Introduction: Previously we reported that CD4+ regulatory T cell (Treg) cell death pathways regulate vascular inflammation. IL-35 is a new anti-inflammatory cytokine originally identified in Treg. We reported that IL-35 is a responsive cytokine that can be induced by proinflammatory stimuli lipopolysaccharide (LPS) and inhibit LPS mediated endothelial cell (EC) activation. However, whether IL-35 is induced during atherogenesis and whether IL-35 inhibits atherogenic lipid-induced EC activation and atherosclerosis is not known. EC activation induced by hyperlipidemic stimuli lysophosphatidylcholine (LPC) initiates monocyte recruitment and atherosclerosis.
Hypothesis: IL-35 expression is increased during early atherogenesis and attenuate atherosclerotic progression by inhibiting EC activation.
Methods & Results: Our microarray and ELISA studies revealed that two IL-35 subunits and their receptor subunits were significantly induced during early atherosclerosis in aortas and blood extracted from hyperlipidemic apolipoprotein E knockout mice (ApoE KO) as well as in plasma of hypercholesterolemic patients. Also, we found that IL-35 suppressed LPC mediated monocyte adhesion to human aortic ECs (HAEC). Mechanistically, our RNA-Seq analysis showed that IL-35 selectively inhibits LPC-induced EC activation-related genes such as ICAM-1, CXCL1, CXCL8, and IL1B. To further discern the molecular pathways mediated by IL-35 during atherosclerosis, we performed flow cytometry, mass spectrometry, electron spin resonance analyses, and CHIP-Seq analyses. We found that IL-35 blocks LPC-induced mitochondrial reactive oxygen species (mtROS) generation, which are required for induction of site-specific histone 3 lysine 14 (H3K14) acetylation. This led to increased binding of pro-inflammatory transcription factor AP-1 to ICAM-1 promoter, thus inducing ICAM-1 transcription in HAEC. Finally, IL-35 cytokine therapy suppressed atherosclerotic lesion development in ApoE KO mice.
Conclusions: Based on our findings, we present a new concept that IL-35 is a newly proposed homeostasis-associated molecular pattern. IL-35 is induced during atherosclerosis and inhibits its progression by suppressing mtROS-H3K14ac-AP-1 mediated EC activation.