Mesenchymal Stem Cells Regulate Lung Inflammation In ARDS Mice By Modulating Dendritic Cells

Objective:To verify mesenchymal stem cells regulate lung inflammation in ARDS mice by modulating dendritic cells.Methods:Sixty-four C57BL/6 mice were randomly (random number)divided into Control group (NS+PBS), ARDS group (LPS+PBS), MSC group (LPS+MSC), DCreg Group (LPS+DCreg).Murine model of ARDS was induced by intratracheal instillation of lipopolysaccharide (LPS 5mg/kg) and blood、spleen and lung were harvested 6h and 24h later after different treatment. (1)Evaluate the frequency and the expressions of CD11c、CD11b、MHCⅡ CD40 of DCs in the lung by flow cytometry; (2) Evaluate the frequency and the expressions of CD11c、CD11b、MHCⅡ、CD40 of DCs in the spleen by flow cytometry; Evaluate the phagocytic ability of DCs in spleen by dextran-FITC phagocytosis; Evaluate the influence of DCs in spleen on lymphocyte proliferation by mixed lymphocyte culture. (3)Assess the Lung injury of ARDS mice:Histopathological analysis of lung tissues were performed by H&E staining and quantified by using Smith lung injury scores to evaluate the severity of lung injury; Calculate lung wet weight/body weight ratio (LWW/BW) to estimated lung edema; and the pulmonary microvascular endothelial permeability was evaluated by Evans blue leakage experimental. (4)Assess the Lung inflammation of ARDS mice:the total cell counts and the differential cell counts in bronchoalveolar lavage fluid (BALF) were measured by staining with Wright’s dye specimens to evaluated the extravasation of inflammatory cell; The levels of IL-4, IL-10 and IL-17、TGF-β、 IFN-y in lung homogenates and blood were measured by ELISA to evaluate the inflammation of lung and whole body.Results:(1) MSC inhibit the increase and mature of DCs in lung:when compared with ARDS group at 24h, MSC significantly decrease the frequency of DCs in lung, as well as the expression of CD11c, CD11b、MHCⅡ CD40 of DCs in lung(p<0.05).(2) MSC inhibit the increase and mature of DCs in spleen:when compared with ARDS group at 6h、24h, MSC significantly decrease the frequency of DCs in spleen, as well as the expression of CD11c、CD11b、MHCⅡ CD40 of DCs in spleen (p<0.05), MSC also enhanced phagocytic ability of spleen DCs(p<0.05), and attenuate the ability of spleen DCs to stimulate T lymphocyte proliferation (p<0.05) at 24h.This indicated that MSC can induce the generation of DCreg in spleen(3) DCreg and MSC inhibit the inflammation of LPS-induced ARDS mice:when compared with ARDS group at 6h、24h, DCreg and MSC can decreased the total nucleated cells and neutrophil counts in BALF, down-regulated the expression levels of the proinflammatory mediators IL-4 and IL-17, and up-regulated the levels of antinflammatory cytokine IL-10、IFN-γand TGF-β in lung tissues and serum (p< 0.05).(4) DCreg and MSC attenuate lung injury of LPS-induced ARDS mice:when compared with ARDS group at 6h、24h, DCreg and MSC can significantly improve lung histopathology, reduce lung injury score and LWW/BW, and decrease pulmonary endothelial permeability of evans blue(p<0.05).Conclusion:Mesenchymal stem cells attenuate lung inflammation in ARDS mice by inhibiting the increase and mature of DCs, and inducing the generation of DCreg.