Abstract Body (Do not enter title and authors here): Background: Clonal hematopoiesis of indeterminate potential (CHIP) refers to somatic mutations in hematopoietic cells with a minimum allele fraction ≥ 2% without hematological disease. CHIP depends on age and increases the risk of coronary artery disease (CAD). In their 70s women’s prevalence of cardiovascular disease catches up to that of men. In Canakinumab Antiinflammatory Thrombosis Outcome Study (CANTOS) neutralizing interleukin-1β (IL-1β) in chronic CAD, subjects with TET2 CHIP showed greater reduction of cardiovascular events. However, this substudy did not investigate sex.

Hypothesis: We hypothesized that sex modifies the association between CHIP mutations and risk of CAD, and the effects of IL-1β inhibition in CHIP-related atherosclerosis in mice.

Methods: Sex differences on the association of CAD and CHIP mutations were investigated using 2 cohorts: the UK and Mass General Brigham Biobanks. CHIP genes showing sex differences in human data were deleted in hematopoietic cells of female and male atherosclerosis-susceptible Ldlr^-/- mice. Mice consumed a 0.2% cholesterol high-fat diet with or without an anti-mouse IL-1β monoclonal antibody for 9 weeks. We assessed atherosclerosis by histology and performed mechanistic studies using single-cell RNA sequencing (scRNAseq) of atherosclerotic aortic arch and in vitro bone-marrow derived macrophages.

Results: Women with TET2 CHIP had larger hazard ratio for CAD than men in both biobanks. Overall CHIP and other mutations did not demonstrate sex differences in the association with CAD. In Ldlr^-/- mice deficient for Tet2 in hematopoietic cells, IL-1β inhibition reduced atherosclerosis in female but not male mice. ScRNAseq revealed more inflammatory cells and more expression of inflammatory genes in hematopoietic Tet2^-/- females than in males. IL-1β inhibition limited these sex differences. In vitro experiments demonstrated that Tet2 deficiency prevents the interaction between estrogen-receptor alpha and histone H3 controlling epigenetic regulation in mouse macrophages.

Conclusions: Females with TET2 CHIP have accentuated CAD risk and IL-1β blockade limits Tet2-augmented atherosclerosis in female but not male mice. These results highlight the importance of study of sex as a biological variable, uncover a new mechanism of Tet2 CHIP on acceleration of atherosclerosis, and inform the design of trials refining allocation of anti-inflammatory therapies in TET2 CHIP.