Hypoxia inducible factor 2α promotes tolerogenic macrophage development during cardiac transplantation through transcriptional regulation of colony stimulating factor 1 receptor.

Solid organ transplantation mobilizes myeloid cells, including monocytes and macrophages, which are central protagonists of allograft rejection. However, myeloid cells can also be functionally reprogrammed by perioperative costimulatory blockade to promote a state of transplantation tolerance. Transplantation tolerance holds promise to reduce complications from chronic immunosuppression and promote long-term survival in transplant recipients. We sought to identify different mediators of transplantation tolerance by performing single-cell RNA sequencing of acute rejecting or tolerized cardiac allografts. This led to the unbiased identification of the transcription factor, hypoxia inducible factor (HIF)-2α, in a subset of tolerogenic monocytes. Using flow cytometric analyses and mice with conditional loss or gain of function, we uncovered that myeloid cell expression of HIF-2α was required for costimulatory blockade-induced transplantation tolerance. While HIF-2α was dispensable for mobilization of tolerogenic monocytes, which were sourced in part from the spleen, it promoted the expression of colony stimulating factor 1 receptor (CSF1R). CSF1R mediates monocyte differentiation into tolerogenic macrophages and was found to be a direct transcriptional target of HIF-2α in splenic monocytes. Administration of the HIF stabilizer, roxadustat, within micelles to target myeloid cells, increased HIF-2α in splenic monocytes, which was associated with increased CSF1R expression and enhanced cardiac allograft survival. These data support further exploration of HIF-2α activation in myeloid cells as a therapeutic strategy for transplantation tolerance.

A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity.

The spleen is a unique lymphoid organ that plays a critical role in the homeostasis of the immune and hematopoietic systems. Patients that have undergone splenectomy regardless of precipitating causes are prone to develop an overwhelming post-splenectomy infection and experience increased risks of deep venous thrombosis and malignancies. Recently, epidemiological studies indicated that splenectomy might be associated with the occurrence of cardiovascular diseases, suggesting that physiological functions of the spleen have not yet been fully recognized. Here, we introduce a mouse model of vascularized heterotopic spleen transplantation, which not only can be utilized to study the function and behavioral activity of splenic immune cell subsets in different biologic processes, but also can be a powerful tool to test the therapeutic potential of spleen transplantation in certain diseases. The main surgical steps of this model include donor spleen harvest, the removal of recipient native spleen, and spleen graft revascularization. Using congenic mouse strains (e.g., mice with CD45.1/CD45.2 backgrounds), we observed that after syngeneic transplantation, both donor-derived splenic lymphocytes and myeloid cells migrated out of the graft as early as post-operative day 1, concomitant with the influx of multiple types of recipient cells, thus generating a unique chimera.  Despite relatively challenging techniques, this procedure can be performed with >90% success rate. This model allows tracking the fate, longevity, and function of splenocytes during steady state and in a disease setting following a spleen transplantation, thereby offering a great opportunity to discover the distinct role for spleen-derived immune cells in different disease processes.

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.

MerTK Cleavage on Resident Cardiac Macrophages Compromises Repair After Myocardial Ischemia Reperfusion Injury.

RATIONALE: Clinical benefits of reperfusion after myocardial infarction are offset by maladaptive innate immune cell function, and therapeutic interventions are lacking. OBJECTIVE: We sought to test the significance of phagocytic clearance by resident and recruited phagocytes after myocardial ischemia reperfusion. METHODS AND RESULTS: In humans, we discovered that clinical reperfusion after myocardial infarction led to significant elevation of the soluble form of MerTK (myeloid-epithelial-reproductive tyrosine kinase; ie, soluble MER), a critical biomarker of compromised phagocytosis by innate macrophages. In reperfused mice, macrophage Mertk deficiency led to decreased cardiac wound debridement, increased infarct size, and depressed cardiac function, newly implicating MerTK in cardiac repair after myocardial ischemia reperfusion. More notably, Mertk(CR) mice, which are resistant to cleavage, showed significantly reduced infarct sizes and improved systolic function. In contrast to other cardiac phagocyte subsets, resident cardiac MHCIILOCCR2- (major histocompatibility complex II/C-C motif chemokine receptor type 2) macrophages expressed higher levels of MerTK and, when exposed to apoptotic cells, secreted proreparative cytokines, including transforming growth factor-ß. Mertk deficiency compromised the accumulation of MHCIILO phagocytes, and this was rescued in Mertk(CR) mice. Interestingly, blockade of CCR2-dependent monocyte infiltration into the heart reduced soluble MER levels post-ischemia reperfusion. CONCLUSIONS: Our data implicate monocyte-induced MerTK cleavage on proreparative MHCIILO cardiac macrophages as a novel contributor and therapeutic target of reperfusion injury.

HIF-2α in Resting Macrophages Tempers Mitochondrial Reactive Oxygen Species To Selectively Repress MARCO-Dependent Phagocytosis.

Hypoxia-inducible factor (HIF)-α isoforms regulate key macrophage (MΦ) functions during ischemic inflammation. HIF-2α drives proinflammatory cytokine production; however, the requirements for HIF-2α during other key MΦ functions, including phagocytosis, are unknown. In contrast to HIF-1α, HIF-2α was not required for hypoxic phagocytic uptake. Surprisingly, basal HIF-2α levels under nonhypoxic conditions were necessary and sufficient to suppress phagocytosis. Screening approaches revealed selective induction of the scavenger receptor MARCO, which was required for enhanced engulfment. Chromatin immunoprecipitation identified the antioxidant NRF2 as being directly responsible for inducing Marco Concordantly, Hif-2α-/- MΦs exhibited reduced antioxidant gene expression, and inhibition of mitochondrial reactive oxygen species suppressed Marco expression and phagocytic uptake. Ex vivo findings were recapitulated in vivo; the enhanced engulfment phenotype resulted in increased bacterial clearance and cytokine suppression. Importantly, natural induction of Hif-2α by IL-4 also suppressed MARCO-dependent phagocytosis. Thus, unlike most characterized prophagocytic regulators, HIF-2α can act as a phagocytic repressor. Interestingly, this occurs in resting MΦs through tempering of steady-state mitochondrial reactive oxygen species. In turn, HIF-2α promotes MΦ quiescence by blocking a MARCO bacterial-response pathway. IL-4 also drives HIF-2α suppression of MARCO, leading to compromised bacterial immunosurveillance in vivo.

Cardiomyocytes induce macrophage receptor shedding to suppress phagocytosis.

BACKGROUND: Mobilization of the innate immune response to clear and metabolize necrotic and apoptotic cardiomyocytes is a prerequisite to heart repair after cardiac injury. Suboptimal kinetics of dying myocyte clearance leads to secondary necrosis, and in the case of the heart, increased potential for collateral loss of neighboring non-regenerative myocytes. Despite the importance of myocyte phagocytic clearance during heart repair, surprisingly little is known about its underlying cell and molecular biology. OBJECTIVE: To determine if phagocytic receptor MERTK is expressed in human hearts and to elucidate key sequential steps and phagocytosis efficiency of dying adult cardiomyocytes, by macrophages. RESULTS: In infarcted human hearts, expression profiles of the phagocytic receptor MER-tyrosine kinase (MERTK) mimicked that found in experimental ischemic mouse hearts. Electron micrographs of myocardium identified MERTK signal along macrophage phagocytic cups and Mertk-/- macrophages contained reduced digested myocyte debris after myocardial infarction. Ex vivo co-culture of primary macrophages and adult cardiomyocyte apoptotic bodies revealed reduced engulfment relative to resident cardiac fibroblasts. Inefficient clearance was not due to the larger size of myocyte apoptotic bodies, nor were other key steps preceding the formation of phagocytic synapses significantly affected; this included macrophage chemotaxis and direct binding of phagocytes to myocytes. Instead, suppressed phagocytosis was directly associated with myocyte-induced inactivation of MERTK, which was partially rescued by genetic deletion of a MERTK proteolytic susceptibility site. CONCLUSION: Utilizing an ex vivo co-cultivation approach to model key cellular and molecular events found in vivo during infarction, cardiomyocyte phagocytosis was found to be inefficient, in part due to myocyte-induced shedding of macrophage MERTK. These findings warrant future studies to identify other cofactors of macrophage-cardiomyocyte cross-talk that contribute to cardiac pathophysiology.

Enhanced efferocytosis of apoptotic cardiomyocytes through myeloid-epithelial-reproductive tyrosine kinase links acute inflammation resolution to cardiac repair after infarction.

RATIONALE: Efficient clearance of apoptotic cells (efferocytosis) is a prerequisite for inflammation resolution and tissue repair. After myocardial infarction, phagocytes are recruited to the heart and promote clearance of dying cardiomyocytes. The molecular mechanisms of efferocytosis of cardiomyocytes and in the myocardium are unknown. The injured heart provides a unique model to examine relationships between efferocytosis and subsequent inflammation resolution, tissue remodeling, and organ function. OBJECTIVE: We set out to identify mechanisms of dying cardiomyocyte engulfment by phagocytes and, for the first time, to assess the causal significance of disrupting efferocytosis during myocardial infarction. METHODS AND RESULTS: In contrast to other apoptotic cell receptors, macrophage myeloid-epithelial-reproductive tyrosine kinase was necessary and sufficient for efferocytosis of cardiomyocytes ex vivo. In mice, Mertk was specifically induced in Ly6c(LO) myocardial phagocytes after experimental coronary occlusion. Mertk deficiency led to an accumulation of apoptotic cardiomyocytes, independently of changes in noncardiomyocytes, and a reduced index of in vivo efferocytosis. Importantly, suppressed efferocytosis preceded increases in myocardial infarct size and led to delayed inflammation resolution and reduced systolic performance. Reduced cardiac function was reproduced in chimeric mice deficient in bone marrow Mertk; reciprocal transplantation of Mertk(+/+) marrow into Mertk(-/-) mice corrected systolic dysfunction. Interestingly, an inactivated form of myeloid-epithelial-reproductive tyrosine kinase, known as solMER, was identified in infarcted myocardium, implicating a natural mechanism of myeloid-epithelial-reproductive tyrosine kinase inactivation after myocardial infarction. CONCLUSIONS: These data collectively and directly link efferocytosis to wound healing in the heart and identify Mertk as a significant link between acute inflammation resolution and organ function.