Mechanisms and Modulation of Accelerated Atherosclerosis in Clonal Hematopoiesis

Project Summary/Abstract With age, humans can accumulate leukocyte clones in blood that arise from somatic mutations in bone marrow stem cells that enhance expansion: clonal hematopoiesis of indeterminate potential (CHIP). Mutations in DNMT3A and TET2 account for the plurality of these clones. CHIP confers highly elevated cardiovascular (CV) risk, independent of traditional risk factors. We have found accelerated atherogenesis and the involvement of IL-1 and IL-6 in mice with myeloid loss of Tet2 or Dnmt3a function and that genetically reduced IL-6 signaling abrogates the elevated CV risk in humans with DNMT3A or TET2 CHIP. Analyses of our CANTOS trial showed greater efficacy of IL-1β inhibition in humans with DNMT3A or TET2 CHIP. These results point the way to a genotype-directed allocation of therapy, an approach that has transformed oncology but remains aspirational in atherosclerosis. The roles of classical vs. trans IL-6 signaling in atherothrombosis requires further study due to conflicting evidence. Specific aim 1 will test the hypothesis that atherosclerotic mice that mimic CHIP due to myeloid deficiency in Dnmt3a treated with an antibody that interrupts global IL-6 signaling by neutralizing IL-6 receptor α (IL-6r, CD126) have decreased atherosclerosis and inflammation within the lesions, blood and other organs as gauged in part by single-cell RNA sequencing (scRNA-seq). We further hypothesize that IL-6r inhibition will limit expansion of the mutant clone. Specific aim 2 will probe the role of classical vs. trans IL-6 signaling in myeloid cells in accelerated atherogenesis in CHIP using Il6rflox/flox/Lyz2-Cre mice bone marrow chimeric LDLR-/- mice (to block leukocyte classical signaling), and administration of a gp130-IgG1-Fc chimeric protein (to block trans signaling) using similar procedures and endpoints. The results will illuminate the controversy and unsettled science regarding the contributions of classical and trans IL-6 signaling to atherogenesis. Specific aim 3. Our preliminary experiments show that female myeloidTet2-/-Ldlr-/- mice have greater acceleration of atherogenesis than Tet2+/+ Ldlr-/- male mice and, unlike their male counterparts, show reduced atherogenesis with IL-1β neutralization. We will localize where this sex difference operates in the inflammasome–IL-1β–IL-6 pathway, and test the hypothesis that female Dnmt3a-/-Ldlr-/- mice have greater response to IL-6r inhibition than males. We will probe mechanisms by gonadal ablation experiments and by analysis of scRNA-seq data, which in preliminary data shows IL-1β expression in resident macrophages cells from atheroma from Tet2-/- females but not males. This work will deepen understanding of the mechanisms of accelerated atherosclerosis in CHIP. Our new pilot clinical data show that an anti-IL-6 antibody can mute inflammation in humans. Thus, the work proposed here will provide an indispensable step toward validating and furnishing the fundamental basis of an immediately translatable targeted therapeutic strategy based on CHIP genotype.