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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL132399-02
Application #
9394817
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Chen, Jue
Project Start
2016-12-12
Project End
2020-11-30
Budget Start
2017-12-01
Budget End
2018-11-30
Support Year
2
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Temple University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
057123192
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
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Virtue, Anthony; Johnson, Candice; Lopez-PastraƱa, Jahaira et al. (2017) MicroRNA-155 Deficiency Leads to Decreased Atherosclerosis, Increased White Adipose Tissue Obesity, and Non-alcoholic Fatty Liver Disease: A NOVEL MOUSE MODEL OF OBESITY PARADOX. J Biol Chem 292:1267-1287
Li, Xinyuan; Fang, Pu; Yang, William Y et al. (2017) IL-35, as a newly proposed homeostasis-associated molecular pattern, plays three major functions including anti-inflammatory initiator, effector, and blocker in cardiovascular diseases. Cytokine :
Li, Ya-Feng; Nanayakkara, Gayani; Sun, Yu et al. (2017) Analyses of caspase-1-regulated transcriptomes in various tissues lead to identification of novel IL-1?-, IL-18- and sirtuin-1-independent pathways. J Hematol Oncol 10:40
Dai, Jin; Fang, Pu; Saredy, Jason et al. (2017) Metabolism-associated danger signal-induced immune response and reverse immune checkpoint-activated CD40+ monocyte differentiation. J Hematol Oncol 10:141
Shao, Ying; Nanayakkara, Gayani; Cheng, Jiali et al. (2017) Lysophospholipids and Their Receptors Serve as Conditional DAMPs and DAMP Receptors in Tissue Oxidative and Inflammatory Injury. Antioxid Redox Signal :
Cheng, Jiali; Nanayakkara, Gayani; Shao, Ying et al. (2017) Mitochondrial Proton Leak Plays a Critical Role in Pathogenesis of Cardiovascular Diseases. Adv Exp Med Biol 982:359-370
Fu, Hangfei; Vadalia, Nish; Xue, Eric R et al. (2017) Thrombus leukocytes exhibit more endothelial cell-specific angiogenic markers than peripheral blood leukocytes do in acute coronary syndrome patients, suggesting a possibility of trans-differentiation: a comprehensive database mining study. J Hematol Oncol 10:74
Xu, Yanjie; Xia, Jixiang; Liu, Suxuan et al. (2017) Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom. Front Biosci (Landmark Ed) 22:1439-1457
Li, Ya-Feng; Li, Rong-Shan; Samuel, Sonia B et al. (2016) Lysophospholipids and their G protein-coupled receptors in atherosclerosis. Front Biosci (Landmark Ed) 21:70-88

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