SUMMARY Diabetes is a major risk factor for atherosclerosis. Although atherosclerosis is initiated by deposition of cholesterol-rich lipoproteins in the artery wall, the entry of inflammatory leukocytes into lesions fuels disease progression and impairs resolution. We show that diabetic mice have increased numbers of circulating neutrophils and Ly6-Chi monocytes, reflecting hyperglycemia-induced proliferation and expansion of bone marrow myeloid progenitors and release of monocytes into the circulation. Increased neutrophil production of S100A8/A9, via an interaction with the receptor for advanced glycation end products on common myeloid progenitor cells, leads to enhanced myelopoiesis. Treatment of hyperglycemia reduces monocytosis, entry of monocytes into atherosclerotic lesions and promotes regression. In patients with type I diabetes plasma S100A8/A9 levels correlate with leukocyte counts and coronary artery disease. Thus, hyperglycemia drives myelopoiesis and thus promotes atherogenesis in diabetes.

Clonal haematopoiesis, which is highly prevalent in older individuals, arises from somatic mutations that endow a proliferative advantage to haematopoietic cells. Clonal haematopoiesis increases the risk of myocardial infarction and stroke independently of traditional risk factors 1 . Among the common genetic variants that give rise to clonal haematopoiesis, the JAK2 V617F (JAK2 VF ) mutation, which increases JAK-STAT signalling, occurs at a younger age and imparts the strongest risk of premature coronary heart disease 1,2 . Here we show increased proliferation of macrophages and prominent formation of necrotic cores in atherosclerotic lesions in mice that express Jak2 VF selectively in macrophages, and in chimeric mice that model clonal haematopoiesis. Deletion of the essential inflammasome components caspase 1 and 11, or of the pyroptosis executioner gasdermin D, reversed these adverse changes. Jak2 VF lesions showed increased expression of AIM2, oxidative DNA damage and DNA replication stress, and Aim2 deficiency reduced atherosclerosis. Single-cell RNA sequencing analysis of Jak2 VF lesions revealed a landscape that was enriched for inflammatory myeloid cells, which were suppressed by deletion of Gsdmd. Inhibition of the inflammasome product interleukin-1β reduced macrophage proliferation and necrotic formation while increasing the thickness of fibrous caps, indicating that it stabilized plaques. Our findings suggest that increased proliferation and glycolytic metabolism in Jak2 VF macrophages lead to DNA replication stress and activation of the AIM2 inflammasome, thereby aggravating atherosclerosis. Precise application of therapies that target interleukin-1β or specific inflammasomes according to clonal haematopoiesis status could substantially reduce cardiovascular risk.Atherosclerotic cardiovascular disease (ACVD) is the major cause of death and disability in the developed world 3 . A large burden of residual ACVD risk remains despite current therapies, including intensive lowering of low-density lipoprotein levels 3 , which highlights the need for new treatments. In the Canakinumab Antiinflammatory Thrombosis Outcomes Study (CANTOS), inhibition of IL-1β reduced cardiovascular events, thereby validating the contribution of inflammation to ACVD 4 . However, canakinumab therapy was associated with a small risk of infections and has not been approved for cardiovascular conditions. Thus, a more precise way to identify patients who may benefit most from anti-inflammatory therapy is required. Clonal haematopoiesis usually arises from somatic mutations in haematopoietic stem and progenitor cells (HSPCs) in one of four genes (TET2, ASXL1, DNMT3A or JAK2), which lead to clonal expansion of haematopoietic cells. The prevalence of clonal haematopoiesis increases with age, and it affects more than 10% of people who are over 70 years old 1 . Although clonal haematopoiesis conferred an increased risk of haematological malignancies of 0.5-1% per year, this modest increase was not nearly enough to account for the 40% incr...

Reverse cholesterol transport (RCT) refers to the mobilization of cholesterol on HDL particles (HDL-C) from extravascular tissues to plasma, ultimately for fecal excretion. Little is known about how HDL-C leaves peripheral tissues to reach plasma. We first used 2 models of disrupted lymphatic drainage from skin -1 surgical and the other genetic -to quantitatively track RCT following injection of [ 3 H]-cholesterol-loaded macrophages upstream of blocked or absent lymphatic vessels. Macrophage RCT was markedly impaired in both models, even at sites with a leaky vasculature. Inhibited RCT was downstream of cholesterol efflux from macrophages, since macrophage efflux of a fluorescent cholesterol analog (BODIPY-cholesterol) was not altered by impaired lymphatic drainage. We next addressed whether RCT was mediated by lymphatic vessels from the aortic wall by loading the aortae of donor atherosclerotic Apoe-deficient mice with [ 2 H] 6 -labeled cholesterol and surgically transplanting these aortae into recipient Apoe-deficient mice that were treated with anti-VEGFR3 antibody to block lymphatic regrowth or with control antibody to allow such regrowth. [ 2 H]-Cholesterol was retained in aortae of anti-VEGFR3-treated mice. Thus, the lymphatic vessel route is critical for RCT from multiple tissues, including the aortic wall. These results suggest that supporting lymphatic transport function may facilitate cholesterol clearance in therapies aimed at reversing atherosclerosis.