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-?, 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-12R?2 (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 IL12R?2 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.

Public Health Relevance

Atherosclerosis and its complications, such as myocardial infarction (MI), stroke, and peripheral artery disease, are still the leading cause of morbidity and mortality in the U.S., regardless of recent progress in lipid lowering therapy including statins. Therefore, novel therapies are urgently needed to inhibit the progression of atherosclerosis. However, the mechanisms underlying the suppression of atherosclerosis by soluble immune factors, called anti-inflammatory cytokines, remain poorly defined. The proposed studies will examine the contribution and mechanism of a new anti- inflammatory cytokine interleukin-35 (IL-35) pathway in atherosclerosis-accelerated vascular disease. Success of this study would characterize IL-35 suppressive molecular signaling in atherosclerosis and lead to the development of new therapeutics for the treatment of cardiovascular disease (MI), cerebrovascular disease (stroke), and peripheral vascular disease.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Chen, Jue
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Temple University
Internal Medicine/Medicine
Schools of Medicine
United States
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