Abstract Body: Inflammasome activation (IA) has been identified as a potential therapeutic target in atherosclerotic cardiovascular disease (CVD). Clonal hematopoiesis (CH), involving somatic mutations in genes driving hematopoiesis, has emerged as an independent CVD risk factor. JAK2V617F (JAK2VF) is a CH variant that is increasingly recognized as a cause of myocardial infarction, ischemic stroke, and thoracic aortic aneurysm in humans. JAK2VF has been shown to promote atherosclerosis via increased macrophage IA. Ninjurin-1 (Ninj1) is a transmembrane protein that mediates plasma membrane rupture (PMR) downstream of IA and other programmed cell death. Ninj1-mediated PMR has been shown to promote aortic aneurysm and liver injury, but its role in atherosclerosis remains poorly understood. By generating a chimeric mouse model with and without hematopoietic Ninj1-deficency, we identified an atheroprotective role of Ninj1 in low allele burden Jak2VF CH but not control mice. Jak2VF exacerbates atherosclerosis by increasing lesion area (50x10⁴ vs 40x10⁴ mm², p=0.04) and Ninj1-deficiency further worsened Jak2VF-mediated lesion progression by increased lesion area (64x10⁴ vs 50x10⁴ mm², p=0.002) and necrotic core area (20x10⁴ vs 13x10⁴ mm², p=0.006). Single-cell RNA sequencing analysis of the aortic intimal-medial layer from different chimeric mice revealed a landscape that was enriched with inflammatory myeloid cells under Jak2VF conditions, which was further exacerbated by Ninj1-deficiency. The deletion of Ninj1 increased the accumulation of inflammatory macrophages enriched with lipid transport (p=0.004) and iron ion transport (p=0.045) genes. Consistently, immunohistochemical staining of Jak2VF;Ninj1^-/- aortic root revealed increased cleaved-gasdermin D (p=0.04), a marker of pyroptosis, and transferrin receptor (p=0.03), a marker of ferroptosis. A similar pro-inflammatory macrophage population was identified in integrated single-cell RNA sequencing analysis of human carotid lesions. The population is also enriched with lipid transport (p=0.018) and iron ion transport genes (p=0.046). Our findings suggest that Ninj1-deficiency promotes atherosclerosis progression under Jak2VF conditions by sustaining pro-inflammatory cell death pathways, like pyroptosis and ferroptosis, and that therapeutic targeting of Ninj1 could potentially increase CVD risk.