1437Single cell RNA sequencing reveals profound changes in monocytic cell clusters in patients with mutations associated with clonal hematopoiesis

Abstract

Abstract Background Monocytes and macrophages have distinct roles in cardiovascular health where they may contribute to beneficial processes like wound healing and cardiac conductance or to pathological processes like inflammation, remodeling and fibrosis. Despite their importance, the specific signatures of circulating monocytes are missing. Single cell RNA sequencing (scRNA-seq) provides a novel opportunity to define subsets of monocytes mediating inflammation in humans. Purpose To detect and study the inflammatory burden driven by subsets of monocytes in healthy individuals and subjects with chronic ischemic heart failure (CHF) using scRNA-seq. Methods and results Circulating CD31+ cells of CHF patients (n=11) along with aged matched (n=3) and young healthy controls (n=5) were sorted and scRNA-seq then performed. Unsupervised clustering of present sequencing data revealed these cells to be comprised of 19 subpopulations (primarily monocytes). Many subpopulations of cells were comprised chiefly or solely by CHF subjects. Dysregulated genes in CHF subjects, relative to healthy controls included interleukin-1b, thrombospondin-1, S100A8 and matrix metalloprotease-1, which were confirmed by FACS and qRT-PCR in a validation cohort. Given the expanded, divergent and highly inflammatory transcriptional populations of monocytes in patients with CHF, we assessed whether occurrence of somatic mutations associated with clonal hematopoiesis of indeterminate potential (CHIP), recently shown to be increased in subjects with atherosclerosis and heart failure, might occur in these subjects. Indeed, we identified patients who revealed mutations in the DNA methyltransferase DNMT3A and other CHIP-driver genes. DNMT3A mutations were associated with changes in known DNMT3A target genes such as the pro-inflammatory genes CXCL1, CXCL2 and IL6 in circulating monocytic cells. Moreover, cell fate trajectory analysis showed shared fates of cells driven by pseudotime-dependent variables from subjects harboring DNMT3A mutations. Regulated genes in this fate were associated with cell survival, migration and inflammation, appropriate for this disease phenotype. Conclusions This is the first study to show scRNA-seq profiles of monocytes in patients with CHF. Healthy subjects displayed remarkable homogeneity in their transcriptional profiles. CHF subjects show changes in inflammatory gene expression. CHF subjects with clonal hematopoiesis share signature gene expression profiles driving similar cell fates. Subjects with CHF of ischemic origin harboring DNMT3A mutations have a worse prognosis than non-CHIP carrier CHF controls. Identification of early alterations in immune cell subsets recognized by single cell sequencing and genetic testing of CHIP mutations may detect subjects with a high risk and allow for a precision treatment of the immune disorders. Acknowledgement/Funding SFB834