Limited proliferation capacity of aortic intima resident macrophages requires monocyte recruitment for atherosclerotic plaque progression.

Early atherosclerosis depends upon responses by immune cells resident in the intimal aortic wall. Specifically, the healthy intima is thought to be populated by vascular dendritic cells (DCs) that, during hypercholesterolemia, initiate atherosclerosis by being the first to accumulate cholesterol. Whether these cells remain key players in later stages of disease is unknown. Using murine lineage-tracing models and gene expression profiling, we reveal that myeloid cells present in the intima of the aortic arch are not DCs but instead specialized aortic intima resident macrophages (MacAIR) that depend upon colony-stimulating factor 1 and are sustained by local proliferation. Although MacAIR comprise the earliest foam cells in plaques, their proliferation during plaque progression is limited. After months of hypercholesterolemia, their presence in plaques is overtaken by recruited monocytes, which induce MacAIR-defining genes. These data redefine the lineage of intimal phagocytes and suggest that proliferation is insufficient to sustain generations of macrophages during plaque progression.

Macrophages Facilitate Electrical Conduction in the Heart.

Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes. Conversely, macrophages render the resting membrane potential of cardiomyocytes more positive and, according to computational modeling, accelerate their repolarization. Photostimulation of channelrhodopsin-2-expressing macrophages improves atrioventricular conduction, whereas conditional deletion of connexin 43 in macrophages and congenital lack of macrophages delay atrioventricular conduction. In the Cd11bDTR mouse, macrophage ablation induces progressive atrioventricular block. These observations implicate macrophages in normal and aberrant cardiac conduction.

Self-renewing resident cardiac macrophages limit adverse remodeling following myocardial infarction.

Macrophages promote both injury and repair after myocardial infarction, but discriminating functions within mixed populations remains challenging. Here we used fate mapping, parabiosis and single-cell transcriptomics to demonstrate that at steady state, TIMD4+LYVE1+MHC-IIloCCR2- resident cardiac macrophages self-renew with negligible blood monocyte input. Monocytes partially replaced resident TIMD4-LYVE1-MHC-IIhiCCR2- macrophages and fully replaced TIMD4-LYVE1-MHC-IIhiCCR2+ macrophages, revealing a hierarchy of monocyte contribution to functionally distinct macrophage subsets. Ischemic injury reduced TIMD4+ and TIMD4- resident macrophage abundance, whereas CCR2+ monocyte-derived macrophages adopted multiple cell fates within infarcted tissue, including those nearly indistinguishable from resident macrophages. Recruited macrophages did not express TIMD4, highlighting the ability of TIMD4 to track a subset of resident macrophages in the absence of fate mapping. Despite this similarity, inducible depletion of resident macrophages using a Cx3cr1-based system led to impaired cardiac function and promoted adverse remodeling primarily within the peri-infarct zone, revealing a nonredundant, cardioprotective role of resident cardiac macrophages.

Publisher Correction: Self-renewing resident cardiac macrophages limit adverse remodeling following myocardial infarction.

In the version of this article initially published, the equal contribution of the third author was omitted. The footnote links for that author should be "Sara Nejat1,11" and the correct statement is as follows: "11These authors contributed equally: Sarah A. Dick, Jillian A. Macklin, Sara Nejat." The error has been corrected in the HTML and PDF versions of the article.

Selective loss of resident macrophage-derived insulin-like growth factor-1 abolishes adaptive cardiac growth to stress.

Hypertension affects one-third of the world's population, leading to cardiac dysfunction that is modulated by resident and recruited immune cells. Cardiomyocyte growth and increased cardiac mass are essential to withstand hypertensive stress; however, whether immune cells are involved in this compensatory cardioprotective process is unclear. In normotensive animals, single-cell transcriptomics of fate-mapped self-renewing cardiac resident macrophages (RMs) revealed transcriptionally diverse cell states with a core repertoire of reparative gene programs, including high expression of insulin-like growth factor-1 (Igf1). Hypertension drove selective in situ proliferation and transcriptional activation of some cardiac RM states, directly correlating with increased cardiomyocyte growth. During hypertension, inducible ablation of RMs or selective deletion of RM-derived Igf1 prevented adaptive cardiomyocyte growth, and cardiac mass failed to increase, which led to cardiac dysfunction. Single-cell transcriptomics identified a conserved IGF1-expressing macrophage subpopulation in human cardiomyopathy. Here we defined the absolute requirement of RM-produced IGF-1 in cardiac adaptation to hypertension.

Resident cardiac macrophages mediate adaptive myocardial remodeling.

Cardiac macrophages represent a heterogeneous cell population with distinct origins, dynamics, and functions. Recent studies have revealed that C-C Chemokine Receptor 2 positive (CCR2+) macrophages derived from infiltrating monocytes regulate myocardial inflammation and heart failure pathogenesis. Comparatively little is known about the functions of tissue resident (CCR2-) macrophages. Herein, we identified an essential role for CCR2- macrophages in the chronically failing heart. Depletion of CCR2- macrophages in mice with dilated cardiomyopathy accelerated mortality and impaired ventricular remodeling and coronary angiogenesis, adaptive changes necessary to maintain cardiac output in the setting of reduced cardiac contractility. Mechanistically, CCR2- macrophages interacted with neighboring cardiomyocytes via focal adhesion complexes and were activated in response to mechanical stretch through a transient receptor potential vanilloid 4 (TRPV4)-dependent pathway that controlled growth factor expression. These findings establish a role for tissue-resident macrophages in adaptive cardiac remodeling and implicate mechanical sensing in cardiac macrophage activation.

Tissue-Resident Macrophages in Pancreatic Ductal Adenocarcinoma Originate from Embryonic Hematopoiesis and Promote Tumor Progression.

Tumor-associated macrophages (TAMs) are essential components of the cancer microenvironment and play critical roles in the regulation of tumor progression. Optimal therapeutic intervention requires in-depth understanding of the sources that sustain macrophages in malignant tissues. In this study, we investigated the ontogeny of TAMs in murine pancreatic ductal adenocarcinoma (PDAC) models. We identified both inflammatory monocytes and tissue-resident macrophages as sources of TAMs. Unexpectedly, significant portions of pancreas-resident macrophages originated from embryonic development and expanded through in situ proliferation during tumor progression. Whereas monocyte-derived TAMs played more potent roles in antigen presentation, embryonically derived TAMs exhibited a pro-fibrotic transcriptional profile, indicative of their role in producing and remodeling molecules in the extracellular matrix. Collectively, these findings uncover the heterogeneity of TAM origin and functions and could provide therapeutic insight for PDAC treatment.

A CD103+ Conventional Dendritic Cell Surveillance System Prevents Development of Overt Heart Failure during Subclinical Viral Myocarditis.

Innate and adaptive immune cells modulate heart failure pathogenesis during viral myocarditis, yet their identities and functions remain poorly defined. We utilized a combination of genetic fate mapping, parabiotic, transcriptional, and functional analyses and demonstrated that the heart contained two major conventional dendritic cell (cDC) subsets, CD103+ and CD11b+, which differentially relied on local proliferation and precursor recruitment to maintain their tissue residency. Following viral infection of the myocardium, cDCs accumulated in the heart coincident with monocyte infiltration and loss of resident reparative embryonic-derived cardiac macrophages. cDC depletion abrogated antigen-specific CD8+ T cell proliferative expansion, transforming subclinical cardiac injury to overt heart failure. These effects were mediated by CD103+ cDCs, which are dependent on the transcription factor BATF3 for their development. Collectively, our findings identified resident cardiac cDC subsets, defined their origins, and revealed an essential role for CD103+ cDCs in antigen-specific T cell responses during subclinical viral myocarditis.

Dissecting and Visualizing the Functional Diversity of Cardiac Macrophages.

Cardiac macrophages represent a functionally diverse population of cells involved in cardiac homeostasis, repair, and remodeling. With recent advancements in single-cell technologies, it is possible to elucidate specific macrophage subsets based on transcriptional signatures and cell surface protein expression to gain a deep understanding of macrophage diversity in the heart. The use of fate-mapping technologies and parabiosis studies have provided insight into the ontogeny and dynamics of macrophages identifying subsets derived from embryonic and adult definitive hematopoietic progenitors that include tissue-resident and bone marrow monocyte-derived macrophages, respectively. Within the heart, these subsets have distinct tissue niches and functional roles in the setting of homeostasis and disease, with cardiac resident macrophages representing a protective cell population while bone marrow monocyte-derived cardiac macrophages have a context-dependent effect, triggering both proinflammatory tissue injury, but also promoting reparative functions. With the increased understanding of the clinical relevance of cardiac macrophage subsets, there has been an increasing need to detect and measure cardiac macrophage compositions in living animals and patients. New molecular tracers compatible with positron emission tomography/computerized tomography and positron emission tomography/ magnetic resonance imaging have enabled investigators to noninvasively and serially visualize cardiac macrophage subsets within the heart to define associations with disease and measure treatment responses. Today, advancements within this thriving field are poised to fuel an era of clinical translation.

Resolvin D1 prevents injurious neutrophil swarming in transplanted lungs.

Neutrophils are the primary cell type involved in lung ischemia-reperfusion injury (IRI), which remains a frequent and morbid complication after organ transplantation. Endogenous lipid mediators that become activated during acute inflammation-resolution have gained increasing recognition for their protective role(s) in promoting the restoration of homeostasis, but their influence on early immune responses following transplantation remains to be uncovered. Resolvin D1, 7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid (RvD1), is a potent stereoselective mediator that exhibits proresolving and anti-inflammatory actions in the setting of tissue injury. Here, using metabololipidomics, we demonstrate that endogenous proresolving mediators including RvD1 are increased in human and murine lung grafts immediately following transplantation. In mouse grafts, we observe lipid mediator class switching early after reperfusion. We use intravital two-photon microscopy to reveal that RvD1 treatment significantly limits early neutrophil infiltration and swarming, thereby ameliorating early graft dysfunction in transplanted syngeneic lungs subjected to severe IRI. Through integrated analysis of single-cell RNA sequencing data of donor and recipient immune cells from lung grafts, we identify transcriptomic changes induced by RvD1. These results support a role for RvD1 as a potent modality for preventing early neutrophil-mediated tissue damage after lung IRI that may be therapeutic in the clinics.

Expansion of Pathogenic Cardiac Macrophages in Immune Checkpoint Inhibitor Myocarditis.

BACKGROUND: Immune checkpoint inhibitors (ICIs), antibodies targeting PD-1 (programmed cell death protein 1)/PD-L1 (programmed death-ligand 1) or CTLA4 (cytotoxic T-lymphocyte-associated protein 4), have revolutionized cancer management but are associated with devastating immune-related adverse events including myocarditis. The main risk factor for ICI myocarditis is the use of combination PD-1 and CTLA4 inhibition. ICI myocarditis is often fulminant and is pathologically characterized by myocardial infiltration of T lymphocytes and macrophages. Although much has been learned about the role of T-cells in ICI myocarditis, little is understood about the identity, transcriptional diversity, and functions of infiltrating macrophages. METHODS: We used an established murine ICI myocarditis model (Ctla4+/-Pdcd1-/- mice) to explore the cardiac immune landscape using single-cell RNA-sequencing, immunostaining, flow cytometry, in situ RNA hybridization, molecular imaging, and antibody neutralization studies. RESULTS: We observed marked increases in CCR2 (C-C chemokine receptor type 2)+ monocyte-derived macrophages and CD8+ T-cells in this model. The macrophage compartment was heterogeneous and displayed marked enrichment in an inflammatory CCR2+ subpopulation highly expressing Cxcl9 (chemokine [C-X-C motif] ligand 9), Cxcl10 (chemokine [C-X-C motif] ligand 10), Gbp2b (interferon-induced guanylate-binding protein 2b), and Fcgr4 (Fc receptor, IgG, low affinity IV) that originated from CCR2+ monocytes. It is important that a similar macrophage population expressing CXCL9, CXCL10, and CD16α (human homologue of mouse FcgR4) was expanded in patients with ICI myocarditis. In silico prediction of cell-cell communication suggested interactions between T-cells and Cxcl9+Cxcl10+ macrophages via IFN-γ (interferon gamma) and CXCR3 (CXC chemokine receptor 3) signaling pathways. Depleting CD8+ T-cells or macrophages and blockade of IFN-γ signaling blunted the expansion of Cxcl9+Cxcl10+ macrophages in the heart and attenuated myocarditis, suggesting that this interaction was necessary for disease pathogenesis. CONCLUSIONS: These data demonstrate that ICI myocarditis is associated with the expansion of a specific population of IFN-γ-induced inflammatory macrophages and suggest the possibility that IFN-γ blockade may be considered as a treatment option for this devastating condition.

Derivation of extra-embryonic and intra-embryonic macrophage lineages from human pluripotent stem cells.

Tissue-resident macrophages are increasingly recognized as important determinants of organ homeostasis, tissue repair, remodeling and regeneration. Although the ontogeny and function of tissue-resident macrophages has been identified as distinct from postnatal hematopoiesis, the inability to specify, in vitro, similar populations that recapitulate these developmental waves has limited our ability to study their function and potential for regenerative applications. We took advantage of the concept that tissue-resident macrophages and monocyte-derived macrophages originate from distinct extra-embryonic and definitive hematopoietic lineages to devise a system to generate pure cultures of macrophages that resemble tissue-resident or monocyte-derived subsets. We demonstrate that human pluripotent stem cell-derived extra-embryonic-like and intra-embryonic-like hematopoietic progenitors differentiate into morphologically, transcriptionally and functionally distinct macrophage populations. Single-cell RNA sequencing of developing and mature cultures uncovered distinct developmental trajectories and gene expression programs of macrophages derived from extra-embryonic-like and intra-embryonic-like hematopoietic progenitors. These findings establish a resource for the generation of human tissue resident-like macrophages to study their specification and function under defined conditions and to explore their potential use in tissue engineering and regenerative medicine applications.

Necroptosis triggers spatially restricted neutrophil-mediated vascular damage during lung ischemia reperfusion injury.

Ischemia reperfusion injury represents a common pathological condition that is triggered by the release of endogenous ligands. While neutrophils are known to play a critical role in its pathogenesis, the tissue-specific spatiotemporal regulation of ischemia-reperfusion injury is not understood. Here, using oxidative lipidomics and intravital imaging of transplanted mouse lungs that are subjected to severe ischemia reperfusion injury, we discovered that necroptosis, a nonapoptotic form of cell death, triggers the recruitment of neutrophils. During the initial stages of inflammation, neutrophils traffic predominantly to subpleural vessels, where their aggregation is directed by chemoattractants produced by nonclassical monocytes that are spatially restricted in this vascular compartment. Subsequent neutrophilic disruption of capillaries resulting in vascular leakage is associated with impaired graft function. We found that TLR4 signaling in vascular endothelial cells and downstream NADPH oxidase 4 expression mediate the arrest of neutrophils, a step upstream of their extravasation. Neutrophil extracellular traps formed in injured lungs and their disruption with DNase prevented vascular leakage and ameliorated primary graft dysfunction. Thus, we have uncovered mechanisms that regulate the initial recruitment of neutrophils to injured lungs, which result in selective damage to subpleural pulmonary vessels and primary graft dysfunction. Our findings could lead to the development of new therapeutics that protect lungs from ischemia reperfusion injury.

Persistent transcriptional changes in cardiac adaptive immune cells following myocardial infarction: New evidence from the re-analysis of publicly available single cell and nuclei RNA-sequencing data sets.

INTRODUCTION: Chronic immunopathology contributes to the development of heart failure after a myocardial infarction. Both T and B cells of the adaptive immune system are present in the myocardium and have been suggested to be involved in post-MI immunopathology. METHODS: We analyzed the B and T cell populations isolated from previously published single cell RNA-sequencing data sets (PMID: 32130914, PMID: 35948637, PMID: 32971526 and PMID: 35926050), of the mouse and human heart, using differential expression analysis, functional enrichment analysis, gene regulatory inferences, and integration with autoimmune and cardiovascular GWAS. RESULTS: Already at baseline, mature effector B and T cells are present in the human and mouse heart, having increased activity in transcription factors maintaining tolerance (e.g. DEAF1, JDP2, SPI-B). Following MI, T cells upregulate pro-inflammatory transcript levels (e.g. Cd11, Gzmk, Prf1), while B cells upregulate activation markers (e.g. Il6, Il1rn, Ccl6) and collagen (e.g. Col5a2, Col4a1, Col1a2). Importantly, pro-inflammatory and fibrotic transcription factors (e.g. NFKB1, CREM, REL) remain active in T cells, while B cells maintain elevated activity in transcription factors related to immunoglobulin production (e.g. ERG, REL) in both mouse and human post-MI hearts. Notably, genes differentially expressed in post-MI T and B cells are associated with cardiovascular and autoimmune disease. CONCLUSION: These findings highlight the varied and time-dependent dynamic roles of post-MI T and B cells. They appear ready-to-go and are activated immediately after MI, thus participate in the acute wound healing response. However, they subsequently remain in a state of pro-inflammatory activation contributing to persistent immunopathology.

Smoking exposure-induced bronchus-associated lymphoid tissue in donor lungs does not prevent tolerance induction after transplantation.

The presence of bronchus-associated lymphoid tissue (BALT) in donor lungs has been suggested to accelerate graft rejection after lung transplantation. Although chronic smoke exposure can induce BALT formation, the impact of donor cigarette use on alloimmune responses after lung transplantation is not well understood. Here, we show that smoking-induced BALT in mouse donor lungs contains Foxp3+ T cells and undergoes dynamic restructuring after transplantation, including recruitment of recipient-derived leukocytes to areas of pre-existing lymphoid follicles and replacement of graft-resident donor cells. Our findings from mouse and human lung transplant data support the notion that a donor's smoking history does not predispose to acute cellular rejection or prevent the establishment of allograft acceptance with comparable outcomes to nonsmoking donors. Thus, our work indicates that BALT in donor lungs is plastic in nature and may have important implications for modulating proinflammatory or tolerogenic immune responses following transplantation.

Transcriptional and Immune Landscape of Cardiac Sarcoidosis.

BACKGROUND: Cardiac involvement is an important determinant of mortality among sarcoidosis patients. Although granulomatous inflammation is a hallmark finding in cardiac sarcoidosis, the precise immune cell populations that comprise the granuloma remain unresolved. Furthermore, it is unclear how the cellular and transcriptomic landscape of cardiac sarcoidosis differs from other inflammatory heart diseases. METHODS: We leveraged spatial transcriptomics (GeoMx digital spatial profiler) and single-nucleus RNA sequencing to elucidate the cellular and transcriptional landscape of cardiac sarcoidosis. Using GeoMX digital spatial profiler technology, we compared the transcriptomal profile of CD68+ rich immune cell infiltrates in human cardiac sarcoidosis, giant cell myocarditis, and lymphocytic myocarditis. We performed single-nucleus RNA sequencing of human cardiac sarcoidosis to identify immune cell types and examined their transcriptomic landscape and regulation. Using multichannel immunofluorescence staining, we validated immune cell populations identified by single-nucleus RNA sequencing, determined their spatial relationship, and devised an immunostaining approach to distinguish cardiac sarcoidosis from other inflammatory heart diseases. RESULTS: Despite overlapping histological features, spatial transcriptomics identified transcriptional signatures and associated pathways that robustly differentiated cardiac sarcoidosis from giant cell myocarditis and lymphocytic myocarditis. Single-nucleus RNA sequencing revealed the presence of diverse populations of myeloid cells in cardiac sarcoidosis with distinct molecular features. We identified GPNMB (transmembrane glycoprotein NMB) as a novel marker of multinucleated giant cells and predicted that the MITF (microphthalmia-associated transcription factor) family of transcription factors regulated this cell type. We also detected additional macrophage populations in cardiac sarcoidosis including HLA-DR (human leukocyte antigen-DR)+ macrophages, SYTL3 (synaptotagmin-like protein 3)+ macrophages and CD163+ resident macrophages. HLA-DR+ macrophages were found immediately adjacent to GPMMB+ giant cells, a distinct feature compared with other inflammatory cardiac diseases. SYTL3+ macrophages were located scattered throughout the granuloma and CD163+ macrophages, CD1c+ dendritic cells, nonclassical monocytes, and T cells were located at the periphery and outside of the granuloma. Finally, we demonstrate mTOR (mammalian target of rapamycin) pathway activation is associated with proliferation and is selectively found in HLA-DR+ and SYLT3+ macrophages. CONCLUSIONS: In this study, we identified diverse populations of immune cells with distinct molecular signatures that comprise the sarcoid granuloma. These findings provide new insights into the pathology of cardiac sarcoidosis and highlight opportunities to improve diagnostic testing.

New Approaches to Target Inflammation in Heart Failure: Harnessing Insights from Studies of Immune Cell Diversity.

Despite mounting evidence implicating inflammation in cardiovascular diseases, attempts at clinical translation have shown mixed results. Recent preclinical studies have reenergized this field and provided new insights into how to favorably modulate cardiac macrophage function in the context of acute myocardial injury and chronic disease. In this review, we discuss the origins and roles of cardiac macrophage populations in the steady-state and diseased heart, focusing on the human heart and mouse models of ischemia, hypertensive heart disease, and aortic stenosis. Specific attention is given to delineating the roles of tissue-resident and recruited monocyte-derived macrophage subsets. We also highlight emerging concepts of monocyte plasticity and heterogeneity among monocyte-derived macrophages, describe possible mechanisms by which infiltrating monocytes acquire unique macrophage fates, and discuss the putative impact of these populations on cardiac remodeling. Finally, we discuss strategies to target inflammatory macrophage populations.

CCL17 Aggravates Myocardial Injury by Suppressing Recruitment of Regulatory T Cells.

BACKGROUND: Recent studies have established that CCR2 (C-C chemokine receptor type 2) marks proinflammatory subsets of monocytes, macrophages, and dendritic cells that contribute to adverse left ventricle (LV) remodeling and heart failure progression. Elucidation of the effector mechanisms that mediate adverse effects of CCR2+ monocytes, macrophages, and dendritic cells will yield important insights into therapeutic strategies to suppress myocardial inflammation. METHODS: We used mouse models of reperfused myocardial infarction, angiotensin II and phenylephrine infusion, and diphtheria toxin cardiomyocyte ablation to investigate CCL17 (C-C chemokine ligand 17). We used Ccl17 knockout mice, flow cytometry, RNA sequencing, biochemical assays, cell trafficking studies, and in vivo cell depletion to identify the cell types that generate CCL17, define signaling pathways that controlled its expression, delineate the functional importance of CCL17 in adverse LV remodeling and heart failure progression, and determine the mechanistic basis by which CCL17 exerts its effects. RESULTS: We demonstrated that CCL17 is expressed in CCR2+ macrophages and cluster of differentiation 11b+ conventional dendritic cells after myocardial infarction, angiotensin II and phenylephrine infusion, and diphtheria toxin cardiomyocyte ablation. We clarified the transcriptional signature of CCL17+ macrophages and dendritic cells and identified granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling as a key regulator of CCL17 expression through cooperative activation of STAT5 (signal transducer and activator of transcription 5) and canonical NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells) signaling. Ccl17 deletion resulted in reduced LV remodeling, decreased myocardial fibrosis and cardiomyocyte hypertrophy, and improved LV systolic function after myocardial infarction and angiotensin II and phenylephrine infusion. We observed increased abundance of regulatory T cells (Tregs) in the myocardium of injured Ccl17 knockout mice. CCL17 inhibited Treg recruitment through biased activation of CCR4. CCL17 activated Gq signaling and CCL22 (C-C chemokine ligand 22) activated both Gq and ARRB (ß-arrestin) signaling downstream of CCR4. CCL17 competitively inhibited CCL22 stimulated ARRB signaling and Treg migration. We provide evidence that Tregs mediated the protective effects of Ccl17 deletion on myocardial inflammation and adverse LV remodeling. CONCLUSIONS: These findings identify CCL17 as a proinflammatory mediator of CCR2+ macrophages and dendritic cells and suggest that inhibition of CCL17 may serve as an effective strategy to promote Treg recruitment and suppress myocardial inflammation.

Donor Macrophages Modulate Rejection After Heart Transplantation.

BACKGROUND: Cellular rejection after heart transplantation imparts significant morbidity and mortality. Current immunosuppressive strategies are imperfect, target recipient T cells, and have adverse effects. The innate immune response plays an essential role in the recruitment and activation of T cells. Targeting the donor innate immune response would represent the earliest interventional opportunity within the immune response cascade. There is limited knowledge about donor immune cell types and functions in the setting of cardiac transplantation, and no current therapeutics exist for targeting these cell populations. METHODS: Using genetic lineage tracing, cell ablation, and conditional gene deletion, we examined donor mononuclear phagocyte diversity and macrophage function during acute cellular rejection of transplanted hearts in mice. We performed single-cell RNA sequencing on donor and recipient macrophages and monocytes at multiple time points after transplantation. On the basis of our imaging and single-cell RNA sequencing data, we evaluated the functional relevance of donor CCR2+ (C-C chemokine receptor 2) and CCR2- macrophages using selective cell ablation strategies in donor grafts before transplant. Last, we performed functional validation that donor macrophages signal through MYD88 (myeloid differentiation primary response protein 88) to facilitate cellular rejection. RESULTS: Donor macrophages persisted in the rejecting transplanted heart and coexisted with recipient monocyte-derived macrophages. Single-cell RNA sequencing identified donor CCR2+ and CCR2- macrophage populations and revealed remarkable diversity among recipient monocytes, macrophages, and dendritic cells. Temporal analysis demonstrated that donor CCR2+ and CCR2- macrophages were transcriptionally distinct, underwent significant morphologic changes, and displayed unique activation signatures after transplantation. Although selective depletion of donor CCR2- macrophages reduced allograft survival, depletion of donor CCR2+ macrophages prolonged allograft survival. Pathway analysis revealed that donor CCR2+ macrophages are activated through MYD88/nuclear factor kappa light chain enhancer of activated B cells signaling. Deletion of MYD88 in donor macrophages resulted in reduced antigen-presenting cell recruitment, reduced ability of antigen-presenting cells to present antigen to T cells, decreased emergence of allograft-reactive T cells, and extended allograft survival. CONCLUSIONS: Distinct populations of donor and recipient macrophages coexist within the transplanted heart. Donor CCR2+ macrophages are key mediators of allograft rejection, and deletion of MYD88 signaling in donor macrophages is sufficient to suppress rejection and extend allograft survival. This highlights the therapeutic potential of donor heart-based interventions.

Ferroptosis in the post-transplantation inflammatory response.

Transplantation is a life-saving therapy for patients with end-stage organ disease. Successful outcomes after transplantation require mitigation of the post-transplant inflammatory response, limiting alloreactivity, and prevention of organ rejection. Traditional immunosuppressive regimens aim to dampen the adaptive immune response; however, recent studies have shown the feasibility and efficacy of targeting the innate immune response. Necroinflammation initiated by donor organ cell death is implicated as a critical mediator of primary graft dysfunction, acute rejection, and chronic rejection. Ferroptosis is a form of regulated cell death that triggers post-transplantation inflammation and drives the activation of both innate and adaptive immune cells. There is a growing acceptance of the clinical relevance of ferroptosis to solid organ transplantation. Modulating ferroptosis may be a potentially promising strategy to reduce complications after organ transplantation.