Anti-inflammatory cytokines IL-35 and IL-10 block atherogenic lysophosphatidylcholine-induced, mitochondrial ROS-mediated innate immune activation, but spare innate immune memory signature in endothelial cells

It has been shown that anti-inflammatory cytokines interleukin-35 (IL-35) and IL-10 could inhibit acute endothelial cell (EC) activation, however, it remains unknown if and by what pathways IL-35 and IL-10 could block atherogenic lipid lysophosphatidylcholine (LPC)-induced sustained EC activation; and if mitochondrial reactive oxygen species (mtROS) can differentiate mediation of EC activation from trained immunity (innate immune memory). Using RNA sequencing analyses, biochemical assays, as well as database mining approaches, we compared the effects of IL-35 and IL-10 in LPC-treated human aortic ECs (HAECs). Principal component analysis revealed that both IL-35 and IL-10 could similarly and partially reverse global transcriptome changes induced by LPC. Gene set enrichment analyses showed that while IL-35 and IL-10 could both block acute EC activation, characterized by upregulation of cytokines/chemokines and adhesion molecules, IL-35 is more potent than IL-10 in suppressing innate immune signatures upregulated by LPC. Surprisingly, LPC did not induce the expression of trained tolerance itaconate pathway enzymes but induced trained immunity enzyme expressions; and neither IL-35 nor IL-10 was found to affect LPC-induced trained immunity gene signatures. Mechanistically, IL-35 and IL-10 could suppress mtROS, which partially mediate LPC-induced EC activation and innate immune response. Therefore, anti-inflammatory cytokines could reverse mtROS-mediated acute and innate immune trans-differentiation responses in HAECs, but it could spare metabolic reprogramming and trained immunity signatures, which may not fully depend on mtROS. Our characterizations of anti-inflammatory cytokines in blocking mtROS-mediated acute and prolonged EC activation, and sparing trained immunity are significant for designing novel strategies for treating cardiovascular diseases, other inflammatory diseases, and cancers. Keywords: Interleukin-35, Interleukin-10, Endothelial cell activation, Mitochondrial ROS, Trained immunity, Atherosclerosis