During the inflammatory response that drives atherogenesis, macrophages accumulate progressively in the expanding arterial wall1,2. The observation that circulating monocytes give rise to lesional macrophages3–9 has reinforced the concept that monocyte infiltration dictates macrophage build-up. Recent work indicates, however, that macrophages do not depend on monocytes in some inflammatory contexts10. We therefore revisited the mechanism of macrophage accumulation in atherosclerosis. We show that murine atherosclerotic lesions experience a surprisingly rapid, 4-week, cell turnover. Replenishment of macrophages in these experimental atheromata depends predominantly on local macrophage proliferation rather than monocyte influx. The microenvironment orchestrates macrophage proliferation via the involvement of scavenger receptor (SR)-A. Our study reveals macrophage proliferation as a key event in atherosclerosis and identifies macrophage self-renewal as a therapeutic target for cardiovascular disease.

Rationale Healing after myocardial infarction (MI) involves the biphasic accumulation of inflammatory Ly-6Chigh and reparative Ly-6Clow monocytes/macrophages (Mo/MΦ). According to one model, Mo/MΦ heterogeneity in the heart originates in the blood and involves the sequential recruitment of distinct monocyte subsets that differentiate to distinct macrophages. Alternatively, heterogeneity may arise in tissue from one circulating subset via local macrophage differentiation and polarization. The orphan nuclear hormone receptor, Nr4a1, is essential to Ly-6Clow monocyte production but dispensable to Ly-6Clow macrophage differentiation; dependence on Nr4a1 can thus discriminate between systemic and local origins of macrophage heterogeneity. Objective This study tested the role of Nr4a1 in MI in the context of the two Mo/MΦ accumulation scenarios. Methods and Results We show that Ly-6Chigh monocytes infiltrate the infarcted myocardium and, unlike Ly-6Clow monocytes, differentiate to cardiac macrophages. In the early, inflammatory phase of acute myocardial ischemic injury, Ly-6Chigh monocytes accrue in response to a brief Ccl2 burst. In the second, reparative phase, accumulated Ly-6Chigh monocytes give rise to reparative Ly-6Clow F4/80high macrophages that proliferate locally. In the absence of Nr4a1, Ly-6Chigh monocytes express heightened levels of Ccr2 on their surface, avidly infiltrate the myocardium, and differentiate to abnormally inflammatory macrophages, which results in defective healing and compromised heart function. Conclusions Ly-6Chigh monocytes orchestrate both inflammatory and reparative phases during MI and depend on Nr4a1 to limit their influx and inflammatory cytokine expression.

The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunologic mechanisms and cell-type-specific pathways, and establishes a functional relevance for lesional leukocytes in human atherosclerosis.

Iron is an essential component of the erythrocyte protein hemoglobin and is crucial to oxygen transport in vertebrates. In the steady state, erythrocyte production is in equilibrium with erythrocyte removal1. In various pathophysiological conditions, however, erythrocyte life span is severely compromised, which threatens the organism with anemia and iron toxicity2,3. Here we identify an on-demand mechanism that clears erythrocytes and recycles iron. We show that Ly-6Chigh monocytes ingest stressed and senescent erythrocytes, accumulate in the liver via coordinated chemotactic cues, and differentiate to ferroportin 1 (FPN1)-expressing macrophages that can deliver iron to hepatocytes. Monocyte-derived FPN1+ Tim-4neg macrophages are transient, reside alongside embryonically-derived Tim-4high Kupffer cells, and depend on Csf1 and Nrf2. The spleen likewise recruits iron-loaded Ly-6Chigh monocytes, but these do not differentiate into iron-recycling macrophages due to the suppressive action of Csf2. Inhibiting monocyte recruitment to the liver leads to kidney and liver damage. These observations identify the liver as the primary organ supporting rapid erythrocyte removal and iron recycling and uncover a mechanism by which the body adapts to fluctuations in erythrocyte integrity.

Recognition and clearance of bacterial infection is a fundamental property of innate immunity. Here we describe an effector B cell population that protects against microbial sepsis. Innate response activator (IRA)-B cells are phenotypically and functionally distinct, develop and diverge from B1a B cells, depend on pattern recognition receptors, and produce GM-CSF. Specific deletion of IRA-B cell activity impairs bacterial clearance, elicits a cytokine storm, and precipitates septic shock. These observations enrich our understanding of innate immunity, position IRA-B cells as gatekeepers of bacterial infection, and identify new treatment avenues for infectious diseases.

Most tissues of the body harbor resident macrophages. Yet, macrophages are phenotypically and functionally heterogeneous, a reflection of the diversity of tissue environments in which they reside. In addition to maintaining tissue homeostasis and responding to invading pathogens, macrophages contribute to numerous pathological processes, making them an attractive potential target for therapeutic intervention. To do so, however, will require a detailed understanding of macrophage origins, the mechanisms that maintain them, and their functional attributes in different tissues and disease contexts.Macrophage ontology has long engendered controversy 1,2 . Nevertheless, the concept that tissue macrophages develop exclusively from circulating bone marrow-derived monocytes has prevailed for nearly a half century 3 . Accumulated evidence, however, including recent studies using sophisticated fate-mapping approaches, have determined that some tissue macrophages and their precursors are established embryonically in the yolk sac (YS) and fetal liver before the onset of definitive hematopoiesis [4][5][6][7][8][9][10][11] . Regardless of their origin, tissue macrophages can maintain themselves in adulthood by self-renewal independent of blood monocytes 12,13 .Gene-expression profiling of macrophage populations from several tissues has established that only a small number of transcripts are expressed by all macrophages 14 , indicating the importance of the context provided by the tissue when studying macrophage function in homeostasis and disease. The normal arterial wall contains many tissue resident macrophages that contribute crucially to immunity, tissue homeostasis and wound healing following injury 15. However, the regulatory networks, ancestry and mechanisms that maintain arterial macrophages remain unknown.Using gene expression analysis, we show that arterial macrophages constitute a distinct population among tissue macrophages. Multiple fate mapping approaches demonstrated that arterial macrophages arise embryonically from CX 3 CR1 + precursors and postnatally from bone marrow-derived monocytes that colonize the tissue during a brief period immediately after birth.In adulthood, arterial macrophages were maintained by CX 3 CR1-CX 3 CL1 interactions and local proliferation without significant further contribution from blood monocytes. Self-renewal also sustained arterial macrophages after severe depletion during polymicrobial sepsis, rapidly restoring them to functional homeostasis. ResultsPhenotype and gene expression profiling of arterial macrophages. (Fig. 1a).Principal component analysis revealed a distinct transcriptome in arterial macrophages, which clustered near other macrophage populations including microglia, alveolar macrophages, and splenic red pulp macrophages, as characterized by the Immunological Genome Consortium (Fig. 1b, Supplementary Fig. 1a) 14. Stringent comparison of gene-expression profiles among arterial, brain, alveolar and splenic red pulp macrophages revealed 212 transcripts that were at ...

Background: Throughout the inflammatory response that accompanies atherosclerosis, autoreactive CD4 + T-helper cells accumulate in the atherosclerotic plaque. Apolipoprotein B 100 (apoB), the core protein of low-density lipoprotein, is an autoantigen that drives the generation of pathogenic T-helper type 1 (T H 1) cells with proinflammatory cytokine secretion. Clinical data suggest the existence of apoB-specific CD4 + T cells with an atheroprotective, regulatory T cell (T reg ) phenotype in healthy individuals. Yet, the function of apoB-reactive T regs and their relationship with pathogenic T H 1 cells remain unknown. Methods: To interrogate the function of autoreactive CD4 + T cells in atherosclerosis, we used a novel tetramer of major histocompatibility complex II to track T cells reactive to the mouse self-peptide apo B 978-993 (apoB + ) at the single-cell level. Results: We found that apoB + T cells build an oligoclonal population in lymph nodes of healthy mice that exhibit a T reg -like transcriptome, although only 21% of all apoB + T cells expressed the T reg transcription factor FoxP3 (Forkhead Box P3) protein as detected by flow cytometry. In single-cell RNA sequencing, apoB + T cells formed several clusters with mixed T H signatures that suggested overlapping multilineage phenotypes with pro- and anti-inflammatory transcripts of T H 1, T helper cell type 2 (T H 2), and T helper cell type 17 (T H 17), and of follicular-helper T cells. ApoB + T cells were increased in mice and humans with atherosclerosis and progressively converted into pathogenic T H 1/T H 17-like cells with proinflammatory properties and only a residual T reg transcriptome. Plaque T cells that expanded during progression of atherosclerosis consistently showed a mixed T H 1/T H 17 phenotype in single-cell RNA sequencing. In addition, we observed a loss of FoxP3 in a fraction of apoB + T regs in lineage tracing of hyperlipidemic Apoe –/– mice. In adoptive transfer experiments, converting apoB + T regs failed to protect from atherosclerosis. Conclusions: Our results demonstrate an unexpected mixed phenotype of apoB-reactive autoimmune T cells in atherosclerosis and suggest an initially protective autoimmune response against apoB with a progressive derangement in clinical disease. These findings identify apoB autoreactive T regs as a novel cellular target in atherosclerosis.

In response to lung infection, pleural innate response activator B cells produce GM-CSF–dependent IgM and ensure a frontline defense against bacterial invasion.