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Recently we reported that IL-35 is an inflammation-induced ant-inflammatory cytokine; thus, function as a HAMP (homeostasis associated molecular patterns). However, whether IL-35 affects endothelial cell activation and subsequent development of atherosclerosis is not known. Hence, we studied the expression of IL-35 during early atherosclerosis; and its roles and mechanisms in suppressing EC activation. Initially, we analyzed whether endogenous IL-35 levels and its receptor expression was increased in early atherosclerosis. Our data revealed that IL-35 level was significantly increased in the plasma of hyperlipidemic patients and apolipoprotein E (ApoE-/-) deficient mice. Further, the expression of IL-35 receptor components were increased in ApoE-/- mouse aortas. IL-35 inhibited monocyte recruitment on to human aortic endothelial cells (EC) activated by pro-atherogenic lysophosphatidylcholine (LPC). Furthermore, our RNA-Seq analysis showed that IL-35 selectively inhibited ICAM-1 expression that mediates EC activation. Further analysis by flowcytometry and electron spin resonance analysis revealed that IL-35 blocked LPC-mediated mitochondrial-reactive oxygen species (mtROS) production. The subsequent mass spectrometry, ChIP-Seq analysis and EMSA showed that LPC-mediated mtROS promotes acetylation of histone 3 lysine 14 (H3K14). Acetylation of histones facilitate the transcription of EC activation-related genes. We found that acetylated H3K14 increased the binding of pro-inflammatory transcription factor Ap-1 on to ICAM-1 promoter region and induction of ICAM-1 gene transcription. IL-35 mediated Inhibition of mtROS attenuated H3K14 acetylation, inhibited AP-1 binding to ICAM-1 promoter and suppressed ICAM-1 transcription. Finally, IL-35 cytokine treatment inhibited the progression and development of atherosclerosis in ApoE-/- mice. Our study concluded that IL-35 is induced during atherosclerosis and plays an anti-atherogenic role by suppressing EC activation through mtROS and H3K14 acetylation dependent mechanism.

Objective— IL-35 (interleukin-35) is an anti-inflammatory cytokine, which inhibits immune responses by inducing regulatory T cells and regulatory B cells and suppressing effector T cells and macrophages. It remains unknown whether atherogenic stimuli induce IL-35 and whether IL-35 inhibits atherogenic lipid-induced endothelial cell (EC) activation and atherosclerosis. EC activation induced by hyperlipidemia stimuli, including lysophosphatidylcholine is considered as an initiation step for monocyte recruitment and atherosclerosis. In this study, we examined the expression of IL-35 during early atherosclerosis and the roles and mechanisms of IL-35 in suppressing lysophosphatidylcholine-induced EC activation. Approach and Results— Using microarray and ELISA, we found that IL-35 and its receptor are significantly induced during early atherosclerosis in the aortas and plasma of ApoE (apolipoprotein E) knockout mice—an atherosclerotic mouse model—and in the plasma of hypercholesterolemic patients. In addition, we found that IL-35 suppresses lysophosphatidylcholine-induced monocyte adhesion to human aortic ECs. Furthermore, our RNA-sequencing analysis shows that IL-35 selectively inhibits lysophosphatidylcholine-induced EC activation-related genes, such as ICAM-1 (intercellular adhesion molecule-1). Mechanistically, using flow cytometry, mass spectrometry, electron spin resonance analyses, and chromatin immunoprecipitation-sequencing analyses, we found that IL-35 blocks lysophosphatidylcholine-induced mitochondrial reactive oxygen species, which are required for the induction of site-specific H3K14 (histone 3 lysine 14) acetylation, increased binding of proinflammatory transcription factor AP-1 in the promoter of ICAM-1, and induction of ICAM-1 transcription in human aortic EC. Finally, IL-35 cytokine therapy suppresses atherosclerotic lesion development in ApoE knockout mice. Conclusions— IL-35 is induced during atherosclerosis development and inhibits mitochondrial reactive oxygen species-H3K14 acetylation-AP-1–mediated EC activation.