The Role And Mechanism Of Mesenchymal Stem Cells-educated Macrophages In Sepsis-induced Lung Injury

Part 1 The role of mesenchymal stem cell-educated macrophages in sepsis-induced lung injuryObjective:Adipose mesenchymal stem cells(ASCs) are pluripotent cells. In addition to owning a strong differentiation and regeneration effect, ASC is also acting as an immuno-modulator. Previous studies have suggested that inflammation was coming up as a cascade chain and monocytes were involved when sepsis occurred. ASC can regulate inflammation-immune condition through educated cells by itself and/or secreting cytokines. Mesenchymal stem cells have been reported to reduce lipopolysaccharide (LPS)-induced inflammatory response. Both bone marrow and adipose-derived mesenchymal stem cells (ASCs) have the capacity to regulate the function of macrophages. The goal of this part was to determine whether ASCs-educated macrophages could directly ameliorate LPS-induced systemic response in a mouse model.Method:Mouse peritoneal macrophages were cocultured with ASCs in a Transwell system for 2 days to educate macrophages. Mice were divided into 5 groups:control, LPS, LPS +ASCs, LPS+untreated macrophages, and LPS+educated macrophages. The weight loss were obtained at 6,24, and 48 h after LPS treatment. Lung, serum, and BAL were harvested at 6,24, and 48 h after LPS treatment. Hematoxylin and eosin (H&E) staining was performed to determine morphology and inflammatory infiltrate, and the wet-dry ratio was determined by dividing the weight before and after desiccating. Also, All the samples from serum and BAL were analyzed for TNF-a、IL-6、IFN-y、IL-4、IL-10、 IL-13 via a Procarta PlexTM Multiplex Immunoassay (eBioscience, San Diego, CA).Results:Educated macrophages had a similar effect as ASCs in reducing weight loss compared to LPS alone at 24 h time point(P<0.05,P<0.05). Significant decreases in the number of neutrophils in animals treated with ASCs or educated macrophages were revealed as compared with LPS alone at 24 h time out(P<0.01, P<0.05). In mice treated with LPS alone, edema reached a peak at 24 h and was largely resolved by 48 h, which is similar to the animals that received LPS+M. No edema developed in the lungs of animals received LPS+ASCs and LPS+EM (P<0.05,P<0.05). Six hours after LPS administration, treatment with ASCs and educated macrophages significantly attenuated the levels of pro-inflammatory cytokine IFN-y (P<0.001,P<0.001) and IL-6 (P<0.01, P<0.001) while increasing the level of anti-inflammatory cytokine IL-10 (P<0.001, P<0.01) in the serum. Meanwhile, ASCs and educated macrophages also significantly decreased IL-6 level (P<0.001,P<0.001) and elevated levels of IL-4 (P<0.01,P<0.001), IL-10 (P<0.001,P<0.001), and IL-13 (P<0.001,P<0.001) in the BAL.Conclusion:Educated macrophages can directly ameliorate LPS-induced systemic response as ASC acts.Part 2 The mechanism of mesenchymal stem cells educated macrophages in sepsis-induced lung injuryObjective:We can conclude that ASCs can educate macrophages from the Part 1. The educated macrophages can defend the acute lung injury by downregulating the level of the proinflammatory factors and upregulating that of anti-inflammatory factors. This part of the study, we want to explore the molecular mechanisms based on the effect of ASCs educated macrophages to lung injury/sepsis.Method:Macrophages were cultured alone or cocultured with ASCs in either a standard 6-well or in a Transwell system for 48 hours. Macrophages were then stimulated with or without LPS (10 ng/ml) for 24 hours. The cell culture supernatants were collected for cytokine analysis. Macrophages were harvested for phenotype analysis and M2 phenotype expression were labeled with FITC anti-mouse CD68 and APC anti-mouse CD206 by flow cytometry and arginase in macrophages was determined using western-blot. Macrophages were treated with LPS or serum from patients with acute respiratory distress syndrome (ARDS). Furthermore, to assess the effects of ASCs on M2 macrophages, the IL-10 experiment was done, macrophages were treated with LPS (10 ng/ml) in the presence of 1 ng/ml or 10 ng/ml of IL-10 for 48 hours. IL-10 antibody or IL-10 receptor antibody was added to the culture. Arginase in macrophages were harvested for western-blot and culture supernatants were harvested for cytokine assay.Results:In vitro, ASCs increased expression of M2 macrophages (P<0.05) and there is no difference between transwell and direct coculture (P>0.05). The levels of pro-inflammatory IL-6 and TNF-α were significantly elevated following LPS treatment (P<0.001,P<0.001) and were significantly decreased by direct coculture (P<0.001, P<0.01) or Transwell with ASCs (P<0.001,P<0.05). In contrast, the levels of anti-inflammatory IL-10 were significantly augmented with both ways of coculture (P<0.01, P<0.05). Furthermore, treatment with 8% ARDS serum reduced the expression of M2 macrophages (P<0.05) and ASC increased the M2 levels either with direct cell-to-cell contact or transwell coculture (P<0.01,P<0.05). IL-10 (1 ng/ml and 10 ng/ml) reduced proinflammatory cytokine IL-6(P<0.001,P<0.001) and TNF-a (P<0.01,P<0.001) expression. IL-10 (1 ng/ml and 10 ng/ml) also induced M2 phenotype (P<0.05,P<0.01). And IL-10 antibody and the antibody of IL-10 receptor revert the effct of ASC coculture with macrophages. The protein Arg decresead (by western-blot) and the IL-6 (*P<0.001, *P<0.001) and TNF-a (*P<0.01,*P<0.05) levels in supernatants increased again.Conclusion:LPS can mimic the effect of ARDS serum to set up the septic micro-environment. Apidose mesenchymal stem cell educated macrophages may exert their anti-inflammatory effects via IL-10 pathway and this effect.Part 3 The effect of mesenchymal stem cells on monocyte of peripheral blood in sepsis childrenObjective:Integrated with the first and second part of the study, we found ASCs can educate macrophages and decrease the inflammation. Previous studies have suggested that inflammation was coming up as a cascade chain and monocytes were involved when sepsis occurred. ASC can regulate inflammation-immune condition through educated cells by itself and/or secreting cytokines. This part of the study was designed to observe submonocytes expression level in peripheral blood from pediatric septic patients and control cases by flow cytometry, and analyzed its expression levels and its correlation with severity of sepsis were determined in pediatric critical ill patients.Method:We enrolled 8 septic and 10 non-septic surgical pediatric patients admitted to university hospital SICU from June 2015 to October 2015. The monocytes were isolated from peripheral whole blood of the enrolled patients which was drawn within 24 hours after diagnosis of sepsis. The expression levels of CD16++CD14+monocytes were measured by flow cytometry.Results:The present study showed that the CD14++CD16+monocytes levels in septic patients was significantly higher compared to that in controls (P<0.05), and positively correlated with Pediatric risk of Mortality III Score (r=0.6064, P=0.0006). In monocytes from pediatric septic patients, CD14++CD16+ levels were also down-regulated when transwell cocultured with ASCs (P<0.05).Conclusion:CD14++CD16+ monocytes expression was upregulated in patients with sepsis, which is positively correlated with clinical outcome score, suggesting CD14++CD16+ monocytes may play a critical role during sepsis, and ASCs can revert this change.