脑损伤后，轴突导向分子 EphA4 通过 ERK/Stat6/MERTK 信号限制噬血细胞作用。

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.

背景：噬作用是一种清除凋亡细胞和细胞碎片的过程。清除这些细胞可以减轻神经炎症，防止炎症分子的释放，并促进抗炎细胞因子的产生，以帮助维持组织内稳态。损伤后大脑中发生这种情况的潜在机制仍未明确。

方法：我们使用 GFP 骨髓嵌合敲除 (KO) 小鼠证明轴突导向分子 EphA4 受体酪氨酸激酶参与抑制大脑中的 MERTK，以限制驻留小胶质细胞和外周衍生的单核细胞/巨噬细胞的噬作用。

结果：单细胞 RNAseq 鉴定了 MERTK 的表达，这是参与噬作用的主要受体，在脑损伤后损伤皮质中的单核细胞、小胶质细胞和一部分星形胶质细胞上表达。浸润性 GFP 表达免疫细胞中 EphA4 的缺失改善了功能结果，同时增强了噬作用和 p-MERTK、p-ERK 和 p-Stat6 的总蛋白表达。在 KO 嵌合小鼠中，GFP 单核细胞/巨噬细胞和驻留小胶质细胞吞噬 NeuN 或 TUNEL 细胞的百分比明显更高。重要的是，与野生型相比，这些小鼠中 Mertk 和其同源配体 Gas6 的 mRNA 表达显著升高。细胞特异性表达分析表明，在脑损伤后皮质损伤区，p-ERK 和 p-Stat6 与表达 MERTK 的 GFP+细胞共定位。使用体外噬作用测定法，共培养 pHrodo 标记的凋亡 Jurkat 细胞和骨髓 (BM) 衍生的巨噬细胞，我们证明在没有 EphA4 的情况下，噬作用效率和 Mertk 和 Gas6 的 mRNA 表达增强。ERK 和 Stat6 的选择性抑制剂减弱了这种作用，证实 EphA4 通过抑制 ERK/Stat6 途径抑制单核细胞/巨噬细胞噬作用。

结论：我们的发现表明，ERK/Stat6/MERTK 轴作为脑损伤中凋亡碎片清除的新调节剂，受外周髓样衍生 EphA4 的限制，以防止炎症的解决。