Introduction: Chronic activation of the innate immune system drives inflammation and contributes directly to obesity, insulin resistance and atherosclerosis. Previously we showed that necroptosis, a pro-inflammatory form of programmed cell death, is activated in the vessel wall and drives atherosclerosis via activation of RIP3 and MLKL. We sought to determine upstream regulators of necroptosis in atherosclerosis and metabolic disease, and hypothesized that RIP1, a key regulatory kinase upstream of NFkB activation, apoptosis and necroptosis, drives macrophage inflammation in cardiometabolic diseases.
Methods: RIP1 anti-sense oligonucleotides (ASOs, 2 unique sequences) were used to reduce RIP1 expression in 2 mouse models: i) atherosclerotic model [ApoE-/- mice fed a western diet for 8wks] and ii) diet-induced obesity (DIO) model [C57Bl/6 male mice fed a high fat diet for 24 wks]. Mice were given weekly injections of RIP1 ASOs (50 or 100mg/kg) or control ASO (non-targeting).
Results: RIP1 ASO treated ApoE-/- mice had a marked 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, IL-17A, p<0.05). RIP1 knockdown in macrophages decreased inflammatory gene expression (NFκB, TNFα, IL-1α) and in vivo LPS-induced NFκB activation. In obese mice, RIP1 ASOs strikingly decreased body weight (25% decrease versus control p<0.001) and total fat mass (50-65% decrease versus control, p<0.001). Further, insulin resistance was improved in RIP1 ASO treated mice (fasted blood glucose: 10.9mM in control versus 8.5±mM in RIP1 ASO, p<0.001; GTT and ITT both p<0.001). In humans, we identified 5 novel SNPs in strong linkage disequilibrium in or nearby RIP1 gene exon 5. Notably, in a cohort of >1,800 people, individuals carrying the minor allele of these RIP1 SNPs have a 75-89% increase in the risk of developing obesity (adjusted odds ratios: 1.75-1.89, p<10-4) and a significant increase in RIP1 mRNA expression in adipose tissue (eQTL association in METSIM cohort, p=10-23).
Conclusions: We have identified RIP1 as central a driver of insulin resistance, obesity, and atherosclerosis and demonstrate the potential of RIP1 as a novel therapeutic target.