The Role And Mechanism Of MSC In Modulating Skewed Treg/Th17 Induced By Lipopolysaccharide And Hypoxia

Part One:Characters of whole blood gene expression involved with T lymphocytes differentiation in patients of severe sepsis with high lipopolysaccharide levels in plasma and hypoxiaObjective:To explore T lymphocytes differentiation-related gene expression in patients of severe sepsis with high lipopolysaccharide(LPS)levels in plasma and hypoxia by whole blood gene transcription analysis.Methods:The clinical study retrospectively included immunocompetent patients with community-acquired severe sepsis.Patients were stratified as control group with LPS?10pg/ml and P/F>200mm Hg,and LPS-hypoxia group were with LPS>10pg/ml and P/F?200mm Hg.The data related to presumed etiology,organ injury and supportive therapy and disease severity were collected.Whole blood samples were collected within 24 hours of admission and microarray assay(Human Lnc RNA Microarray V4.0)was performed.The database of NCBI,GO and STRING were utilized for differentially expression(DE)analysis and enrichment analysis involved in T cell related biological process.Results:(1)Baseline information:Patients in LPS-hypoxia group(n=9)were characterized with high lipopolysaccharide(LPS)levels in serum and hypoxia(LPS:66.25[16.05,97.48]vs.5.00[5.00,6.38]pg/ml,P=0.003;Pa O₂/Fi O₂:129±62 vs.346±55 mm Hg,P<0.001),compared to those of control group(n=6);(2)Clinical presentation and organ injury:LPS-hypoxia patients presented significantly higher levels of serum creatine(164[79,332]vs.60[55,105],P=0.017),higher levels of serum lactate(2.2[1.5,3.4]vs.1.2[0.8,2],P=0.039)and higher SOFA scores than those without at admission(11±4 vs.7±1,P=0.046),indicating a higher illness severity;(3)Inflammatory and immune indicators:Compared to control group,LPS-hypoxia patients held a lower lymphocyte count(0.4[0.3,0.6]vs.0.8[0.5,1.2],P=0.020)and higher N/L ratio(17.9[7.1,23.4]vs.6.8[2.9,12.3],P=0.050).There was no statistical difference between two groups in terms with systemic inflammatory response presentations(heart rate,respiratory rate,white blood cell count or systolic blood pressure)as well as neutrophil count,hs-CRP,PCR and IL-6;(4)Go functions related to cell response to hypoxia were found to be significantly up-regulated in septic patients with LPS-hypoxia,compared to controls(P<0.05).(5)Bio informative analysis of whole blood gene transcription involved with T lymphocytes differentiation:Compared to control group,LPS-hypoxia group held altered gene expression,favouring Th17 cell differentiation,rather than that of Treg,which included significant upregulation of STAT3(P=0.006),BATF(P=0.014),TLR2(0.002),FABP5(P=0.016)and IL6ST(P=0.046)along with the decreased expression of negative gene signatures:STAT5A(P=0.045)and PPARG(P=0.049)while TET3,as one of Treg gene signatures was observed pronouncedly inhibited(P=0.004).Conclusion:Patients characterized with high plasma LPS and hypoxia among severe sepsis held a higher disease severity,more complicated organ injuries and decreased peripheral lymphocyte count,of which population activation of genes expression related to Th17differentiation and inhibition of those related to Treg differentiation indicated differentiation favouring to Th17 subpopulation,rather than Treg subset,leading to the Treg/Th17 skewing.Part Two:Effects of mesenchymal stem cells treatment on Treg/Th17 differentiation under hypoxic and LPS-stimulated conditionObjective:To set up an ex-vivo CD4⁺T cells model of LPS-hypoxia-induced Treg/Th17skewing and explore effects of mesenchymal stem cells treatment on Treg/Th17 skewing under hypoxic and LPS-stimulated condition.Methods:Purified CD4⁺T cells(5×10⁶)isolated by positive CD4 selection from mice spleen were seeded into a well of the 24-well plate pre-coated with anti-CD3(5ug/ml)and anti-CD28(2ug/ml)in an atmosphere of 5%CO2 at 37?and treated overnight.RAW264.7 cells(10⁶)were added as antigen presenting cells(APCs).T cells with and without RAW264.7 cells were treated with various LPS concentrations of 0,10,100,1000ng/ml or/and at hypoxia condition of5%O2.CD4⁺T and RAW264.7 cells were seeded in a 24-well plate precoated with anti-CD3and anti-CD28,set as the control group.Based on LPS(100ng/ml)and hypoxia condition(5%O2)as stimuli,treatment groups were set with MSCs in direct coculture or indirect coculture methods by transwell system.After 48 hours,cells were collected for the following measurements:(1)T cells survival rate by Annexin V staining assay;(2)Treg frequency identified with positive expression of CD25 and Foxp3 and T helper 17(Th17)frequency identified with negative expression of CD8 and positive expression of IL-17A by flowcytometry.Results:(1)Effects of LPS stimulation on CD4⁺T cells:Compared with controls,CD4⁺T cells survival rates and Treg frequency were decreased significantly(P<0.05)while Th17frequency was increased markedly in groups with LPS stimulation at 10 and 100ng/ml(P<0.05).In groups of LPS stimulation at 100ng/ml and 100ng/ml,no differences were detected in terms of T cell survival rate and frequency of Treg and Th17.Treg/Th17 ratio values were significantly higher in groups with LPS stimulation than that of control group(P<0.05).(2)Effects of hypoxia stimulation on CD4⁺T cells:There was no difference in terms of CD4⁺T cells survival rates between control and hypoxia groups.Compared with control group,Treg frequency was decreased significantly while that of Th17 held a pronounce increase(P<0.05)in hypoxia group.(3)Effects of LPS stimulation in combination with hypoxia on CD4⁺T cells:Compared with control group,T cells survival rates and Treg frequency were decreased significantly in groups with LPS stimulation combined with hypoxia(P<0.05)while Th17percentage was increased significantly(P<0.05),which were lower than that in groups with LPS stimulation alone.Compared to control group,Treg/Th17 ratio values were significantly evaluated in groups with LPS stimulation in combination with hypoxia(P<0.001).At LPS dose of 10ng/ml,Treg/Th17 ratio values were significantly lower in group with LPS stimulation combined with hypoxia than that with LPS alone while no differences were observed between groups with LPS alone versus with LPS combined with hypoxia at other doses.(4)Effects of MSCs on the skewed Treg/Th17 induced by LPS and hypoxia:Compared to LPS-hypoxia stimulated group,MSCs treatment could significantly restore the Treg/Th17 differentiation with increasing Treg frequencies and decreasing Th17 frequencies well as decreased values of Treg/Th17 induced by LPS and hypoxia(P<0.05).No differences were observed between groups with direct and indirect MSCs treatment.Conclusion:LPS-hypoxia stimulation could induce Treg/Th17 skewing.MSCs could restore the skewed Treg/Th17 induced by LPS and hypoxia,which was independent on cell-to-cell contact mechanism.Part Three:The mechanism of mesenchymal stem cells on restoring the skewed Treg/Th17 induced by LPS and hypoxiaObjective:To explore the mechanism of mesenchymal stem cells modulating the skewed Treg/Th17 induced by LPS and hypoxiaMethods:CD4⁺T cells in the lower chamber of trans well insert were cocultured with MSCs on the upper layer,receiving LPS(100ng/ml)and hypoxia(5%O2)stimulation.Anti-TGF-?1neutralization antibody was added to explore the role of TGF-?1 among the soluble factors secreted by MSCs;In addition,mi R-155 overexpression of CD4⁺T cells were performed by transfection for 4 hours and then were added to the MSCs-CD4⁺T cells coculture system in hypoxic and LPS-stimulated condition.After 48 hours,cells or supernatant were collected for measurement as followings:(1)Levels of pro-inflammatory cytokines of INF-?,IL-17A and IL-21 and anti-inflammatory cytokines of TGF-?1 and IL-10 in culture medium by ELISA;(2)Levels of mi R-155,Rorc,Foxp3 and Ptpn2 m RNA expression of CD4⁺T cells by RT-PCR;(3)Frequency of Treg subset identified with positive expression of CD25 and Foxp3 and of Th17with negative expression of CD8 and positive expression of IL-17A by flowcytometry;(4)T cells survival rates by Annexin V/PI staining;(5)Cell proliferation capacity by CTV staining.Results:(1)Effects of MSCs on the soluble factors secretion in hypoxic and LPS-stimulated condition:Compared to groups without MSCs,the levels of cytokines of TGF-?1,IL-10 and IL-17A were significantly increased following MSCs treatment(P<0.05);(2)Effects of TGF-?1 neutralization on MSCs regulating the skewed Treg/Th17 induced by LPS and hypoxia stimulation:Compared to group of MSC treatment,TGF-?1 neutralization could significantly inhibit the effects of MSCs restoring the skewed Treg/Th17(P<0.05),reversing Th17 phenotypic suppression and induction of Treg phenotype by MSCs.(3)Effects of MSCs-secreted TGF-?1 on mi R-155 expression of CD4⁺T cells in LPS and hypoxia-stimulated condition:MSCs treatment could attenuate the increased expression of mi R-155 in hypoxic and LPS-stimulated CD4⁺T cells(P<0.05)while the addition of TGF-?1 neutralization abolished the effect of MSCs treatment.(4)Effects of mi R-155 expression of CD4⁺T cells on MSCs-secreted TGF-?1 regulating Treg/Th17 skew in hypoxic and LPS-stimulated condition:Mi R-155 overexpression inhibited effects of MSCs-TGF-?1 restoring Treg/Th17skew induced by LPS and hypoxia.Moreover,upregulation of Th17 phenotypic transcriptional factor Rorc m RNA(P<0.05)and downregulation of Treg phenotypic transcriptional factor Foxp3 and Ptpn2 m RNA(P<0.05)were observed in group of mir-155 overexpression in comparison with control.(5)Effects of modulating mi R-155 expression on CD4⁺T cells with MSCs-secreted TGF-?1 treatment in hypoxic and LPS-stimulated condition:The proliferation capacity and survival rates of T cells was comparable in various treatment groups regardless of transfection with mi R-155 mimic or inhibitors(P>0.05).Conclusion:TGF-?1 is one of mediators underlying paracrine mechanism of MSCs restoring the skewed Treg/Th17 induced by LPS and hypoxia stimulation,which suppressed mi R-155expression of CD4⁺T cells,increased expression of Treg phenotypic transcriptional factor Foxp3 and Ptpn2 m RNA and inhibited expression of Th17 phenotypic transcriptional factor Rorc m RNA.