The Role Of TRPM2 In Bacterial Clearance Of Macrophage And Its Molecular Mechanism

Part 1 The effect of TRPM2 on bacterial clearance of macrophage and sepsisObjectives:Transient Receptor Potential Melastatin 2?TRPM2?is a nonselective,calcium-permeable nonselective cation channel widely expressed in immune cells and tissues such as brain and lung.TRPM2 channels have been implicated in the development of various inflammatory diseases such as ulcerative colitis,myocardial ischemia/reperfusion injury,neuropathic pain,and autoimmune encephalomyelitis.This contributes to multiple cellular functions including cytokine production,insulin release,cell motility,and cell death.However,the role of TRPM2 regulation in the degradation of engulfed bacteria in macrophages remains unknown.Thus this part of the study was designed to investigate the effects of TRPM2 on the outcome of E.coli sepsis and bacterial clearance of macrophage.Methods:Sepsis was induced by intraperitoneal injection of E.coli on WT(Trpm2^+/+)and Trpm2^-/-mice.Mortality was assessed every 12 hours.To determine the bacterial burden,peritoneal lavage fluids were obtained at 2h,6h and 12h after E.coli challenged and detected by using agar plating method.Trpm2^-/-peritoneal macrophages and WT cohorts were cultured in vitro and then bacterial uptakes and intracellular bacterial burdens of peritoneal macrophages were detected.PBMCs were obtained and TRPM2 mRNA levels were quantified by qPCR.Bacterial uptakes and intracellular bacterial burdens of PBMCs were detected.Results:In a E.coli model,most of the Trpm2^-/-mice?85.0%?succumbed to sepsis within 36h after E.coli infection,while 45.5%of the wild-type(Trpm2^+/+)mice survived over 72h?**P<0.01?.Trpm2^-/-mice displayed a significant increased bacterial counts in peritoneal lavage fluids at 2h,6h and 12h after E.coli challenged?*P<0.05.**P<0.01?**p<0.01?.The gentamicin protection assay showed that after infection with live E.coli for 2 and 6 h,there were around 2.5-and 4-fold more bacteria,respectively,left in Trpm2^-/-peritoneal macrophages than in WT peritoneal macrophages.The defect in bacterial killing in Trpm2^-/-peritoneal macrophages could not be attributed to a difference in bacterial uptake,since intracellular bacterial burdens were comparable in Trpm2^-/-and WT peritoneal macrophages at 1h after E.coli infection.Moreover,PBMCs with higher TRPM2 mRNA level showed higher bacterial clearance,while lower TRPM2 mRNA level led to bad bacterial clearance.Conclusions:TRPM2 plays a critical role in improving the septic outcome via preventing the pathogen from spreading by macrophages.Part 2 The molecular mechanism of TRPM2 in facilitating bacterial clearanceObjectives:In the first part of study,we found that TRPM2 plays a critical role in improving the septic outcome via preventing the pathogen from spreading by macrophages.Thus,this part we want to explore the molecular mechanisms of eliminateing invaded E.coli involved in TRPM2 of macrophages.Methods:pHrodo-conjugated E.coli were used to monitored phagosomal pH until 100min in every other 20min after stimulation.Acidification of lysosomes in Trpm2^-/-peritoneal macrophages and WT cohorts were checked by using acridine orange.The maturation of the lysosomal hydrolase cathepsin D?CTSD?with or without E.coli stimulation were investigated by western blot analysis.Lysosomal acidification and fusion were further assessed by staining the cells with another fluorescent dye,Lyso-Tracker Red DND-99.Primary peritoneal macrophages were treated with Alexa Fluor???-conjugated E.coli,then colocalization of E.coli-containing phagosomes with lysosome marker LAMP-1 was evaluated using confocal microscopy.LAMP-1,CTSD and Rab7 stainings and protein expressions in isolated bead-containing phagosomes were evaluated by fluorescence confocal microscopy and western blot analysis.The dynamic fluorescence intensity changes of Rab5 and EEA-1 were observed by fluorescence confocal microscopy.With or without E.coli stimulation for 30min,immunoprecipitation of Rab5 and EEA-1 were performed in Trpm2^-/-peritoneal macrophages and WT cohorts to detected protein interaction.Results:There was no difference in phagosomal pH between Trpm2^-/-and WT peritoneal macrophages in the early stages of phagosomes.In contrast,60min later,the phagosomal pH in Trpm2^-/-peritoneal macrophages was lower than that in Trpm2^+/+peritoneal macrophages,and significant differences were observed at 80min and 100min?*P<0.05,**P<0.01?.No differences were observed in the number of red dots between Trpm2^-/-and WT peritoneal macrophages in steady-state conditions of acridine orange staining.The expression levels of mature CTSD showed no obvious differences between Trpm2^-/-and WT peritoneal macrophages with or without E.coli stimulation.No differences in Lysotracker-positive dots were detected between Trpm2^-/-and WT peritoneal macrophages under basic conditions.When the macrophages were incubated with Alexa Fluor???-conjugated E.coli,the majority of engulfed E.coli were contained inside phagosomes which efficiently fused with lysosomes in WT peritoneal macrophages.However,in Trpm2^-/-macrophages,only a minority of the E.coli-containing vacuoles acquired the Lysotracker dye.In Trpm2^-/-peritoneal macrophages,only 24.6%of the E.coli-containing phagosomes co-localized with LAMP1,significantly lower than that in WT peritoneal macrophages?66.0%,P<0.001?.Bead-containing phagosomes isolated from Trpm2^-/-peritoneal macrophages had lower LAMP-1,CTSD and Rab7 contents than those isolated from WT peritoneal macrophages analyzed by using fluorescence confocal microscopy or western blot.In the very early stage of phagosome maturation,phagosomes isolated from both WT and Trpm2^-/-peritoneal macrophages possess similar abilities to activate Rab5.During phagosome maturation,phagosomes isolated from WT peritoneal macrophages displayed a time-course dependent decrease in the level of Rab5,which paralleled a similar gradual reduction in the level of EEA1.On the contrary,in Trpm2^-/-peritoneal macrophages,a persistently high level of active Rab5 was detected in isolated phagosomes,which was accompanied by a steadily lowered level of EEA1.Interestingly,in Trpm2^-/-peritoneal macrophages,immunoprecipitation of Rab5 resulted in an obvious decrease in coimmunoprecipitation of endogenous EEA1 upon E.coli stimulation for 30 min.Conclusions:Disruption of TRPM2 did not directly affect lysosome acidification;Disruption of TRPM2 impaired phagosome-lysosome fusion;Disruption of TRPM2 impeded phagosome maturation via inhibiting the interaction of Rab5 and EEA-1.All the above indicated that TRPM2 absent impaired phagosome-lysosome fusion may via impeding phagosome maturation.Part 3 The Ca2+ influx regulated by TRPM2 is required for phagosome-lysosome fusionObjectives:Integrated with the previous two parts studies,we found that TRPM2 absent impeded phagosome maturation,increased bacterial burden in peritoneal lavage fluid,thereby aggravating the outcome of sepsis.We next to further explore whether TRPM2 could be a promising therapeutic target for treatment of sepsis.We adopted ionomycin to increased intracellular[Ca2+]i restricted by TRPM2 absent,and then observed the effects of phagosome-lysosome fusion snd the outcome of sepsis.Methods:Peritoneal macrophages were first cultured with medium containmng Ca2+ in the presence of 2?M ionomycin.Five minutes later,Ca2+ influx was recorded using the calcium indicator Fluo 3-AM.Plated macrophages were incubated with 3-?m latex beads coated with a crude preparation of E.coli outer membrane extracts for 60 min,then treated with 2?M ionomycin or vehicle for 5min.Additional 55min later,phagosomes were isolated by the density gradient ultracentrifugation.LAMP-1 staining and protein expression in isolated bead-containing phagosomes were evaluated by fluorescence confocal microscopy and western blot analysis.After treated with 2?M ionomycin or vehicle for 5min,immunoprecipitation of Rab5 and EEA-1 were performed in Trpm2^-/-peritoneal macrophages and WT cohorts to detected protein interaction with or without E,coli stimulation for 30min.Peritoneal macrophages were incubated with E.coli for 60min,then treated with 2?M ionomycin or vehicle for 5min.Bacterial killing by peritoneal macrophages were assessed using the gentamycin assay.Trpm2^-/-peritoneal macrophages were treated with 2?M ionomycin or vehicle control DMSO for 5min and then injected into the peritoneal cavity of Trpm2^-/-mice?5×105 cells per mice?2h before intraperitoneal challenge of E.coli.The 72h survival rates were assessed and bacterial burdens in the peritoneal cavity were determined 12h after injection of E.coli.Results:Ionomycin treatment elevated intracellular Ca2+ influx.Moreover,ionomycin treatment induced a strongly rising level of LAMP-1 in isolated phagosomes than that in vehicle treated macrophages.Increased intracellular Ca2+ of Trpm2^-/-peritoneal macrophages facilitated EEA1 recruitment to Rab5 after E.coli infection,as manifested by coimmunoprecipitation analysis.The inefficient bactericidal ability of Trpm2^-/-peritoneal macrophages was rescued following ionomycin treatment.Ionomycin-treated Trpm2^-/-peritoneal macrophages protected 66.7%of the mice against E.coli-induced sepsis while vehicle-treated Trpw2^-/-peritoneal macrophages only protected 28.6%of the mice?P<0.001?.Furthermore,as expected,the bacterial burden in the peritoneal cavity was significantly lower in mice adoptive transfer of ionomycin-treated Trpm2^-/-peritoneal macrophages than that in control mice at 12h after sepsis onset.Conclusions:Increased intracellular Ca2+ of Trpm2^-/-peritoneal macrophages contributed to phagosome-lysosome fusion;Increased intracellular Ca2+ of Trpm2^-/-peritoneal macrophages facilitated the interaction of Rab5 and EEA-1;Increased intracellular Ca2+of Trpm2^-/-peritoneal macrophages enhanced the bactericidal activity;Adoptive transfer of ionomycin-treated Trpm2^-/-peritoneal macrophages increased the survival rate and reduced bacterial burden.All the above conclusions inllustrated that TRPM2 promote phagosome maturation and phagosome-lysosome fusion through its calcium channel activity and hence degradate pathogens.