Extracellular vesicles derived from TNF-α-preconditioned mesenchymal stem cells mitigate inflammatory retinal injury

Aim: To evaluate the therapeutic efficacy and mechanisms of tumor necrosis factor-alpha (TNF-α) preconditioned mesenchymal stem cells (MSCs)-derived small extracellular vesicles (hereafter abbreviated as T-sEV), along with the control small extracellular vesicles (sEV, representing vesicles from naive/unstimulated MSCs), in mitigating inflammatory retinal injury. Methods: T-sEV were isolated from TNF-α-preconditioned MSCs and systematically characterized. Small RNA sequencing was performed to identify the microRNA (miRNA) cargo of T-sEV. The effect of T-sEV on lipopolysaccharide (LPS)-induced M1 macrophage polarization was assessed by flow cytometry. Integrated bioinformatic analysis linked T-sEV miRNAs to macrophage transcriptome changes. T-sEV were administered intravitreally in a murine sodium iodate (NaIO₃)-induced retinal degeneration model. Electroretinography (ERG), optical coherence tomography (OCT), flow cytometry, intraocular pressure (IOP) and systemic examinations were conducted. Results: T-sEV exhibited an enrichment of anti-inflammatory miRNAs, notably miR-146a-5p. They were efficiently internalized by macrophages, significantly suppressing M1 polarization, as evidenced by the decreased percentage of cluster of differentiation (CD)11b⁺/CD86⁺ cells (29.60% ± 2.30%) compared to the sEV group (34.90% ± 1.57%, P < 0.05). Analysis showed T-sEV miRNAs targeted and downregulated key pro-inflammatory genes such as Cd86 and Il1r1. In vivo, T-sEV treatment significantly preserved retinal a- and b-wave amplitudes and structural integrity. T-sEV treatment markedly reduced retinal macrophage infiltration, decreasing the proportion of F4/80⁺CD11b⁺ cells to 0.38% ± 0.13%, significantly lower than in the sEV group (1.66% ± 0.47%, P < 0.01). No adverse effects on IOP or systemic markers were observed. Conclusion: TNF-α preconditioning enhances MSC-derived sEV therapeutic capacity by enriching their anti-inflammatory miRNAs. T-sEV suppress pro-inflammatory macrophage activation and provide superior neuroprotection in a retinal degeneration model, indicating a safe and promising cell-free therapeutic strategy.