Abstract 4142847: <i>Dnmt3a</i> modifies the anti-inflammatory effect of the IL-6 classical pathway in macrophages to change the atherosclerosis burden.

Background: Clonal hematopoiesis of indeterminate potential (CHIP) can contribute to cardiovascular risk. The Interleukin-6 receptor (IL6R) polymorphism (D358A) modifies the risk of coronary artery diseases among CHIP carriers in human cohorts. However, the effects of the IL6R D358A on IL-6 pathways are discordant with the canonical expectation, that the classical IL-6 signaling pathway is anti-inflammatory while the trans-signaling pathway is pro-inflammatory. Hypothesis: DNMT3A, the most frequent driver gene for CHIP, changes the inflammatory consequence of IL-6 classical signaling from anti-inflammatory to pro-inflammatory to worsen atherosclerosis. Method: We investigated if an anti-IL6R antibody modifies atherosclerosis associated with myeloid Dnmt3a deficiency in vivo . Ldlr -/- mice were transplanted with 10% bone marrow cells from Dnmt3a -/- mice/90% of wild-type (WT) cells compared with 100% WT transfer. After engraftment, mice consumed a high-fat diet, and received an anti-IL6R antibody for 10 weeks to assess aortic root atherosclerosis vs. control antibody treatment. We also investigated the differences between IL-6 classical- vs trans-signaling pathways and whether Dnmt3a deficiency in bone marrow-derived macrophages (BMDM) modifies the IL-6 pathways. RNA-seq was performed in BMDM stimulated with IL-6, which activates the classical IL-6 pathway, or IL-6/IL6R conjugate, which activates trans-signaling. We further compared IL-6 vs control stimulation between Dnmt3a -/- and WT BMDM using RNA-seq, and the major finding was replicated by quantitative PCR. In addition, IL-1β was quantified in the conditioned medium. Result: Anti-IL6R antibody treatment reduced atherosclerosis in the mice with myeloid Dnmt3a deficiency. IL4R expression rose significantly in BMDM stimulated by IL-6 compared to IL-6/IL6R conjugate. IL4R induction by IL-6, but not the IL-6/IL6R conjugate, fell significantly in Dnmt3a -/- BMDM compared to WT. IL-1β secretion declined with IL-6 stimulation and was higher in IL6R deficient BMDM, but IL-6 stimulation and deletion of IL6R did not affect IL-1β secretion in Dnmt3a -/- BMDMs. Conclusion: These data suggest that DNMT3A tonically limits the IL-6 classical pathway to limit atherogenesis in an IL-4 signaling-dependent manner. These findings promote understanding of the CHIP-atherosclerosis association and inform the development of management strategies for CVD risk in CHIP carriers.