Impact Of S1PR2on The Initiation And Development Of Sepsis Induced Acute Lung Injury And Its Molecular Mechanism

Part1Impact of S1FR2on the initiation and development of sepsis induced acute lung injuryObjectives:Sphingosine1-phosphate receptor2(S1PR2) is belonged to the seven transmembrane G protein coupled receptor family, which is involved in many critical cellular processes after binding with Sphingosine1-phosphate (SIP). Recently, it’s reported that S1PR2modulates endotoxin induced inflammation in endothelium. However, its role in regulating macrophage response to bacterial infection remains unclear. So, this part of the study was designed to investigate the impact of S1PR2in sepsis induced acute lung injury.Methods:Sepsis was induced by CLP on WT (S1pr2+/+) and S1pr2-/-mice. Mortality was assessed daily. To determine the bacterial burden, peritoneal lavage fluid, blood samples, and lung homogenate were obtained at72h after CLP challenge and were detected using agar plating method. Lung morphology changes were assessed by HE staining. To further investigate whether S1PR2participates in lung immune defense against bacterial infection, mice with different genotypes (WT, S1pr2+-/or S1pr2-/-) were challenged with E. coli intratracheal inoculation. Mortality was assessed hourly. Bronchoalveolar lavage fluid (BALF) and blood were obtained at0h,4h and18h after infection and bacterial burdens were assayed using agar plating method. Lung morphology changes were assessed by HE staining. Pulmonary oedema and pulmonary vascular permeability were assessed by using lung wet/dry weight ratios and measuring the BALF protein accumulation separately. The TNF-a and IL-6levels in the blood or BALF were determined by ELISA. Pharmaceutical inhibition of S1PR2before and after intratracheal instillation of E. coli, and mortality was assessed hourly. BALF and blood were obtained at4h after infection and bacterial burdens were assayed using agar plating method.Results:In a sublethal CLP model, the survival rate of the S1pr2-/-mice was66.7%, while only28.6%of WT mice survived more than10days (*P<0.05). S1PR2deficiency led to a significant decrease in bacterial counts within the peritoneal lavage fluid and peripheral blood at72h following CLP. Furthermore, a decreased pulmonary bacterial burden was also observed in the S1pr2-/-mice, which was concomitant with alleviated lung injury in these mice. After the E, coli intratracheal inoculation, there was no survival of WT and heterozygote mice beyond32h. Remarkably,80of S1pr2-/-mice were still alive48h after E. coli challenge (***P<0.001). Four hours after inoculation of E. coli, bacterial counts in the blood collected from the WT mice were higher than those from the S1pr2-/-mice by10folds. Bacterial counts continued to rise at18h after E. coli inoculation in the WT mice. In contrast, bacterial counts in the blood samples taken from the S1pr2-/-mice remained very low from4h to18h. Consistent with the serological findings, bacterial counts in BALF from the WT mice were significantly higher than those from the S1pr2-/-mice at both time points. Along with reduced pulmonary bacterial load, diminished lung injury and permeability were observed in the Slpr2-/-group. In addition, we found largely increased early-stage inflammation in S1pr2-/-mice compared with WT mice documented by IL-6and TNF-a levels in BALF at4h following E. coli administration. While, the plasma IL-6and TNFa levels in E. coli treated mice were comparably low in S1pr2-/-mice. Following JTE-013pretreatment,50%of the mice survived for at least36h after bacterial infection, but only10%of the mice treated with vehicle survived for this length of time. Moreover, the bacterial burdens measured within the blood and BALF of these mice were also significantly reduced following JTE-013treatment.Conclusions:The absence of S1PR2enhances lung immune defense to eliminate invading E. coli and prevents the pathogen from spreading, ultimately improving the outcome. Part2Impact of S1PR2on phagocytosis function of macrophageObjectives:As the absence of S1PR2enhances lung immune defense to eliminate invading E. coli and prevents the pathogen from spreading, ultimately improving the outcome. We next to identify which cells were responsible for the different rates of bacterial clearance observed above.Methods:We created reciprocal BM transplantation between WT and Slpr2’A mice as follows:WT→WT, S1pr2-/-→S1pr2-/-, WT→S1pr2-/-, and S1pr2-/-→T. The chimeras were then subjected to intratracheal E. coli challenge. Bronchoalveolar lavage fluid (BALF) were obtained at4h after infection and bacterial burdens were assessed using agar plating method. Also, mortality of the WT→WT and S1pr2-/-→WT chimeras were observed hourly. The kind of BALF cell types at different times after intratracheal E. coli challenge were assessed using FACS. In addition, the S1PR2mRNA levels expressed on the alveolar macrophages (AMs) and neutrophils were further detected by qPCR. We depleted AMs populations via intratracheal administration of liposome-encapsulated clodronate prior to E. coli challenge, and the bacterial burdens in BALF and the mice mortality were assessed as above. Furthermore, the Phagocytosis and bactericidal ability of AMs from WT and S1pr2-/-mice were assessed ex vivo.Results:Replacement of WT mouse BM with that from S1pr2-/-mice mirrored the phenotype observed in the S1pr2-/-mice, but engraftment of WT BM into the S1pr2-/- recipients restored the WT phenotype. The S1pr2-/-→WT chimeric mice survived from bacterial infection better than the WT→WT chimeras (*P<0.05). AMs were dominant in the BALF samples at0h and4h after infection. The recruitment of neutrophils dramatically increased18h post infection. However, far fewer neutrophils were present in S1pr2-/-mice BALF samples. The mRNA level of S1PR2was～25-fold higher in AMs than in neutrophils, regardless of E. coli stimulation (***P<0.001). Clodronate treatment abrogated the difference of bacterial loads and survival between the S1pr2-/-and WT cohorts. S1pr2-/-AMs exhibited increased rates of bacterial engulfinent. Opsonizing E, coli using rabbit IgG increased ingestion of the bacteria by-3-fold in WT AMs but did not increase phagocytosis in S1pr2-/-AMs. The bactericidal rate of AMs is varied in the short term depending on the quantity of ingested bacteria, but presence of S1PR2does not affect bactericidal capacity in the long run.50%of WT mice survived at33h post-challenge with opsonized E. coli, which is15h longer than when they were challenged with unopsonized E. coli.Conclusions:S1PR2deficiency enhances the phagocytic function of AMs in a substrate-independent manner, thereby improving pulmonary defense and survival rates following bacterial infection. Part3The downsteam signaling of S1PR2in inhibiting macrophage phagocytosisObjectives:Integrated with the first and second part of the study, we found S1PR2deficiency enhances the phagocytic function of macrophage, thereby improving pulmonary defense and alleviating lung injury. This part of the study, we want to explore the molecular mechanisms based on different the S1P concentration.Methods:Bronchoalveolar lavage fluid (BALF) was obtained at0h,4h and18h after intratracheal E. coli challenge. Secretion of endogenous S1P from WT and S1pr2-/-BMDMs after E. coli incubation (0min,30min and90min) and S1P concentration within collected BALFs were assessed by HPLC-ESI-MS/MS. The influence of exogenous S1P concentration on the phagocytosis abilities of WT and S1pr2-/-BMDMs were detected by fluorescence microscope. We applied exogenous S1P (100nM) to mimic an in vivo environment representative of later stages of the disease (18h), the actin reorganization and phagocytosis abilities of macrophages during this condition was determined using confocal microscopy. RhoA activity was assessed by pull down. In addition, we used only E. coli infection in vitro to represent early stage bacterial infection, Racl location and activity in WT and S1pr2-/-BMDMs were determined using confocal microscopy and pull down. F-actin content was assessed by western blot. Binding of IQGAP1to Racl was detected using confocal microscopy and co-immunoprecipitation. The Racl activity and phagocytosis ability changes in IQGAP1siRNA silenced BMDMs were also assessed by pull down and fluorescence microcopy. Results:During acute lung infection, SIP concentration within collected BALFs increased from0h to18h after bacterial infection. In contrast, secretion of endogenous SIP from WT and Slpr2-/-BMDMs before and after E. coli infection alone was very limited. We further found that incubation with exogenous SIP (from100nM to5μM) reduced phagocytosis by～40%in WT BMDMs. However, phagocytosis by S1pr2-/-BMDMs was not inhibited (***P<0.001). Exogenous SIP treatment stimulated rapid redistribution of actin in WT BMDMs leading to the cellular adoption of a round-shaped morphology within10min of SIP application; however, no such morphological changes were observed in S1pr2-/-cells. When incubating cells with E. coli alone, autocrine signaling of S1PR2via the macrophage-derived S1P ligand failed to induce cellular contraction; instead, it was found that S1PR2signaling decreased the presence of prey-stimulated formation of lamellipodia in WT cells. However, the presence of prey did not induce lamellipodiual formationin WT cells pretreated with SIP. Exogenous SIP treatment increased RhoA-GTP levels in WT cells but not in S1pr2-/-cells (*P<0.05). Incubation with E. coli alone did not significantly affect the levels of RhoA-GTP. Immunofluorescent staining revealed that Racl was primarily localized within the perinuclear area of the cytoplasm in both WT and S1pr2-/-BMDMs in the absence of bacterial stimulation. However, when S1pr2-/-cells were challenged with E. coli, Racl almost completely relocated from the perinuclear area to the cell periphery. In contrast, Racl localization did not substantially change in WT BMDMs following cellular exposure to E. coli. As expected, incubation with E. coli stimulated a dramatic elevation of Racl-GTP levels in S1pr2-/-BMDMs, but only a mild increase of Racl activity in WT cells. Consistent with Racl-GTP levels in these cells, bacterial incubation increased the presence of F-actin slightly in WT BMDMs and greatly in the S1pr2-/-BMDMs (*P<0.05). Similar to Racl, IQGAP1was found to be located in the cytosol of both WT and S1pr2-/-BMDMs. E. coli incubation efficiently induced membrane localization of IQGAP1in S1pr2-/-BMDMs but not in WT cells. Furthermore, there was a greater degree of co-localization between IQGAP1and Racl in S1pr2-/-cells after bacterial stimulation, consistent with the co-immunoprecipitation result that bacterial stimulation led to a greater quantity of Racl pulled down with IQGAP1in S1pr2-/-cells. Interestingly, a greater degree of IQGAP1tyrosine phosphorylation was also observed in S1pr2-/-cells. To determine the degree to which IQGAP1is required for enhanced phagocytosis in Slpr2-/-macrophages, IQGAP1was silenced with siRNA. Knockdown of IQGAP1resulted in negligible levels of Racl-GTP in both WT and S1pr2-/-BMDMs after E. coli stimulation, and reduced bacterial phagocytosis to a similar degree in both cell types.Conclusions:Our data demonstrate a ligand dose-dependent S1PR2signaling mechanism for regulating actin rearrangement and phagocytosis in macrophages that can account for the severity of pulmonary infection observed in WT mice during different stages of E. coli challenge. RhoA-dependent cell contraction was induced in high S1P concentration, while at low S1P concentration, IQGAPl-Racl-dependent lamellipodial protrusion was inhibited. Part4S1PR2expression levels and its correlation with severity of sepsisObjectives:Integrated with the above three parts’study, the absence of S1PR2enhances the phagocytosis of macrophages, ultimately improving the outcome. We next to identify whether S1PR2signaling may offer a promising therapeutic approach for the prevention and/or treatment of sepsis. S1PR2expression levels and its correlation with severity of sepsis were determined in critical ill patients.Methods:From Augest,2013to January,2015,28patients were admitted to the intensive care unit of the First Affiliated Hospital, College of Medicine, Zhejiang University and were considered for inclusion in this study if they met the criteria for severe sepsis or septic shock. Exclusion criteria for the current study included the following:with human immunodeficiency virus infection, treatment with corticosteroids or chemotherapy. Ten patients in the intensive care unit who did not show features of systemic inflammatory response syndrome or any evidence of infection were enrolled as nonseptic control patients. Clinical and demographic data were recorded within the first24h after diagnosis of severe sepsis or septic shock by two senior intensivists. Acute Physiologic and Chronic Health Evaluation Ⅱ (APACHE Ⅱ) scores, Sequential Organ Failure Assessment (SOFA) scores, and28-day mortalities were recorded for all patients. PBMCs were obtained by Ficoll density gradient centrifugation, and S1PR2mRNA levels were assessed by qPCR. Phagocytosis ability of PBMCs were also determined by fluorescence microcopy. Results:S1PR2mRNA levels were significantly higher in septic patients compared to those in non-septic controls (*P<0.05). Furthermore, increased S1PR2expression was positively correlated to the severity of sepsis, evaluated by Acute Physiologic and Chronic Health Evaluation Ⅱ (APACHE Ⅱ) scores (r=0.845). The relationship between S1PR2expression level and phagocytic function of PBMCs was also assayed in one non-septic control and three septic patients newly recruited. Significantly decreased phagocytosis of red fluorescent labeled E. coli was found in PBMCs collected from septic patients. Moreover, PBMCs with higher expression levels of S1PR2showed lower phagocytic ability, which further supports the positive relationship between S1PR2expression and severity of sepsis.Conclusions:In septic patients, elevated S1PR2levels in PBMCs were positively correlated with the severity of sepsis. Interventions targeting S1PR2signaling may serve as promising therapeutic approaches for sepsis.