Efferocytosis is restricted by axon guidance molecule EphA4 via ERK/Stat6/MERTK signaling following brain injury.

BACKGROUND: Efferocytosis is a process that removes apoptotic cells and cellular debris. Clearance of these cells alleviates neuroinflammation, prevents the release of inflammatory molecules, and promotes the production of anti-inflammatory cytokines to help maintain tissue homeostasis. The underlying mechanisms by which this occurs in the brain after injury remain ill-defined. METHODS: We used GFP bone marrow chimeric knockout (KO) mice to demonstrate that the axon guidance molecule EphA4 receptor tyrosine kinase is involved in suppressing MERTK in the brain to restrict efferocytosis of resident microglia and peripheral-derived monocyte/macrophages. RESULTS: Single-cell RNAseq identified MERTK expression, the primary receptor involved in efferocytosis, on monocytes, microglia, and a subset of astrocytes in the damaged cortex following brain injury. Loss of EphA4 on infiltrating GFP-expressing immune cells improved functional outcome concomitant with enhanced efferocytosis and overall protein expression of p-MERTK, p-ERK, and p-Stat6. The percentage of GFP+ monocyte/macrophages and resident microglia engulfing NeuN+ or TUNEL+ cells was significantly higher in KO chimeric mice. Importantly, mRNA expression of Mertk and its cognate ligand Gas6 was significantly elevated in these mice compared to the wild-type. Analysis of cell-specific expression showed that p-ERK and p-Stat6 co-localized with MERTK-expressing GFP + cells in the peri-lesional area of the cortex following brain injury. Using an in vitro efferocytosis assay, co-culturing pHrodo-labeled apoptotic Jurkat cells and bone marrow (BM)-derived macrophages, we demonstrate that efferocytosis efficiency and mRNA expression of Mertk and Gas6 was enhanced in the absence of EphA4. Selective inhibitors of ERK and Stat6 attenuated this effect, confirming that EphA4 suppresses monocyte/macrophage efferocytosis via inhibition of the ERK/Stat6 pathway. CONCLUSIONS: Our findings implicate the ERK/Stat6/MERTK axis as a novel regulator of apoptotic debris clearance in brain injury that is restricted by peripheral myeloid-derived EphA4 to prevent the resolution of inflammation.

Efferocytosis is restricted by axon guidance molecule EphA4 via ERK/Stat6/Mertk signaling following brain injury.

Background: Efferocytosis is a process that removes apoptotic cells and cellular debris. Clearance of these cells alleviates neuroinflammation and prevents the release of inflammatory molecules and promotes the production of anti-inflammatory cytokines to help maintain tissue homeostasis. The underlying mechanisms by which this occurs in the brain after injury remains ill-defined. Methods: We demonstrate using GFP bone marrow chimeric knockout (KO) mice, that the axon guidance molecule EphA4 receptor tyrosine kinase is involved in suppressing Mertk signaling in the brain to restrict the function of efferocytosis on resident microglia and peripheral-derived monocyte/macrophages. Results: Single-cell RNAseq identified Mertk expression, the primary receptor involved in efferocytosis, on monocytes, microglia, and a subset of astrocytes in the damaged cortex following brain injury. Loss of EphA4 on infiltrating GFP-expressing immune cells improved functional outcome concomitant with enhanced efferocytosis, and overall protein expression of p-Mertk, p-ERK, and p-Stat6. The percentage of GFP+ monocyte/macrophages and resident microglia engulfing NeuN+ or TUNEL+ cells was significantly higher in KO chimeric mice. Importantly, mRNA expression of Mertk and its cognate ligand Gas6 was significantly elevated in these mice compared to wild-type. Analysis of cell-specific expression showed that p-ERK and p-Stat6 co-localized with Mertk-expressing GFP + cells in the peri-lesional area of the cortex following brain injury. Using an in vitro efferocytosis assay, co-culturing pHrodo-labeled apoptotic Jurkat cells and bone marrow (BM)-derived macrophages, we demonstrate that efferocytosis efficiency and mRNA expression of Mertk and Gas6 was enhanced in the absence of EphA4. Select inhibitors of ERK and Stat6 attenuated this effect confirming that EphA4 suppresses monocyte/macrophage efferocytosis via inhibition of the ERK/Stat6 pathway. Conclusions: Our findings implicate the Mertk/ERK/Stat6 axis as a novel regulator of apoptotic debris clearance in brain injury that is restricted by peripheral myeloid-derived EphA4 to prevent the resolution of inflammation.

Conditional Deletion of EphA4 on Cx3cr1-Expressing Microglia Fails to Influence Histopathological Outcome and Blood Brain Barrier Disruption Following Brain Injury.

Erythropoietin-producing human hepatocellular receptors play a major role in central nervous system injury. Preclinical and clinical studies revealed the upregulation of erythropoietin-producing human hepatocellular A4 (EphA4) receptors in the brain after acute traumatic brain injury. We have previously reported that Cx3cr1-expressing cells in the peri-lesion show high levels of EphA4 after the induction of controlled cortical impact (CCI) injury in mice. Cx3cr1 is a fractalkine receptor expressed on both resident microglia and peripheral-derived macrophages. The current study aimed to determine the role of microglial-specific EphA4 in CCI-induced damage. We used Cx3cr1 CreER/+ knock-in/knock-out mice, which express EYFP in Cx3cr1-positive cells to establish microglia, EphA4-deficient mice following 1-month tamoxifen injection. Consistent with our previous findings, induction of CCI in wild-type (WT) Cx3cr1 CreER/+ EphA4 +/+ mice increased EphA4 expression on EYFP-positive cells in the peri-lesion. To distinguish between peripheral-derived macrophages and resident microglia, we exploited GFP bone marrow-chimeric mice and found that CCI injury increased EphA4 expression in microglia (TMEM119+GFP-) using immunohistochemistry. Using Cx3cr1 CreER/+ EphA4 f/f (KO) mice, we observed that the EphA4 mRNA transcript was undetected in microglia but remained present in whole blood when compared to WT. Finally, we found no difference in lesion volume or blood-brain barrier (BBB) disruption between WT and KO mice at 3 dpi. Our data demonstrate a nonessential role of microglial EphA4 in the acute histopathological outcome in response to CCI.