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

Abstract

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.

Skull bone marrow-derived immune cells infiltrate the damaged cortex and exhibit anti-inflammatory properties

“…Efferocytosis, the process of clearing dead cells, is crucial for resolving inflammation and promoting tissue repair after TBI [6]. Our previous studies have shown an upregulation of "find-me" signal receptors (S1pr1 and Cx3cr1), engulfment receptor (MerTK), and bridging molecules (Gas6 and Pros1) in the damaged cortex, indicating active efferocytosis after trauma.…”

“…This emerging concept suggests that skull-bone marrow cells may be crucial in modulating brain homeostasis and neuroinflammation, particularly following injuries. While it is well-established that TBI disrupts the blood-brain barrier, leading to immune cell infiltration from the bloodstream [6,22], the proportion and the identity of immune cells that migrate from skull bone marrow to brain parenchyma after injury remain uncertain. Using the calvarium bone-flap transplantation technique and controlled cortical impact (CCI) model of TBI, our study aimed to investigate whether calvarium-derived immune cells infiltrate the brain following TBI and differentiate them from their blood-derived counterparts.…”

“…Studying the effect of skull bone marrow in TBI poses significant challenges due to the intricate nature of immune cell trafficking and the diverse responses elicited by different TBI models. The controlled cortical impact (CCI) model is commonly used to study TBI, as it allows for precise control over the location and severity of the injury [6,11,12,22,23]. However, this model involves direct manipulation of the skull tissue, which can pose challenges, particularly in understanding the influence of proximal skull tissue on injury progression and recovery [21].…”

“…Controlled cortical impact (CCI) injury. At 14 days post-calvarium transplantation, CCI was carried out as previously described [6,[15][16][17]. Briefly, mice were anesthetized using a subcutaneous injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and then positioned in a stereotaxic frame.…”

“…Once matured, these macrophages engage in a range of activities mediating both protection and neuronal dysfunction. The neuroprotective or pro-resolving function of these phagocytes may be attributed to their ability to clear apoptotic cell debris formed following tissue damage via a process called efferocytosis [5,6]. The detrimental effects, on the other hand, have been attributed to the production of pro-inflammatory and cytotoxic mediators [4].…”