Rationale: Monocyte infiltration into the subintimal space and their intracellular lipid accumulation are the most prominent features of atherosclerosis. To understand the pathophysiology of atherosclerotic disease, we need to understand the characteristics of lipid-laden foamy macrophages in the subintimal space during atherosclerosis. Objective: We sought to examine the transcriptomic profiles of foamy and non-foamy macrophages isolated from atherosclerotic intima. Methods and Results: Single-cell RNA-sequencing analysis of CD45+ leukocytes from murine atherosclerotic aorta revealed that there are macrophage subpopulations with distinct differentially expressed genes involved in various functional pathways. To specifically characterize the intimal foamy macrophages of plaque, we developed a lipid staining-based flow cytometric method for analyzing the lipid-laden foam cells of atherosclerotic aortas. We employed the fluorescent lipid probe BODIPY493/503 and assessed side-scattered light (SSC) as an indication of cellular granularity. BODIPYhiSSChi foamy macrophages were found residing in intima and expressing CD11c. Foamy macrophage accumulation determined by flow cytometry was positively correlated with the severity of atherosclerosis. Bulk RNA-seq analysis showed that compared with non-foamy macrophages, foamy macrophages expressed few inflammatory genes but many lipid-processing genes. Intimal non-foamy macrophages formed the major population expressing interleukin-1β and many other inflammatory transcripts in atherosclerotic aorta. Conclusions: RNA-seq analysis of intimal macrophages from atherosclerotic aorta revealed that lipid-loaded plaque macrophages are not likely the plaque macrophages that drive lesional inflammation.

Atopic asthma is an inflammatory pulmonary disease associated with Th2 adaptive immune responses triggered by innocuous antigens. While dendritic cells (DCs) are known to shape the adaptive immune response, the mechanisms by which DCs promote Th2 differentiation remain elusive. Herein we demonstrate that Th2-promoting stimuli induce DC expression of IRF4. Mice with conditional deletion of Irf4 in DCs show a dramatic defect in Th2-type lung inflammation, yet retain the ability to elicit pulmonary Th1 anti-viral responses. Using loss- and gain-of-function analysis, we demonstrate that Th2 differentiation is dependent on IRF4 expression in DCs. Finally, IRF4 directly targets and activates the Il10 and Il33 genes in DCs. Reconstitution with exogenous IL-10 and IL-33 recovers the ability of Irf4 deficient DCs to promote Th2 differentiation. These findings reveal a regulatory module in DCs by which IRF4 modulates IL-10 and IL-33 cytokine production to specifically promote Th2 differentiation and inflammation.

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 ...

The diverse leukocyte infiltrate in atherosclerotic mouse aortas was recently analyzed in 9 single cell RNA-Seq (scRNA-Seq) and 2 mass cytometry (CyTOF) studies. In a comprehensive meta-analysis, we demonstrate four macrophage subsets: resident, inflammatory, IFNIC and Trem2 foamy macrophages. We also find that monocytes, neutrophils, dendritic cells, natural killer cells, innate lymphoid cells-2 (ILC2) and CD8 T cells form prominent and separate populations. The CD4 T cells show a large population of Th17-like cells, which also contain γδ T cells. A small number of Tregs and Th1 cells is also identified. The present meta-analysis overcomes limitations of individual studies that, because of their experimental approach, overor under-represent certain cell populations. CyTOF identifies an even larger number of clusters, suggesting that surface markers provide more discriminatory information than transcriptomes. The present analysis provides evidence to further resolve some long-standing controversies in the field. First, Trem2 + foamy macrophages are not pro-inflammatory, but interferon-inducible cell (IFNIC) and inflammatory macrophages are. Second, about half of all foam cells are smooth muscle cell-derived, retaining smooth muscle cell transcripts rather than transdifferentiating to macrophages. Third, Pf4, which had been considered specific for platelets and megakaryocytes, is also prominently expressed in resident vascular macrophages. Finally, the discovery of a prominent ILC2 cluster links the scRNA-Seq work to recent flow cytometry data suggesting a strong atheroprotective role of ILC2 cells. This resolves apparent discrepancies regarding the role of Th2 cells in atherosclerosis based on studies that pre-dated the discovery of ILC2 cells.

Gata6 regulates differentiation, metabolism and survival of peritoneal macrophages.

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 (DC) 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 aorta intima resident macrophages (Mac AIR ) that depend upon Csf-1 and sustained by local proliferation. Although Mac AIR comprise the earliest foam cells in plaques, their proliferation during plaque progression is limited. After months of hypercholesterolemia, their presence in plaque is overtaken by recruited monocytes, which induce Mac AIR -defining genes. These data redefine the lineage of intimal phagocytes and suggest that proliferation is insufficient to sustain generations of macrophages during plaque progression.

Randolph and colleagues describe the ontogenic origin and developmental program of a distinct resident peritoneal macrophage population.

No abstract