IL-35 suppression of endothelial cell activation and atherosclerosis

动脉粥样硬化及其并发症，如心肌梗死、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。 来抑制动脉粥样硬化的发展CD 4 + Foxp 3+调节性T细胞（Treg）抑制 血管炎症和动脉粥样硬化。然而， Treg对动脉粥样硬化的抑制作用仍不明确。白细胞介素-35（IL-35）是一种新近发现的 抗炎细胞因子，主要由Treg分泌，其抑制由各种炎症引起的炎症。 条件，包括自身免疫性糖尿病和关节炎等，与其他相比， 抗炎细胞因子，IL-10和TGF-β，IL-35更强大，并且IL-35显著转化 将效应T细胞和B细胞转化为诱导性Treg和调节性B细胞。 IL-35是由p35和EB病毒诱导基因3（EBI 3）亚基组成的异源二聚体。 两种IL-35亚基均在患者的内皮细胞（EC）和巨噬细胞（M β）中强烈表达。 动脉粥样硬化斑块，表明IL-35，而不是IL-27，在动脉粥样硬化病变中上调。 与健康对照组相比，冠心病患者血浆中IL-35降低 (CAD)而相关细胞因子IL-12和IL-27在CAD患者中升高。然而，IL- 35在抑制EC活化和动脉粥样硬化形成中的作用尚未研究。 目的是确定IL-35抑制的作用和机制。 EC激活和动脉粥样硬化。我们有很长的研究EC激活的出版记录， 功能障碍、单核细胞（MC）募集、动脉粥样硬化和Treg在血管加速中的减少 炎症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） 显示1）IL-35是应答性细胞因子，其在大多数细胞类型中不是组成型表达， 在脂多糖（LPS）诱导的炎症和致动脉粥样硬化的载脂蛋白 E（ApoE-/-）小鼠; 2）IL-35与其相关细胞因子IL-27共享EBI 3亚基。与IL-27相反，IL-35 抑制EC活化诱导的促动脉粥样硬化Toll样受体4（TLR 4）配体，LPS，通过抑制 通过丝裂原活化蛋白激酶（MAPK）表达血管细胞粘附分子1（VCAM-1）- 人主动脉EC中的AP-1依赖性途径; 3）IL-35抑制溶血磷脂酰胆碱（lysoPC，一种oxLDL-1受体拮抗剂）。 衍生的致动脉粥样硬化脂质刺激）诱导的EC活化; 4）在机制上，IL-35抑制lysoPC-1。 诱导人主动脉EC线粒体活性氧（mtROS）的产生; 5）我们 在ApoE-/-小鼠中建立IL-35治疗模型，发现IL-35治疗抑制动脉粥样硬化; 最后，6）我们已经产生了EBI 3-/-/ApoE-/-双敲除（KO）小鼠，并且也在进行中。 产生另外两只双KO小鼠，包括IL-12 R β2（IL-35受体（IL 35 R）亚基）-/- /ApoE-/-双KO小鼠和p35（IL-35亚基）-/-/ApoE-/-双KO小鼠。这些强有力的初步数据 并且出版物表明IL-35可以抑制EC活化和动脉粥样硬化。 f t是项目 中央 假设 IL-35抑制EC活化和促炎性反应， ly 6C高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 利用下文简述的三个相互关联的具体目标。目标1将确定表达式和 IL-35/IL-35 R亚单位在致动脉粥样硬化刺激激活的人主动脉EC中的抑制功能 ApoE-/-小鼠的睾丸炎（相关研究）。目的2将研究IL-35的潜在机制 动脉粥样硬化过程中EC活化和单核细胞/巨噬细胞募集的抑制（机制研究）。 目的3探讨IL-35亚单位p35、EBI 3和IL-35 R亚单位IL 12 R β2在IL-12介导的细胞凋亡中的作用。 ApoE-/-小鼠的动脉粥样硬化形成（治疗研究）。这项建议的成功将导致确定 IL-35抑制EC活化和MC//M β募集的作用和机制， 动脉粥样硬化这些结果将为基于IL-35的治疗方法的未来发展带来巨大希望 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.