4) Mechanistically, IL-35 inhibits lysoPC- induced generation of mitochondrial reactive oxygen species (mtROS) in human aortic EC; 5) We established an IL-35 therapy model in ApoE-/- mice and found that IL-35 therapy inhibits atherosclerosis; and finally, 6) we have generated EBI3-/-/ApoE-/- double knock-out (KO) mice and are also in the process of generating two additional double KO mice, including IL-12Rβ2 (an IL-35 receptor (IL35R) subunit)-/- /ApoE-/- double KO mice and p35 (IL-35 subunit)-/-/ApoE-/- double KO mice. These strong preliminary data and publications suggest that IL-35 may suppress EC activation and atherosclerosis. f h t is project The central hypothesis to be examined is that IL-35 inhibits EC activation and proinflammatory ly6Chigh MC recruitment, thereby contributing to suppression of atherosclerosis. We will test this hypothesis using three linked, specific aims briefly described below. Aim 1 will determine the expressions and suppressive function of IL-35/IL-35R subunits in human aortic EC activated by proatherogenic stimuli and aortas of ApoE-/- mice (relevant studies). Aim 2 will examine the mechanisms underlying IL-35 suppression of EC activation and monocyte/M recruitment during atherosclerosis (mechanistic studies). Aim 3 will determine the mediating roles of IL-35 subunits p35, EBI3 and IL-35R subunit IL12Rβ2 in atherogenesis in ApoE-/- mice (therapeutic studies). Success of this proposal will lead to the identification of the role and mechanisms underlying IL-35 suppression of EC activation and MC//M recruitment and atherogenesis. The results will hold great promise for the future development of IL-35-based therapeutics for suppression of vascular inflammation and atherosclerosis.