Three tissue resident macrophage subsets coexist across organs with conserved origins and life cycles.

Resident macrophages orchestrate homeostatic, inflammatory, and reparative activities. It is appreciated that different tissues instruct specialized macrophage functions. However, individual tissues contain heterogeneous subpopulations, and how these subpopulations are related is unclear. We asked whether common transcriptional and functional elements could reveal an underlying framework across tissues. Using single-cell RNA sequencing and random forest modeling, we observed that four genes could predict three macrophage subsets that were present in murine heart, liver, lung, kidney, and brain. Parabiotic and genetic fate mapping studies revealed that these core markers predicted three unique life cycles across 17 tissues. TLF+ (expressing TIMD4 and/or LYVE1 and/or FOLR2) macrophages were maintained through self-renewal with minimal monocyte input; CCR2+ (TIMD4−LYVE1−FOLR2−) macrophages were almost entirely replaced by monocytes, and MHC-IIhi macrophages (TIMD4−LYVE1−FOLR2−CCR2−), while receiving modest monocyte contribution, were not continually replaced. Rather, monocyte-derived macrophages contributed to the resident macrophage population until they reached a defined upper limit after which they did not outcompete pre-existing resident macrophages. Developmentally, TLF+ macrophages were first to emerge in the yolk sac and early fetal organs. Fate mapping studies in the mouse and human single-cell RNA sequencing indicated that TLF+ macrophages originated from both yolk sac and fetal monocyte precursors. Furthermore, TLF+ macrophages were the most transcriptionally conserved subset across mouse tissues and between mice and humans, despite organ- and species-specific transcriptional differences. Here, we define the existence of three murine macrophage subpopulations based on common life cycle properties and core gene signatures and provide a common starting point to understand tissue macrophage heterogeneity.

Dynamic CD4+ T cell heterogeneity defines subset-specific suppression and PD-L1-blockade-driven functional restoration in chronic infection.

Inhibiting PD-1:PD-L1 signaling has transformed therapeutic immune restoration. CD4+ T cells sustain immunity in chronic infections and cancer, yet little is known about how PD-1 signaling modulates CD4+ helper T (TH) cell responses or the ability to restore CD4+ TH-mediated immunity by checkpoint blockade. We demonstrate that PD-1:PD-L1 specifically suppressed CD4+ TH1 cell amplification, prevents CD4+ TH1 cytokine production and abolishes CD4+ cytotoxic killing capacity during chronic infection in mice. Inhibiting PD-L1 rapidly restored these functions, while simultaneously amplifying and activating TH1-like T regulatory cells, demonstrating a system-wide CD4-TH1 recalibration. This effect coincided with decreased T cell antigen receptor signaling, and re-directed type I interferon (IFN) signaling networks towards dominant IFN-γ-mediated responses. Mechanistically, PD-L1 blockade specifically targeted defined populations with pre-established, but actively suppressed proliferative potential, with limited impact on minimally cycling TCF-1+ follicular helper T cells, despite high PD-1 expression. Thus, CD4+ T cells require unique differentiation and functional states to be targets of PD-L1-directed suppression and therapeutic restoration.

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.

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.

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.

Fzd4 Haploinsufficiency Delays Retinal Revascularization in the Mouse Model of Oxygen Induced Retinopathy.

Mutations in genes that code for components of the Norrin-FZD4 ligand-receptor complex cause the inherited childhood blinding disorder familial exudative vitreoretinopathy (FEVR). Statistical evidence from studies of patients at risk for the acquired disease retinopathy of prematurity (ROP) suggest that rare polymorphisms in these same genes increase the risk of developing severe ROP, implying that decreased Norrin-FZD4 activity predisposes patients to more severe ROP. To test this hypothesis, we measured the development and recovery of retinopathy in wild type and Fzd4 heterozygous mice in the absence or presence of ocular ischemic retinopathy (OIR) treatment. Avascular and total retinal vascular areas and patterning were determined, and vessel number and caliber were quantified. In room air, there was a small delay in retinal vascularization in Fzd4 heterozygous mice that resolved as mice reached maturity suggestive of a slight defect in retinal vascular development. Subsequent to OIR treatment there was no difference between wild type and Fzd4 heterozygous mice in the vaso-obliterated area following exposure to high oxygen. Importantly, after return of Fzd4 heterozygous mice to room air subsequent to OIR treatment, there was a substantial delay in retinal revascularization of the avascular area surrounding the optic nerve, as well as delayed vascularization toward the periphery of the retina. Our study demonstrates that a small decrease in Norrin-Fzd4 dependent retinal vascular development lengthens the period during which complications from OIR could occur.

IFN-gamma and Fas/FasL pathways cooperate to induce medial cell loss and neointimal lesion formation in allograft vasculopathy.

Using a clinically relevant, fully disparate, allogeneic aortic transplant mouse model of allograft vasculopathy, we have demonstrated that neointimal proliferation is dependent on CD8(+) T cell effector pathways in the presence of therapeutic doses of calcineurin inhibitor (CNI) immunosuppression. CD4(+) T cell pathways are ablated by CNI immunosuppression. In the current study, we examined the relationship between CD8(+) T cell activities, medial SMC loss and neointimal hyperplasia. We demonstrate that at 5-6wk post transplantation in a wild type/wild type transplant CD8(+) T cell infiltration, CD8(+) CTL effector cell mediator expression and medial SMC loss all occur within aortic interposition grafts in the face of CNI immunosuppression. Both IFN-gamma and CTL mediated effector function is required for SMC loss and lesion formation under these conditions. Using strain combinations and reconstitution models, we provide data that blockade of the perforin/granzyme pathway does not prevent lesion formation but that blockade of the Fas/FasL pathway of cytotoxicity dramatically reduces SMC loss and prevents neointimal lesion formation. Both of these blockade strategies are in the face of an active IFN-gamma pathway. These data suggest a cooperative role between Fas/FasL and IFN-gamma mediated effector functions in medial SMC loss and neointimal lesion formation.

CD8(+) T cells mediate aortic allograft vasculopathy under conditions of calcineurin immunosuppression: role of IFN-gamma and CTL mediators.

We have developed a model of aortic allograft vasculopathy (AV) that uses mouse strains that are fully disparate at Class I, Class II and minor histocompatibility antigens. Acute rejection is ablated with therapeutic doses of the calcineurin inhibitor Cyclosporine A (CyA). In this way we successfully mimic human disease. Using this model we have demonstrated, with cell transfer models using highly purified T cell populations, that calcineurin inhibitors ablate CD4(+) T cell effector mechanisms. As such, in the presence of calcineurin inhibition, graft vasculopathy is dependent on CD8(+) T cell effector mechanisms. In this study we examine the etiology of graft vasculopathy by these CD8(+) T cells in the presence of calcineurin inhibition. We transferred CD8(+) T cells from CyA treated IFN-gamma deficient mice into immunodeficient mouse recipients of aortic allografts to demonstrate that IFN-gamma production by CD8(+) T cells is essential for the development of AV in the presence of calcineurin inhibition. Using two models of CTL ablation we also demonstrated that CTL activity by CD8(+) T cells is essential for the development of AV in the presence of calcineurin inhibition. This is in contrast to models without calcineurin inhibitor immunosuppression where either pathway is capable, by itself, of inducing AV. These data indicate that although calcineurin inhibition ablates CD4(+) T cell effects and weakens CD8(+) T cell pathways, the antigenic challenge of the graft is enough to induce sufficient responsiveness from CD8(+) T cells to induce robust AV.

Immunologic targets in the etiology of allograft vasculopathy: endothelium versus media.

The respective roles of the endothelium and the media as allo-immune targets in the generation of allograft vasculopathy (AV) have yet to be clearly defined. Although endothelial damage has been implicated in the progression of AV, evidence from mechanical vascular injury models suggests that medial injury may play a more dominant role. The overall objective of this research was to determine the relative importance of the endothelium versus the media as a target for immune injury and induction of AV. To investigate this we developed a novel model which involved the creation of chimeric aortic segments. To accomplish this we removed aortic segments from C3H/HeJ (C3H) mice and stripped them of endothelium by a short pulse with EDTA. The stripped C3H grafts were implanted into immunodeficient C57BL/6 (B6) RAG1(-/-) mice for a period of 21 days. As the immunodeficient mice did not mount an allo-immune response to the grafts, the endothelium was renewed by normal repair mechanisms. The new endothelium was recipient in origin, resulting in a chimeric graft with C3H media and B6 endothelium. We confirmed complete denudement by immunocytochemistry for endothelial specific markers, as well as by transmission and scanning electron microscopy. Replacement of endothelium with recipient endothelial cells was confirmed by immunocytochemistry, electron microscopy and by using a green fluorescent protein mouse transplant combination. Subsequent re-transplantation of the chimeric grafts into either B6 or C3H recipients demonstrated that an allogeneic media is more important than an allogeneic endothelium in inducing robust AV.