sFgl2‑enriched MSC-EVs attenuate colitis by enhancing immunoregulatory capacity.

Dendritic cell (DC) hyperactivation drives pathogenic Th1/Th17 immune responses and contributes to the progression of inflammatory bowel disease (IBD). Although mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) represent promising cell-free immunomodulatory agents, strategies to enhance their tolerogenic DC-reprogramming capacity remain limited. Here, we investigated whether soluble fibrinogen-like protein 2 (sFgl2), an immunoregulatory protein, could function as a therapeutic cargo of MSC-EVs and explored its receptor-associated signaling mechanism in experimental colitis. sFgl2-enriched MSC-EVs were generated by lentiviral engineering of MSCs and characterized by morphological, physicochemical, molecular, and quality-control analyses. EV particle concentration and sFgl2 abundance were quantified by nanoparticle tracking analysis and ELISA, respectively, allowing estimation of sFgl2 copies per EV. The surface accessibility of EV-associated sFgl2 was evaluated by proteinase K protection assays. Therapeutic efficacy was assessed in DSS-induced colitis. Co-immunoprecipitation was performed to investigate the association between sFgl2 and CD32b, and CD32b blockade was used to evaluate pathway involvement in vitro and in vivo. BMDC-based studies further assessed DC maturation, cytokine production, and CD4⁺ T-cell responses. sFgl2-MSC-EVs exhibited increased sFgl2 abundance while retaining characteristic EV properties, with detectable EV-associated markers and no detectable endotoxin contamination or residual lentiviral signals. Proteinase K protection assays supported substantial surface accessibility of EV-associated sFgl2. In DSS-induced colitis, sFgl2-MSC-EVs produced greater therapeutic benefit than NC-MSC-EVs, as evidenced by improved body weight, reduced disease activity, preservation of colon length, attenuation of histopathological injury, and reduced levels of IL-6, TNF-α, and IL-1β together with increased IL-10. sFgl2-MSC-EVs also more effectively suppressed CD11c⁺ DC accumulation and maturation in mesenteric lymph nodes and spleen and promoted a more regulatory CD4⁺ T-cell profile characterized by reduced Th1/Th17 responses and increased Treg frequencies. Co-immunoprecipitation supported an association between sFgl2 and CD32b in BMDCs. Mechanistically, sFgl2-MSC-EVs enhanced ERK1/2-STAT3 activation, whereas CD32b blockade or ERK1/2 inhibition attenuated their immunoregulatory effects. Importantly, in vivo CD32b blockade weakened the therapeutic effects of sFgl2-MSC-EVs and partially reversed their inhibitory effects on DC accumulation and maturation. These findings identify EV-associated sFgl2 as a functional immunoregulatory cargo that enhances the therapeutic activity of MSC-EVs through CD32b-associated ERK1/2-STAT3 signaling and tolerogenic DC reprogramming. sFgl2-engineered MSC-EVs represent a promising cell-free strategy for immunomodulatory therapy in IBD and provide a potential platform for functionalized EV-based therapies.