RIPK1
Expression Associates With Inflammation in Early Atherosclerosis in Humans and Can Be Therapeutically Silenced to Reduce NF-κB Activation and Atherogenesis in Mice
Authors: Karunakaran Denuja , Nguyen My-Anh , Geoffrion Michele , Vreeken Dianne , Lister Zachary , Cheng Henry S. , Otte Nicola , Essebier Patricia , Wyatt Hailey , Kandiah Joshua W. , Jung Richard , Alenghat Francis J. , Mompeon Ana , Lee Richard , Pan Calvin , Gordon Emma , Rasheed Adil , Lusis Aldons J. , Liu Peter , Matic Ljubica Perisic , Hedin Ulf , Fish Jason E. , Guo Liang , Kolodgie Frank , Virmani Renu , van Gils Janine M. , Rayner Katey J. 
Published: November 2020

Background:

Chronic activation of the innate immune system drives inflammation and contributes directly to atherosclerosis. We previously showed that macrophages in the atherogenic plaque undergo RIPK3 (receptor-interacting serine/threonine-protein kinase 3)-MLKL (mixed lineage kinase domain-like protein)–dependent programmed necroptosis in response to sterile ligands such as oxidized low-density lipoprotein and damage-associated molecular patterns and that necroptosis is active in advanced atherosclerotic plaques. Upstream of the RIPK3-MLKL necroptotic machinery lies RIPK1 (receptor-interacting serine/threonine-protein kinase 1), which acts as a master switch that controls whether the cell undergoes NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells)–dependent inflammation, caspase-dependent apoptosis, or necroptosis in response to extracellular stimuli. We therefore set out to investigate the role of RIPK1 in the development of atherosclerosis, which is driven largely by NF-κB–dependent inflammation at early stages. We hypothesize that, unlike RIPK3 and MLKL, RIPK1 primarily drives NF-κB–dependent inflammation in early atherogenic lesions, and knocking down RIPK1 will reduce inflammatory cell activation and protect against the progression of atherosclerosis.

Methods:

We examined expression of RIPK1 protein and mRNA in both human and mouse atherosclerotic lesions, and used loss-of-function approaches in vitro in macrophages and endothelial cells to measure inflammatory responses. We administered weekly injections of RIPK1 antisense oligonucleotides to
Apoe
−/−
mice fed a cholesterol-rich (Western) diet for 8 weeks.

Results:

We find that RIPK1 expression is abundant in early-stage atherosclerotic lesions in both humans and mice. Treatment with RIPK1 antisense oligonucleotides led to a reduction in aortic sinus and en face lesion areas (47.2% or 58.8% decrease relative to control,
P
<0.01) and plasma inflammatory cytokines (IL-1α [interleukin 1α], IL-17A [interleukin 17A],
P
<0.05) in comparison with controls.
RIPK1
knockdown in macrophages decreased inflammatory genes (NF-κB, TNFα [tumor necrosis factor α], IL-1α) and in vivo lipopolysaccharide- and atherogenic diet–induced NF-κB activation. In endothelial cells, knockdown of
RIPK1
prevented NF-κB translocation to the nucleus in response to TNFα, where accordingly there was a reduction in gene expression of
IL1B
,
E-selectin
, and monocyte attachment.

Conclusions:

We identify RIPK1 as a central driver of inflammation in atherosclerosis by its ability to activate the NF-κB pathway and promote inflammatory cytokine release. Given the high levels of RIPK1 expression in human atherosclerotic lesions, our study suggests RIPK1 as a future therapeutic target to reduce residual inflammation in patients at high risk of coronary artery disease.