间充质基质细胞外囊泡通过调控巨噬细胞炎症小体改善免疫检查点抑制剂相关肺炎OA

Objective:To investigate the therapeutic effect and molecular mechanism of mesenchymal stromal cell-derived extracellular vesicles(MSC-EV)on immune checkpoint inhibitor-related pneumonitis(CIP)in a mouse model.Methods:Foxp3DTR mice were inoculated with MC38 mouse colon cancer cells and randomly divided into 3 groups(n=6 per group):① control group(MC38 inoculation only);② CIP model group(inoculated with MC38 cells,intraperitoneally injected with diphtheria toxin[DT]and anti-PD-1 antibody);③ MSC-EV intervention group(based on the CIP model,intranasally administered with MSC-EV[dose 109 particles/mouse,every 3 days,2 times in total]).A mouse CIP model induced by Treg depletion combined with anti-PD-1 antibody was established.In vivo,H-E staining was used to observe lung pathological injury,lung wet/dry ratio was measured to assess pulmonary edema,ELISA was used to detect IL-1β,IL-6,and TNF-α levels in bronchoalveolar lavage fluid(BALF),and flow cytometry was used to detect the proportion of Ly6G ⁺ granulocyte infiltration in BALF.Tumor volume was monitored to evaluate antitumor efficacy.In vitro,DiO fluorescent labeling was used to observe the uptake of extracellular vesicles(EVs)by bone marrow-derived macrophages,and WB assay was used to detect the expression levels of key NLRP3 inflammasome proteins(cleaved GSDMD and mature IL-1β).Potential mechanisms were explored using GEO database miRNA sequencing data(GSE69909),Dicer knockdown,and miR-21/miR-125 inhibitor intervention.Results:Intranasal administration of MSC-EV alleviated lung inflammatory injury in CIP model mice,reduced IL-1β,IL-6,and TNF-α levels and the proportion of Ly6G⁺ granulocyte infiltration in BALF(P<0.05),without compromising the antitumor efficacy of anti-PD-1 antibody therapy.In vitro DiO labeling tracing showed that MSC-EV could be taken up by macrophages.WB assay showed that MSC-EV decreased the levels of key activated proteins(cleaved GSDMD and mature IL-1β)of the NLRP3 inflammasome(P<0.05).In vitro,after Dicer knockdown,the MSC-EV showed a weakened downregulation effect on IL-1β in macrophages(P<0.05).miRNA sequencing(GEO database,GSE69909)showed high expression of miR-21 and miR-125 in MSC-EV,and inhibition of miR-21 or miR-125 reduced the inhibitory effect of the corresponding MSC-EV on IL-1β cleavage in macrophages(P<0.05).Conclusion:MSC-EV exert anti-inflammatory effects through their carried miRNAs such as miR-21 and miR-125,and the mechanism may be related to inhibition of NLRP3 inflammasome activation in macrophages,thereby effectively alleviating CIP without compromising the antitumor efficacy of immune checkpoint inhibitor.