Bone Marrow-derived Mesenchymal Stem Cells Attenuate Sepsis By Inhibiting The Activity Of NLRP3 Inflammasome

Background and objectiveSepsis is the leading cause of death in the intensive care unit and ranks among the top 10 causes of death worldwide. Septic shock are progressively severe stages of the host’s systemic inflammatory response to infection. This stage carries increasing rates of end-organ failure and death. Since the inflammatory response to infection is complex, independent efforts have been made in the past two decades to inhibit the activity of such likely biochemical triggers as endotoxin, tumor necrosis factor α, interleukin-1, and others. Lots of clinical trials designed to investigate new agents to interfere with these pathways have not shown a benefit. Many studies underlie not only the failing of immune cell responses and cell-specific apoptosis, but also the endothelial and epithelial dysfunction in disease severity and the related multiple organ failure. The therapeutic and supportive alterations are exceedingly complex because of the interconnection among the multiple biological pathways involved in the septic process. The existing knowledge supports the idea that bone marrow-derived mesenchymal stem cells(BMSCs)might be beneficial in sepsis treatment. In studies involving in vivo animal models, these cells were capable of reducing mortality and organ dysfunction-related markers.Notably, immunomodulation, anti-apoptosis, increased bacterial clearance, and the protection from endothelial/epithelial cell dysfunction have been postulated as possible mechanisms of action describing how BMSCs may improve septic outcomes. Nevertheless, the functional mechanism and therapeutic efficacy of these properties of BMSCs on the potential treatment of sepsis remain unclear.The present study was designed to investigate the effects and mechanistic insight of BMSCs on sepsis-induced inflammation and survival in a mouse model of sepsis. MethodsIn thefirst part of the study, BMSCs were isolated from B6-e GFP mice, which constitutively expressed enhanced green fluorescent protein(e GFP). Cardiac fibroblasts(Fbs) were isolated from 1- or 3-day-old C57BL/6 mice. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture(CLP), followed 1 hour later by the tail vein injection of BMSCs, Fbs or saline. 24 hours after CLP, left ventricular systolic function and hemodynamic parameters were obtained by transthoracic echocardiography. Sera and tissues were collected for analysing the density of neutrophil and macrophages infiltration, the secretion of proinflammatory cytokines and the expression of proteins. We assessed survival after surgery every 6 h for 96 h.In thesecond part of the study, bone marrow-derived macrophages(BMDMs) were isolated from C57BL/6 mice. BMSCs and BMDMs were co-cultured after stimulated by LPS and ATP. The culture supernatants of BMDMs was collected to detect the released proinflammatory cytokines. Mitochondrial reactive oxygen species(ROS) were measured by flow cytometry. Mitochondrial morphology were examined by transmission electron microscopy(TEM). The subcellular localization of target proteins were analysed by immunofluorescence staining. The level of protein expressions were examined by western blot analysis.Results 1. Survival rate at 96 hours after CLP was recorded. The survival rate of saline-treated CLP group was 10% at 96 hours after CLP, which was reduced to nearly 40% by BMSCs-treatment, and this different was significant(P<0.05). The BMSCs-treated CLP mice also had significantly increased survival rate compared with the Fbs-treated CLP group(P<0.05).There was no significant difference in survival rate between saline-treated CLP group and Fbs-treated CLP group.2. BMSCs were detected in the blood up to 1 h after intravenous injections and mainly localized in the liver. Histologic assessment of organ sections from mice with CLP-induced sepsis revealed evidence of inflammatory infiltration and interstitial edema, whereas inflammation was decreased in mice that received BMSCs. Organ dysfunction was determined by measurement of biochemical indicators of organ function in serum samples. Administration of BMSCs reduced systemic levels of biochemical indicators significantly in septic mice. Left ventricular cardiac function was assessed in vivo using two-dimensional M-mode echocardiography in all mice. Cardiac function was significantly improved in BMSCs-treated CLP group versus saline-treated or Fbs-treated CLP group as evidenced by a rise in EF and FS. Maximal velocity of pressure development and decline(±LVd P/dt max) also revealed a significant improvement in response to BMSCs treatment in septic mice. The ±LVd P/dt max was significantly higher in the BMSCs-treated CLP group compared with saline-treated or Fbs-treated CLP group. BMSCs treatment significantly inhibited inflammatory cell infiltration as evidenced by decreased macrophage(Mac-3) and neutrophil(Ly6G) infiltration. To evaluate levels of systemic inflammation, multiplex analysis of cytokines was performed on serum samples. Compared with saline-treated or Fbs-treated CLP group, serum concentrations of proinflammatory cytokines, such as TNF-a、IL-6 and IL-1β, were significantly reduced and the antiinflammatory cytokine IL-10 were markedly elevated in mice with BMSCs treatment. Caspase-1 and IL-1β activation was markedly suppressed inliver tissues of mice with BMSCs treatment as assessed by Western blot.3. We observed that BMDMs isolated from C57BL/6 mice generated more mitochondrial superoxide anion radical(O2–) after being stimulated by LPS and ATP, as detected by the fluorescence of Mito SOX, indicative of more mitochondrial production of ROS and mitochondrial dysfunction. But co-cultured with BMSCs suppressed the generation of mitochondrial ROS. We further observed that treatment with LPS and ATP increased the percentage of damaged mitochondria(positive for Mito Tracker Deep Red and Mito Tracker Green) in BMDMs, from 4.64% to 11.9% of the total population. In contrast, co-culture with BMSCs decreased damaged mitochondria from 11.9% to 6.19% of the total mitochondria. Using confocal microscopy, the localization of NLRP3 in BMDMs was examined. We found co-culture with BMSCs may change the localization of NLRP3 protein in BMDMs after being stimulated by LPS and ATP. Treatment of BMDMs with the mitochondria-targeted antioxidant Mito-TEMPO, a scavenger specific for mitochondrial ROS, caspase-1 and IL-1β activation was markedly suppressed in BMDMs by transwell co-culture with BMSCs as assessed by Western blot in cell lysates. Consistent with the reduction in caspase-1 activity, the levels of IL-1β and IL-18 in the supernatants were significantly reduced as measured by ELISA.4. To avoid cellular damage, ROS-generating mitochondria are constantly removed by mitophagy, and we investigated the ultrastructure of BMDMs from all groups We observed more swollen mitochondria in BMSCs-treated group than those in treatment with LPS and ATP by transmission electron microcopy.In agreement with this established mechanism of mitochondrial disposal, autophagy-associated LC3 puncta were found to accumulate around mitochondria in BMDMs cocultured with BMSCs after stimulated by LPS and ATP. Treatment of BMDMs with the mitophagy/autophagy inhibitor 3-methyladenine(3-MA), enhanced the activation of caspase-1, IL-1β and the secretion of IL-1β, IL-18 in BMDMs cocultured with BMSCs after being stimulated by LPS and ATP.5. Treatment of BMDMs with Mito-TEMPO increased the expression of PINK1, Parkin and attenuated the ratio of LC3-I to LC3-II in BMDMs coculture with BMSCs after being stimulated by LPS and ATP. 3-MA was added to BMDMs, which resulted in the accumulation of damaged mitochondria and increased concentrations of mitochondrial ROS in BMDMs cocultured with BMSCs after being stimulated by LPS and ATP.Conclusion These data of this study demonstrate that BMSCs have beneficial effects on experimental sepsis, possibly by inhibiting NLRP3 inflammasome activation in macrophages primarily by increasing mitophagy of macrophages and thus decreasing mitochondrial ROS. Our fingings suggest that immunomodulatory cell therapymay be an effective adjunctive treatment to reduce sepsis-related morbidity and mortality.