Single-cell profiling reveals inflammatory polarization of human carotid versus femoral plaque leukocytes.

Femoral atherosclerotic plaques are less inflammatory than carotid plaques histologically, but limited cell-level data exist regarding comparative immune landscapes and polarization at these sites. We investigated intraplaque leukocyte phenotypes and transcriptional polarization in 49 patients undergoing femoral (n = 23) or carotid (n = 26) endarterectomy using single-cell RNA-Seq (scRNA-Seq; n = 13), flow cytometry (n = 24), and IHC (n = 12). Comparative scRNA-Seq of CD45+-selected leukocytes from femoral (n = 9; 35,265 cells) and carotid (n = 4; 30,655 cells) plaque revealed distinct transcriptional profiles. Inflammatory foam cell-like macrophages and monocytes comprised higher proportions of myeloid cells in carotid plaques, whereas noninflammatory foam cell-like macrophages and LYVE1-overexpressing macrophages comprised higher proportions of myeloid cells in femoral plaque (P < 0.001 for all). A significant comparative excess of CCR2+ macrophages in carotid versus plaque was observed by flow cytometry in a separate validation cohort. B cells were more prevalent and exhibited a comparatively antiinflammatory profile in femoral plaque, whereas cytotoxic CD8+ T cells were more prevalent in carotid plaque. In conclusion, human femoral plaques exhibit distinct macrophage phenotypic and transcriptional profiles as well as diminished CD8+ T cell populations compared with human carotid plaques.

Macrophage-produced VEGFC is induced by efferocytosis to ameliorate cardiac injury and inflammation.

Clearance of dying cells by efferocytosis is necessary for cardiac repair after myocardial infarction (MI). Recent reports have suggested a protective role for vascular endothelial growth factor C (VEGFC) during acute cardiac lymphangiogenesis after MI. Here, we report that defective efferocytosis by macrophages after experimental MI led to a reduction in cardiac lymphangiogenesis and Vegfc expression. Cell-intrinsic evidence for efferocytic induction of Vegfc was revealed after adding apoptotic cells to cultured primary macrophages, which subsequently triggered Vegfc transcription and VEGFC secretion. Similarly, cardiac macrophages elevated Vegfc expression levels after MI, and mice deficient for myeloid Vegfc exhibited impaired ventricular contractility, adverse tissue remodeling, and reduced lymphangiogenesis. These results were observed in mouse models of permanent coronary occlusion and clinically relevant ischemia and reperfusion. Interestingly, myeloid Vegfc deficiency also led to increases in acute infarct size, prior to the amplitude of the acute cardiac lymphangiogenesis response. RNA-Seq and cardiac flow cytometry revealed that myeloid Vegfc deficiency was also characterized by a defective inflammatory response, and macrophage-produced VEGFC was directly effective at suppressing proinflammatory macrophage activation. Taken together, our findings indicate that cardiac macrophages promote healing through the promotion of myocardial lymphangiogenesis and the suppression of inflammatory cytokines.

Bone marrow-derived AXL tyrosine kinase promotes mitogenic crosstalk and cardiac allograft vasculopathy.

Cardiac Allograft Vasculopathy (CAV) is a leading contributor to late transplant rejection. Although implicated, the mechanisms by which bone marrow-derived cells promote CAV remain unclear. Emerging evidence implicates the cell surface receptor tyrosine kinase AXL to be elevated in rejecting human allografts. AXL protein is found on multiple cell types, including bone marrow-derived myeloid cells. The causal role of AXL from this compartment and during transplant is largely unknown. This is important because AXL is a key regulator of myeloid inflammation. Utilizing experimental chimeras deficient in the bone marrow-derived Axl gene, we report that Axl antagonizes cardiac allograft survival and promotes CAV. Flow cytometric and histologic analyses of Axl-deficient transplant recipients revealed reductions in both allograft immune cell accumulation and vascular intimal thickness. Co-culture experiments designed to identify cell-intrinsic functions of Axl uncovered complementary cell-proliferative pathways by which Axl promotes CAV-associated inflammation. Specifically, Axl-deficient myeloid cells were less efficient at increasing the replication of both antigen-specific T cells and vascular smooth muscle cells (VSMCs), the latter a key hallmark of CAV. For the latter, we discovered that Axl-was required to amass the VSMC mitogen Platelet-Derived Growth Factor. Taken together, our studies reveal a new role for myeloid Axl in the progression of CAV and mitogenic crosstalk. Inhibition of AXL-protein, in combination with current standards of care, is a candidate strategy to prolong cardiac allograft survival.

Macrophage AXL receptor tyrosine kinase inflames the heart after reperfused myocardial infarction.

Tyro3, AXL, and MerTK (TAM) receptors are activated in macrophages in response to tissue injury and as such have been proposed as therapeutic targets to promote inflammation resolution during sterile wound healing, including myocardial infarction. Although the role of MerTK in cardioprotection is well characterized, the unique role of the other structurally similar TAMs, and particularly AXL, in clinically relevant models of myocardial ischemia/reperfusion infarction (IRI) is comparatively unknown. Utilizing complementary approaches, validated by flow cytometric analysis of human and murine macrophage subsets and conditional genetic loss and gain of function, we uncover a maladaptive role for myeloid AXL during IRI in the heart. Cross signaling between AXL and TLR4 in cardiac macrophages directed a switch to glycolytic metabolism and secretion of proinflammatory IL-1ß, leading to increased intramyocardial inflammation, adverse ventricular remodeling, and impaired contractile function. AXL functioned independently of cardioprotective MerTK to reduce the efficacy of cardiac repair, but like MerTK, was proteolytically cleaved. Administration of a selective small molecule AXL inhibitor alone improved cardiac healing, which was further enhanced in combination with blockade of MerTK cleavage. These data support further exploration of macrophage TAM receptors as therapeutic targets for myocardial infarction.

Acute and chronic phagocyte determinants of cardiac allograft vasculopathy.

Post-transplant immunosuppression has reduced the incidence of T cell-mediated acute rejection, yet long-term cardiac graft survival rates remain a challenge. An important determinant of chronic solid organ allograft complication is accelerated vascular disease of the transplanted graft. In the case of cardiac allograft vasculopathy (CAV), the precise cellular etiology remains inadequately understood; however, histologic evidence hints at the accumulation and activation of innate phagocytes as a causal contributing factor. This includes monocytes, macrophages, and immature dendritic cell subsets. In addition to crosstalk with adaptive T and B immune cells, myeloid phagocytes secrete paracrine signals that directly activate fibroblasts and vascular smooth muscle cells, both of which contribute to fibrous intimal thickening. Though maladaptive phagocyte functions may promote CAV, directed modulation of myeloid cell function, at the molecular level, holds promise for tolerance and prolonged cardiac graft function.