Abstract Body: Introduction
Chronic innate immune activation drives atherosclerosis. Pro-inflammatory agents (oxidised or aggregated LDL; oxLDL, aggLDL) promote lesion growth, whereas anti-inflammatory cues [IL-10, HDL, Apolipoprotein-AI (ApoAI)] support resolution and lesion reduction. RIPK1 (Receptor-interacting protein kinase 1) controls inflammation, cell survival and death via its phosphorylation or ubiquitination states. Complete macrophage Ripk1 ablation worsens atherosclerosis. We hypothesize that anti-inflammatory cues suppress RIPK1, and partial Ripk1 loss is atheroprotective.

Methods
Bone marrow (BM) from Ripk1^+/- or WT mice was transplanted into irradiated female and male Ldlr^-/- mice (n ≥ 8/group) and fed a western diet (WD; 0.2% cholesterol) for 12-16 weeks. In vitro, BM-derived macrophages were treated with oxLDL or aggLDL (50-100 μg/mL), reconstituted discoidal HDL (rHDL) or ApoAI (500 μg/mL), porphyrin-lipid HDL nanoparticles (Por-HDL-NPs; 600 μg/mL) or IL-10/IL-13 (100 ng/mL). RIPK1 levels and signaling were assessed. ApoE^-/- mice on WD received Por-HDL-NPs (40 mg/kg) for 6 weeks.

Results
In Ldlr^-/- mice, transplantation of Ripk1^+/- BM reduced aortic sinus lesions (-68.9% at 12 weeks; -49.7% at 16 weeks, p<0.05), necrotic core size and serum IL-1α (-45.9%, p<0.05) relative to controls, with unchanged HDL-C or LDL-C. oxLDL and aggLDL increased Ripk1 mRNA by 2-fold in WT macrophages but not in Ripk1^+/- cells. Ripk1^+/- macrophages had reduced mitochondrial superoxide, IL-1α/β secretion and cell death, but showed ~2-fold increase in CCL2-induced migration in response to oxLDL/aggLDL versus WT cells in a scratch assay (p<0.001). ApoAI, rHDL, IL-10 and IL-13 reduced RIPK1 mRNA and protein by ~50% in WT macrophages (p<0.05). Por-HDL-NPs rescued IFN-γ-induced Ripk1 mRNA increase (1.7-fold to -0.6-fold, p<0.05). In ApoE^-/- mice, Por-HDL-NPs reduced aortic sinus lesional area by 22% and RIPK1 protein by 11-fold versus controls (p<0.0001). Mechanistically, in macrophages, ApoAI increased Il10, which ‘switched off’ RIPK1 via 2 STAT3-dependent actions: (i) RIPK1 phosphorylation at its inhibitory site Ser321 at 30 mins, promoting cell survival; and (ii) RIPK1 protein reduction at 24h.

Conclusion
Partial Ripk1 loss and anti-inflammatory cues limit atherosclerosis by suppressing endogenous RIPK1. ApoAI increased IL-10 and suppressed RIPK1 via STAT3, while Por-HDL-NPs lowered RIPK1 and reduced lesions in vivo. Targeted RIPK1 modulation may dampen atherosclerotic inflammation.