sTNFR1 from UCMSCs promotes post-MCAO recovery by driving macrophage reparative polarization via NF-κB/SOCS3 signaling

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.