Epigenetic Regulation of Trained Immunity in Thoracic Aortic Aneurysms and Dissections

Profound accumulation of macrophages (MΦs) and persistent inflammation are prominent features in as- cending thoracic aortic aneurysms and dissections (ATAAD), and the underlying mechanisms are poorly under- stood. Recent studies suggest that innate immune cells, including monocytes/MΦs, can differentiate into a pro- inflammatory phenotype and conserve this memory after exposure to stimuli, a process known as trained im- munity, thereby enhancing the subsequent inflammatory response. In our preliminary studies, scRNA-seq analysis revealed several MΦ subpopulations. Importantly, aortic MΦs in sporadic ATAAD patients and in an- giotensin (Ang II)-induced ATAAD mice exhibited pro-inflammatory status with enhanced differentiation to pro- inflammatory MΦs and suppressed differentiation to phagocytic/anti-inflammatory MΦs. Intriguingly, this pro- inflammatory status can be observed in monocytes/MΦs isolated from bone marrow of mice, and the memory can stably last for 5 days in ex vivo culture. Furthermore, scATAC-seq suggested that the training of the mono- cytes/MΦs occurred at the epigenetic level, and several TFs, including IRF3 and surprisingly IRF4 (known as an anti-inflammatory lineage-determining TF), may drive the induction of pro-inflammatory differentiation. In cul- tured cells, DNA from damaged aortic cells induced monocytes/MΦs toward a pro-inflammatory phenotype through cGAMP-STING-IRF3/IRF4 signaling. Finally, Sting-deficient mice showed preservation of phago- cytic/anti-inflammatory MΦs and reduction of pro-inflammatory MΦs. The objective of this project is to under- stand the training of monocytes/MΦs to different functional phenotypes in the aortic wall, and to identify key pathways driving monocytes/MΦs to a pro-inflammatory direction in ATAAD. In Aim 1, we will determine the sites of the immunity training, the epigenetic signatures of the trained monocytes/MΦs, and the duration of the memory in ATAAD by performing integrated omics analyses (scRNA-seq, scATAC-seq, and spatial RNA-seq) of monocytes/MΦs in bone marrow and the aortic wall in mice and will confirm the epigenetic signatures of the trained monocytes/MΦs in circulation and the aortic wall in ATAAD patients. In Aim 2, we will determine the causal role of the STING-IRF3/IRF4 circuitry in training monocytes/MΦs during ATAAD development by per- forming omics and functional analyses of aortic tissues and bone marrow monocytes from MΦ-Sting-/-, MΦ-Irf3- /-, MΦ-Irf4-/-, and WT mice, and dissect the link between dsDNA/cGAMP-STING-IRF3/IRF4, chromatin remodel- ing, gene expression, and MΦ phenotypes in cultured monocytes/MΦs. In Aim 3, we will determine the role of MΦ-specific STING-IRF3/IRF4 in ATAAD development in mice. The impact of this work will be an improved understanding of the molecular mechanisms that cause persistent aortic inflammation and degeneration, provid- ing a new direction for treatment strategies for preventing ATAAD and its fatal sequelae. Elucidating the immunity training of monocytes/MΦs is likely to have broad implications for many other cardiovascular diseases in which persistent inflammation plays a critical role in tissue destruction and disease progression.