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Immune checkpoint inhibitor (ICI) therapies can increase the risk of cardiovascular events in survivors of cancer by worsening atherosclerosis. Here we map the expression of immune checkpoints (ICs) within human carotid and coronary atherosclerotic plaques, revealing a network of immune cell interactions that ICI treatments can unintentionally target in arteries. We identify a population of mature, regulatory CCR7 + FSCN1 + dendritic cells, similar to those described in tumors, as a hub of IC-mediated signaling within plaques. Additionally, we show that type 2 diabetes and lipid-lowering therapies alter immune cell interactions through PD-1, CTLA4, LAG3 and other IC targets in clinical development, impacting plaque inflammation. This comprehensive map of the IC interactome in healthy and cardiometabolic disease states provides a framework for understanding the potential adverse and beneficial impacts of approved and investigational ICIs on atherosclerosis, setting the stage for designing ICI strategies that minimize cardiovascular disease risk in cancer survivors., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Atherosclerosis results from lipid-driven inflammation of the arterial wall that fails to resolve. Imbalances in macrophage accumulation and function, including diminished migratory capacity and defective efferocytosis, fuel maladaptive inflammation and plaque progression. The neuroimmune guidance cue netrin-1 has dichotomous roles in inflammation partly due to its multiple receptors; in atherosclerosis, netrin-1 promotes macrophage survival and retention via its receptor Unc5b. To minimize the pleiotropic effects of targeting netrin-1, we tested the therapeutic potential of deleting Unc5b in mice with advanced atherosclerosis. We generated Unc5b fl/fl Cx3cr1 creERT2/WT mice, which allowed conditional deletion of Un5b (∆ Unc5b MØ ) in monocytes and macrophages by tamoxifen injection. After inducing advanced atherosclerosis by hepatic PCSK9 overexpression and western diet feeding for 20 wk, Unc5b was deleted and hypercholesterolemia was normalized to simulate clinical lipid management. Deletion of myeloid Unc5b led to a 40% decrease in atherosclerotic plaque burden and reduced plaque complexity compared to Unc5b fl/fl Cx3cr1 WT/WT littermate controls (Ctrl MØ ). Consistently, plaque macrophage content was reduced by 50% in ∆ Unc5b MØ mice due to reduced plaque Ly6C hi monocyte recruitment and macrophage retention. Compared to Ctrl MØ mice, plaques in ∆ Unc5b MØ mice had reduced necrotic area and fewer apoptotic cells, which correlated with improved efferocytotic capacity by Unc5b -deficient macrophages in vivo and in vitro. Beneficial changes in macrophage dynamics in the plaque upon Unc5b deletion were accompanied by an increase in atheroprotective T cell populations, including T-regulatory and Th2 cells. Our data identify Unc5b in advanced atherosclerosis as a therapeutic target to induce pro-resolving restructuring of the plaque immune cells and to promote atherosclerosis regression., Competing Interests: Competing interests statement:K.J.M. is on the scientific advisory board of Beren Therapeutics and Bitterroot Bio. The other authors declare no conflict of interest. E.A.F. and K.J.M. have a patent on the use of inhibitors of Unc5b for treating inflammatory arthritis and KJM has a patent on the use of inhibitors of Unc5b for osteolysis.

Atherosclerosis is fueled by a failure to resolve lipid-driven inflammation within the vasculature that drives plaque formation. Therapeutic approaches to reverse atherosclerotic inflammation are needed to address the rising global burden of cardiovascular disease (CVD). Recently, metabolites have gained attention for their immunomodulatory properties, including itaconate, which is generated from the tricarboxylic acid-intermediate cis-aconitate by the enzyme Immune Responsive Gene 1 (IRG1/ACOD1). Here, we tested the therapeutic potential of the IRG1-itaconate axis for human atherosclerosis. Using single-cell RNA sequencing (scRNA-seq), we found that IRG1 is up-regulated in human coronary atherosclerotic lesions compared to patient-matched healthy vasculature, and in mouse models of atherosclerosis, where it is primarily expressed by plaque monocytes, macrophages, and neutrophils. Global or hematopoietic Irg1 -deficiency in mice increases atherosclerosis burden, plaque macrophage and lipid content, and expression of the proatherosclerotic cytokine interleukin (IL)-1β. Mechanistically, absence of Irg1 increased macrophage lipid accumulation, and accelerated inflammation via increased neutrophil extracellular trap (NET) formation and NET-priming of the NLRP3-inflammasome in macrophages, resulting in increased IL-1β release. Conversely, supplementation of the Irg1 -itaconate axis using 4-octyl itaconate (4-OI) beneficially remodeled advanced plaques and reduced lesional IL-1β levels in mice. To investigate the effects of 4-OI in humans, we leveraged an ex vivo systems-immunology approach for CVD drug discovery. Using CyTOF and scRNA-seq of peripheral blood mononuclear cells treated with plasma from CVD patients, we showed that 4-OI attenuates proinflammatory phospho-signaling and mediates anti-inflammatory rewiring of macrophage populations. Our data highlight the relevance of pursuing IRG1-itaconate axis supplementation as a therapeutic approach for atherosclerosis in humans., Competing Interests: Competing interests statement:K.J.M. is on the scientific advisory Board of Beren Therapeutics and Bitterroot Bio. K.J.M. and A.M.S. have patents and patent applications through NYU Grossman School of Medicine that have been submitted/published and that are not related to the work detailed in this manuscript. The other authors declare no conflict of interest.

Histone variants and the associated post-translational modifications that govern the stemness of haematopoietic stem cells (HSCs) and differentiation thereof into progenitors (HSPCs) have not been well defined. H3.3 is a replication-independent H3 histone variant in mammalian systems that is enriched at both H3K4me3- and H3K27me3-marked bivalent genes as well as H3K9me3-marked endogenous retroviral repeats. Here we show that H3.3, but not its chaperone Hira, prevents premature HSC exhaustion and differentiation into granulocyte-macrophage progenitors. H3.3-null HSPCs display reduced expression of stemness and lineage-specific genes with a predominant gain of H3K27me3 marks at their promoter regions. Concomitantly, loss of H3.3 leads to a reduction of H3K9me3 marks at endogenous retroviral repeats, opening up binding sites for the interferon regulatory factor family of transcription factors, allowing the survival of rare, persisting H3.3-null HSCs. We propose a model whereby H3.3 maintains adult HSC stemness by safeguarding the delicate interplay between H3K27me3 and H3K9me3 marks, enforcing chromatin adaptability., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)