Expression Of Neuropilin-1Contributes To The Immunosuppressive Property Of CD4~+CD25~+Regulatory T Cells In Septic Mouse

Objective:l.To investigate the effect of different ratio of cecum ligation on neuropilin-1,which expressed on splenic regulatory T cell in mouse.2.To investigate the effect of lipopolysaccharides on the expression of splenic regulatory T cells’neuropilin-1,membrane-associated transforming growth factor-β,cytotoxic T lymphocyte-associated antigen-4and forkhead box-p3transcription factor,which is followed by various doses of neuropilin-1antibody.3. To investigate the effect of various doses of neuropilin-1antibody on CD4+CD25-T lymphocytes’proliferative activity,apoptosis,the expression and phosphorylation of Smad2Smad2transcription factor,whitch is mediated by regulatory T cell.Methords:1.60healthy male BALB/c mice were randomly divided into three different groups,according to the ratio of cecum ligation. After24hours, the expression of neuropilin-1and Foxp3were determined by FACS Calibur flow cytometry.2. CD4+CD25+Tregs were blocked with various doses of neuropilin-1antibody (0.5,5,10μg/ml) for24hours with anti-CD3/28and various doses of lipopolysaccharides(10,100,1000ng/ml)stimulation,the expression of neuropilin-1, membrane associated transforming growth factor-β,cytotoxic T lymphocyte-associated antigen-4and forkhead box-p3transcription factor were determined by FACS Calibur flow cytometry.3.CD4+CD25+Tregs were cultured with different doses of neuropilin-1antibody(0.5,5,10μg/ml)for1h, and co-cultured with CD4+CD25T cell for12,24and48h respectively, the proliferative activity of CD4+CD25-T cells was analysised by Spectra MR microplate reader, the apoptosis of CD4+CD25-T cells was analysised by FACS Calibur flow cytometry, the expression and phosphorylation of Smad2was analysised via Western blot.Results:1. Compared to control and sham groups,the survival rate of CLP mouse is becoming worse, along with the ratio of cecum ligation (P<0.01),under24hours;the expression of Foxp3is gradually increasing (P<0.01).compared to low-grade sepsis group, the expression of neuropilin-1was significantly increased in medium-and severe sepsis(P<0.01),there is no difference between medium-and severe sepsis(P<0.05).2.Compared to control and anti-CD3/CD28groups,the expression of neuropilin-1,membrane associated TGF-β,CTLA-4and Foxp3were significantly increased,along with the doses of LPS,and100ng/ml of LPS already has the maximal efficacy(P<0.05or0.01).compared to LPS+anti-CD3/CD28groups,the expression of neuropilin-1and membrane associated TGF-β were significantly reduced,along with the doses of anti-neuropilin-1(P<0.05or0.01),but there are no difference among CTLA-4and Foxp3(P>0.05).3.Ccompared to LPS+anti-CD3/CD28group, The normal Treg had the potential to inhibit the proliferation of CD4+CD25-T cells (P<0.05), while various doses of neuropilin-1antibody had the ability to reverse the immunosuppressive function of CD4+CD25+Treg, showing a dose-dependent response pattern,and5μg/ml of neuropilin-1antibody already has the maximal efficacy (P<0.05).compared to LPS+anti-CD3/CD28group, the normal Treg had the potential to promote the apoptosis of CD4+CD25-T cells at12hours,but there is no difference between them(P>0.05),while at24hours,further increasing the apoptosis of CD4+CD25-T cells(P<0.01),5μg/ml of neuropilin-1antibody had the ability to reverse the apoptosis of CD4+CD25-T cells(P<0.01).the level of β-actin was also designed as an internal control,the expression and phosphorylation of Smad2have no difference among control,sham and low-grade sepsis groups,but further increasing the expression and phosphorylation of Smad2in medium-and severe sepsis,the vitro exprement further prove that the normal Treg had the potential to promote the expression and phosphorylation of Smad2of CD4+CD25" T cells at48hours,and5μg/ml of neuropilin-1antibody had the ability to reverse this affection.Conclusions:1.The activity of Treg gradually increased,along with the ratio of cecum ligation,and the expression of neuropilin-1was significantly enhanced.2.The expression of membrane-associated TGF-β was significantly decreased,along with various doses of neuropilin-1antibody,but there are no effection on CTLA-4and Foxp3.3.Neuropilin-1involved in Treg mediated negative immuneregulation by effecting membrane associated TGF-β mediated immunosuppression.