Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by fibroblast activation and collagen deposition. While ANTXR1 is a transmembrane protein implicated in various pathologies including lung cancer, its specific expression patterns and molecular mechanisms within the context of IPF remain elusive. In this study, we show that ANTXR1 expression is markedly elevated in IPF lung tissues, in bleomycin (BLM)-induced pulmonary fibrosis mouse models, and in TGF-β1-stimulated fibroblasts. Functionally, ANTXR1 was found to drive the proliferation and differentiation of TGF-β1-treated human and mouse fibroblasts. Mechanistically, in TGF-β1-treated fibroblasts, we identified that the transcription factor SOX8 directly binds to the ANTXR1 promoter to transcriptionally activate its expression. Furthermore, ANTXR1 promotes IPF progression by upregulating GLI1, a critical downstream effector of the Hedgehog signaling pathway. Therapeutically, intratracheal delivery of Antxr1-targeting shRNA via adeno-associated virus (AAV) significantly ameliorated the fibrotic phenotype in BLM-induced mice. Our findings suggest that SOX8 drives fibroblast activation and fibrotic progression in idiopathic pulmonary fibrosis by elevating GLI1 expression via ANTXR1, and identify ANTXR1 as a promising therapeutic target for IPF.
Retinal pigment epithelium (RPE) dysfunction is central to age-related macular degeneration (AMD), yet the early cellular alterations that precede overt degeneration remain incompletely understood. In this study, we investigated the impact of subtoxic hypoxia-mimetic stress (STH) on the polarized secretion of small extracellular vesicles (sEVs) by human iPSC-RPE monolayers, using this system to model early AMD-relevant RPE stress. The STH regimen induced hypoxia-mimetic stress without causing overt cytotoxicity, while preserving epithelial barrier integrity. However, it markedly remodeled the sEV secretome and disrupted apicobasal cargo allocation, as exemplified by basolaterally biased SERPINA5 enrichment and apically biased CTSC reduction. Proteomic analysis further revealed increased AMD-relevant cargo, such as APOE, VEGFA, and ANGPTL4, while reducing lysosomal hydrolases. Concurrently, STH impaired RPE phagocytic and degradative function. Notably, these sEV changes were largely uncoupled from bulk intracellular proteomic alterations, suggesting that polarized sEV profiling may serve as a sensitive indicator of RPE dysfunction under AMD-relevant stress. At the structural level, STH disrupted apical microvilli and reduced microtubule-associated cytoskeletal stability. Importantly, CoCl₂ washout partially reversed these secretory, structural, and functional abnormalities. Taken together, our findings suggest that early and reversible remodeling of polarized sEV secretion reflects a subtoxic AMD-related stress state in RPE cells, which provides a potential opportunity to detect and modulate RPE dysfunction before overt degeneration.
Atherosclerosis is a chronic inflammatory disease of the arterial wall and remains the leading cause of cardiovascular events worldwide. Schaftoside is a naturally occurring flavone C-glycoside that improves lipid metabolism and exerts anti-inflammatory effects. However, its atheroprotective effects and anti-inflammatory mechanisms remain unclear. This study explores the therapeutic potential of schaftoside in atherosclerosis and its ability to inhibit NLRP3 inflammasome activation through a LAMP2A-dependent CMA-related lysosomal pathway. We established an high-fat diet (HFD)-induced atherosclerosis model in ApoE⁻/⁻ mice, along with ox-LDL-induced foam cell formation and LPS/ATP-induced NLRP3 inflammasome activation models in primary peritoneal macrophages (PMs). Atherosclerosis was assessed by lipid deposition and plaque progression in the artery. Foam cell formation was determined by measuring intracellular lipid accumulation and inflammatory responses. The molecular mechanisms by which schaftoside inhibits NLRP3 inflammasome assembly were investigated using Western blotting, immunofluorescence, cycloheximide (CHX) chase assays, molecular docking, molecular dynamics simulations, surface plasmon resonance (SPR), and cell transfection. Schaftoside treatment attenuated atherosclerotic lesions in ApoE⁻/⁻ mice, as evidenced by reduced plaque formation, diminished lipid deposition, and improved serum lipid profiles. Concurrently, NLRP3 inflammasome activation during atherogenesis was suppressed by schaftoside. This inhibition occurred at the post-translational level of NLRP3 protein and prior to ASC oligomerization. In vitro validation revealed that schaftoside exerted its anti-atherosclerotic effects by promoting NLRP3 protein degradation. CHX chase assays demonstrated that the lysosomal inhibitor NH₄Cl reversed the schaftoside-induced NLRP3 protein degradation. Molecular docking and SPR analyses revealed direct interactions between schaftoside and two key proteins of the CMA pathway, LAMP2A and HSC70. Both in vivo and in vitro experiments supported the involvement of a LAMP2A-dependent CMA-related lysosomal pathway in schaftoside-induced NLRP3 degradation and inhibition of inflammasome assembly. Knockdown of LAMP2A reversed the inhibitory effects of schaftoside on foam cell formation and NLRP3 inflammasome assembly. Schaftoside alleviates HFD-induced atherosclerosis and macrophage-derived foam cell formation while improving lipid metabolism. Moreover, schaftoside suppresses NLRP3 inflammasome activation in atherosclerotic progression through a LAMP2A-dependent CMA-related lysosomal pathway.
The post-ischemic immune microenvironment critically balances neuroinflammation and tissue repair, making macrophage polarization a pivotal therapeutic target in cerebral infarction. Umbilical cord mesenchymal stromal cells (UCMSCs) modulate macrophage polarization primarily through paracrine signaling, while the key mediators and underlying mechanisms remain incompletely elucidated. Using proteomic array-based screening of co-culture supernatants, we identified soluble factors secreted by UCMSCs that regulate macrophage polarization. Stable soluble tumor necrosis factor receptor 1 (sTNFR1)-overexpressing (oe-sTNFR1) UCMSCs were generated via lentiviral transduction. The mechanisms by which sTNFR1 regulates macrophage polarization were investigated in vitro using immunoblotting, RT-qPCR, immunofluorescence, and flow cytometry. The therapeutic efficacy was evaluated in a rat model of middle cerebral artery occlusion (MCAO) via intracranial delivery of oe-sTNFR1 UCMSCs, with outcomes assessed via histological (Nissl and 2,3,5-triphenyltetrazolium chloride) staining, behavioral tests, and magnetic resonance imaging. To circumvent risks of genetic modification, a TNF-α-targeting nanobody (TNF-α Nb) was developed and tested in combination with wild-type UCMSCs. Proteomic analysis identified sTNFR1 as a key mediator secreted by UCMSCs for reprogramming macrophage polarization. oe-sTNFR1 UCMSCs enhanced macrophage chemotaxis and promoted reparative polarization in a paracrine-dependent manner. Mechanistically, UCMSC-derived sTNFR1 suppressed the NF-κB axis, which not only attenuated pro-inflammatory polarization but also derepressed SOCS3 signaling, thereby promoting the reparative phenotype. In MCAO rats, intracranial delivery of oe-sTNFR1 UCMSCs reshaped the post-ischemic brain microenvironment toward an anti-inflammatory and pro-repair state, improving neurological recovery through reduced infarct volume and improved functional outcomes. Furthermore, co-administration of the TNF-α Nb with wild-type UCMSCs enhanced a reparative myeloid phenotype and improved neurological outcomes compared to UCMSCs alone. This study establishes UCMSC-derived sTNFR1 as a key paracrine mediator of macrophage polarization via the sTNFR1/NF-κB/SOCS3 axis, facilitating functional recovery after ischemic stroke. The strategy combining a TNF-α Nb with UCMSCs offers a novel and clinically promising immunotherapeutic approach.
The NLRP3 inflammasome is a central innate immune sensor that matures pro‑inflammatory cytokines and can drive neuroinflammation. Dysregulated activation contributes to tissue injury and neuronal damage. This review systematically examines pathogen-specific mechanisms of NLRP3 activation in central nervous system (CNS) infections caused by bacteria, viruses, parasites, and fungal pathogens. These pathogens engage markedly heterogeneous upstream triggers, most commonly lysosomal cathepsin B release, reactive oxygen species generation, and potassium efflux, alongside less broadly documented mechanisms including mitochondria-associated membrane signaling and direct pathogen–protein interactions. Despite this diversity, these pathways ultimately converge on a common downstream axis defined by caspase-1-dependent maturation of IL-1β and IL-18, followed by pyroptosis. Evidence is well substantiated for certain pathogens but remains largely inferential for others, and host-protective patterns of NLRP3 activity are distinguished from immunopathogenic ones. Finally, the therapeutic promise and translational challenges of NLRP3-targeted interventions in the infected CNS are evaluated, indicating that inhibition strategies must be tailored to the pathogen-specific context, as balancing antimicrobial defense against collateral neuronal injury remains a central challenge in this field.
