Endocytosis

BackgroundNeutrophil extracellular traps (NETs) contribute to atherosclerosis progression and are linked to adverse clinical outcomes such as myocardial infarction and stroke. Although the triggers of NET formation in plaques are known, the mechanisms governing DNase-mediated NET clearance and how these are disrupted during atherosclerosis remain unclear. Moreover, the consequences of impaired NET clearance on disease progression are not known.MethodsLow-density lipoprotein receptor knockout (Ldlr-/-) mice with hematopoietic cell-specific deletion of DNase1 and DNase1L3 were fed a Western-type diet for 16 weeks to examine the impact of loss of DNase activity and the subsequent NET accumulation on advanced atherosclerosis. The effect of NETs on macrophage efferocytosis was examined in vitro and in the mouse peritoneal cavity and atherosclerotic plaque in vivo. To identify the signaling pathway impairing the NET-induced DNase response, in vitro assays were performed using selective endoplasmic reticulum stress pathway inhibitors, and the findings were validated in murine and human atherosclerotic tissues.ResultsLack of DNase secretion by macrophages led to accumulation of NETs in local tissues, including atherosclerotic plaques. Persisting NETs in turn promoted cleavage of the efferocytosis receptor MerTK (c-mer proto-oncogene tyrosine kinase), resulting in defective macrophage efferocytosis and increased atherosclerotic plaque necrosis. In vitro screening identified endoplasmic reticulum stress-induced activation of the PERK (protein kinase R-like endoplasmic reticulum kinase)-ATF (activating transcription factor) 4 signaling axis in atherogenic macrophages as a key driver of impaired DNase secretion, leading to delayed NET clearance and their pathological persistence. Treatment of human atherosclerotic plaques and Ldlr-/- mice with integrated stress response inhibitor, a selective PERK inhibitor, restored vascular DNase secretion and facilitated NET clearance.ConclusionsMacrophages play a key role in clearing NETs from tissues. Endoplasmic reticulum stress suppresses macrophage DNase secretion, leading to NET accumulation in atherosclerotic plaques, which triggers efferocytosis impairment and plaque progression. Targeting the PERK-ATF4 axis to restore DNase release and NET clearance represents a promising therapeutic strategy to promote plaque stabilization.

Microglia are the resident immune cells of the CNS, maintaining brain homeostasis partially through phagocytosis. In Alzheimer's disease (AD), microglial phagocytosis is significantly impaired, contributing to the accumulation of pathological aggregates. Microglial phenotypes are dynamic and can shift depending on the disease stage and local environment. While some subpopulations retain or enhance phagocytic activity, especially under inflammatory conditions, others lose their capacity to clear toxic debris effectively. This variability underscores the need for a more nuanced understanding of microglial regulation and function. This paper explores the dual role of microglial phagocytosis in AD and discusses the emerging insights into microglial heterogeneity and how phenotypic shifts affect phagocytic capacity throughout disease progression. A comprehensive understanding of microglial phagocytosis and its dysregulation in AD is essential for designing targeted treatments. Modulating microglial activity to enhance their protective roles without triggering harmful inflammation represents a promising direction for therapeutic intervention in AD.

Spinal cord injury (SCI) is a devastating condition characterized by the accumulation of myelin debris (MD), persistent neuroinflammation, and impaired neural regeneration. Although macrophages are pivotal for MD clearance, the impact of excessive MD phagocytosis on macrophage phenotype and function remains poorly understood. Building upon our prior evidence that exendin-4 (Ex-4), a glucagon-like peptide-1 receptor (GLP-1R) agonist, mitigates microglia-driven neuroinflammation post-SCI, this study elucidates the therapeutic efficacy and underlying mechanisms of Ex-4 in alleviating macrophage senescence, restoring efferocytotic capacity, and facilitating neural repair. Employing a T10 contusive SCI model in male C57BL/6 mice, in vivo administration of Ex-4 was combined with macrophage-specific knockdown of growth arrest-specific 6 (Gas6) via AAV-shRNA. Complementary in vitro assays involved bone marrow-derived macrophages (BMDMs) challenged with MD in the presence or absence of Ex-4 or AMP-activated protein kinase (AMPK) inhibition. Cellular senescence and efferocytosis were comprehensively assessed through live-cell imaging, immunofluorescence, senescence-associated β-galactosidase staining, quantitative PCR, and western blotting. Molecular docking and dynamics simulations elucidated GLP-1R-AMPK interactions, corroborated by in vivo validation. Results demonstrate that MD-engulfing macrophages exhibit foam cell-like morphology and upregulated senescence markers, including increased β-galactosidase activity and senescence-associated secretory phenotype, concomitant with diminished efferocytosis via downregulation of the Axl receptor. Senescent macrophages were shown to exacerbate neuronal apoptosis and astrocytic scar formation in co-culture systems. Ex-4 treatment significantly attenuated macrophage senescence, restored efferocytotic function, and reduced neuronal injury and astrocyte activation, effects contingent upon AMPK/Gas6/Axl pathway activation and abrogated by Gas6 knockdown. In vivo, Ex-4 administration enhanced remyelination, axonal regeneration, and functional recovery, while attenuating glial scar formation following SCI. Collectively, these findings identify macrophage senescence induced by excessive MD phagocytosis as a novel pathological contributor to SCI progression and establish Ex-4 as a promising therapeutic agent that restores macrophage homeostasis and promotes neural repair via GLP-1R/AMPK/Gas6/Axl signaling.

The regulation of macrophage function, particularly that of monocyte-derived macrophages (MoMs), by mechanical forces during myocardial infarction (MI) remains poorly understood. Consistently upregulated Piezo1 expression in cardiac macrophages and MoMs post-MI is found. Elevated Piezo1 expression in MoMs directly contributes to increased Piezo1 levels in cardiac macrophages. Myeloid cell-specific Piezo1-deficient mice (Piezo1Lyz2) exhibit significant improvements in ventricular function/remodeling after MI, accompanied by decreased apoptotic cardiomyocytes and decreased inflammation, increased numbers of macrophages, and increased border zone efferocytosis. In vitro, Piezo1 activation by Yoda1 increased oxygen-glucose deprivation (OGD)-induced ferroptosis and impaired MoM efferocytosis. Conversely, Piezo1 deficiency in MoMs decreases ferroptosis and increases efferocytosis. SLC7A11 is shown to mediate Piezo1-induced defective efferocytosis in MoMs. Piezo1 activation aggravated OGD-induced macrophage ferroptosis via Ca2+ influx followed by SLC15A3 upregulation. Piezo1 upregulated SLC7A11 in macrophages via a Ca2+/ATF4-dependent pathway. MoM-specific SLC7A11 knockdown significantly increases efferocytosis, reduces cardiomyocyte apoptosis and inflammation, and ameliorates post-MI left ventricular remodeling and function. In conclusion, early Piezo1 activation in MoMs is identified during MI, which governs the fate and function of recruited macrophages. These data establish an ischemic heart-bone marrow functional network and provide a novel therapeutic strategy in which MoM Piezo1 is targeted for post-MI heart repair.