Mechanisms of Atherogenesis in Clonal Hematopoiesis

1克隆性造血(CH)是一种在老年人中高度流行的疾病，源于赋予 2造血细胞亚群的增殖优势。先天性心脏病增加心肌梗死和中风的风险 3不受传统风险因素影响。相对于引起CH的其他常见遗传变异，JAK2V617F(JAK2VF) 4增加JAK/STAT信号的突变发生在较年轻的年龄，并赋予过早冠状动脉的巨大风险 5心脏病(CHD)。然而，在CH中，突变的细胞只占血细胞的一小部分，然而，患者保持 6患冠心病的风险增加，这表明突变细胞可能对它们的环境产生了过大的影响。使用小鼠模型 7Jak2VF CH我们发现IL-1特异性地促进Jak2VF巨噬细胞的细胞内源性增殖，但不能促进野生型 8(WT)巨噬细胞，即使在相同的动脉粥样硬化病变内。应用抗IL-1抗体降低Jak2VF 9巨噬细胞增殖，斑块稳定性改善。Jak2VF小鼠皮损的scRNA-Seq分析 10在所有骨髓细胞中的表达证实了一个显著的炎性髓系群体，其基因丰富。 11与IL-1信号转导相关。我们发现炎性髓系细胞的丰度在缺乏 12上睑下垂执行剂Gasdermin D(Gsdmd)，可能是由于抑制了IL-1的分泌。因此，具体目标1 13将通过以下方式研究Jak2VF细胞在同一病变中促进WT细胞炎症表型的能力 14 IL-1信号转导。从机制上讲，我们已经证明了AIM2炎症体在Jak2VF小鼠的皮损中被激活。 15从Jak2VF细胞中缺失Gsdmd会导致皮损中DNA双链断裂增加。骨髓源 表达Jak2VF但缺乏Caspase1/11的16个巨噬细胞增加了活性氧，提示DNA损伤 17发生在炎性小体激活的上游。因此，特异性目标2将确定Jak2VF中氧化DNA是否增加 巨噬细胞促进炎症小体的激活和皮损中的炎症。特定目标3随后将调查是否 导致PPM1D失活的CH变异蛋白磷酸酶镁/锰依赖1D基因的19个截断突变 20种DNA修复蛋白，促进动脉粥样硬化。这里提出的研究将通过扩展我的 21巨噬细胞DNA损伤和动脉粥样硬化的概念性知识，并将通过培训补充 22细胞测序技术，以了解免疫细胞群如何根据CH变异状态在病变中发生变化。这 23培训将在K99阶段得到严格的指导和与我的科学顾问的合作支持。 24拟议的研究和培训将阐明DNA损伤和炎症体激活的机制 25推动CH加速动脉粥样硬化，这将为我提供成功过渡到 26名独立学术研究员。 27 28 29 30

1 Clonal hematopoiesis (CH), a highly prevalent condition in the elderly, arises from somatic mutations that endow 2 a proliferative advantage to a subset of hematopoietic cells. CH increases the risk of myocardial infarction and stroke 3 independent of traditional risk factors. Relative to other common genetic variants giving rise to CH, the JAK2V617F (JAK2VF) 4 mutation that increases JAK/STAT signaling occurs at a younger age and imparts a substantial risk of premature coronary 5 heart disease (CHD). In CH mutated cells make up only a small fraction of blood cells, however, patients maintain an 6 elevated risk of CHD, suggesting mutant cells may have an outsized impact on their environment. Using murine models of 7 Jak2VF CH we found that IL-1 specifically promotes cell intrinsic proliferation of Jak2VF macrophages, but not wild type 8 (WT) macrophages even within the same atherosclerotic lesions. Administration of antibodies to IL-1 reduced Jak2VF 9 macrophage proliferation and improved features of plaque stability. scRNA-Seq analysis of lesion from mice with Jak2VF 10 expression in all bone marrow cells identified a prominent inflammatory myeloid population that was enriched for genes 11 associated with IL-1 signaling. We found that inflammatory myeloid cell abundance was decreased in the absence of the 12 pyroptosis executioner Gasdermin D (Gsdmd), potentially due to suppressed IL-1 secretion. Therefore, Specific Aim 1 13 will investigate the ability of Jak2VF cells to promote inflammatory phenotypes in WT cells within the same lesion through 14 IL-1 signaling. Mechanistically we have shown that the AIM2 inflammasome is activated in lesions from Jak2VF mice. 15 Deletion of Gsdmd from Jak2VF cells leads to increased DNA double strand breaks in lesions. Bone marrow derived 16 macrophages expressing Jak2VF, but lacking Caspase 1/11 have increased reactive oxygen species, suggesting DNA damage 17 occur upstream of inflammasome activation. Thus, Specific Aim 2 will determine if increased oxidized DNA in Jak2VF 18 macrophages promotes inflammasome activation and inflammation in lesions. Specific Aim 3 will then investigate if 19 truncation mutations in the CH variant protein phosphatase Mg2+/Mn2+ dependent 1D (PPM1D) that leads to inactivation of 20 DNA repair proteins, promotes atherosclerosis. The research proposed here will be accomplished by expanding my 21 conceptual knowledge of DNA damage in macrophages and atherosclerosis and will be complemented by training in single 22 cell sequencing technology to understand how immune cell populations change in lesions based on CH variant status. This 23 training will be supported by rigorous mentoring and collaboration with my scientific advisors during the K99 phase. 24 Together the proposed research and training will elucidate mechanism by which DNA damage and inflammasome activation 25 drive accelerated atherosclerosis in CH which will provide me the knowledge and skills to successfully transition into an 26 independent academic researcher. 27 28 29 30