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

Abstract

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.

Interleukin 35 Delays Hindlimb Ischemia-Induced Angiogenesis Through Regulating ROS-Extracellular Matrix but Spares Later Regenerative Angiogenesis

“…However, mitochondrial oxidants can induce phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2), resulting in activation of the receptor and subsequent angiogenesis. Similar to those findings, our previous reports showed that i) IL-35 inhibits lipopolysaccharide (LPS) and proatherogenic lipids LPC induced HAEC activation ( 5 , 7 ); ii) IL-35 suppresses vascular inflammation and atherosclerosis via inhibiting mitochondrial ROS( 8 , 9 , 34 – 37 ); iii) hypoxia may induce thrombus leukocyte transdifferentiation by upregulating endothelial cell-specific angiogenic markers ( 38 ); and iv) inhibition of caspase-1/inflammasome activation in EC improves ischemia-triggered angiogenesis ( 39 , 40 ). However, the question remained whether IL-35 modulates ischemia-triggered angiogenesis potentially via a ROS-related mechanism.…”

“…We reported that IL-35 is a new responsive heterodimeric anti-inflammatory cytokine ( 4 ). IL-35 belongs to the IL-12 cytokine family and is potent in inhibiting various inflammatory diseases including lipopolysaccharide (LPS)-induced lung inflammation, human aortic endothelial cell (HAEC) activation ( 5 ), atherogenic lipids lysophosphatidylcholine (LPC) ( 6 – 8 ) -induced HAEC activation and atherosclerosis ( 9 ) but sparing innate immune memory (trained immunity) in HAEC ( 8 , 10 – 12 ) as we reported. IL-35 α -chain p35 (encoded by IL12A) is shared with IL-12; and β -chain EBI3 (encoded by Epstein–Barr virus-induced 3, EBI3) is shared with IL-27 and IL-39.…”

“…The induction of both IL-35 subunits, IL-12A and EBI3, implicated that exogenous IL-35 could stimulate these immune cells ( 14 , 50 ) and/or ECs in the ischemic muscle to secrete more IL-35. In our previous findings, IL-35 can directly signal through EC and inhibit LPC- or LPS-induced EC activation ( 5 , 8 ). Therefore, we treated HMVEC with IL-35 for 6 h under normoxia or hypoxia to examine the direct effect on EC.…”

“…These reports demonstrated that ROS and RNS play a significant role in modulating the pathophysiological processes of HLI. Recently, we reported that IL-35 inhibits human aortic EC activation via suppressing ROS generation ( 8 , 9 ), mitogen-activated protein kinase pathway ( 5 ), epigenetic ( 9 ) and gene transcription ( 8 ). Of note, our experimental data have demonstrated for the first time that IL-35 not only suppresses inflammation but also inhibits ROS-mediated processes.…”

Interleukin-35 pretreatment attenuates lipopolysaccharide-induced heart injury by inhibition of inflammation, apoptosis and fibrosis

“…IL-35 protected against acute liver and kidney injury caused by LPS through inhibiting inflammatory responses (27,28). IL-35 also inhibited lysophosphatidylcholine-induced mitochondrial reactive oxygen species (mtROS) production and human aortic endothelial activation (29,30). Furthermore, IL-35 was reported to exert anti-apoptotic effects that prevent diabetic neuropathic pain and doxorubicin-induced cardiac injury (24,31).…”