Tim-3/Gal-9 Pathway On MDSCs In Normal Pregnancy And Pre-Eclampsia

Introduction:Maternal-fetal immune tolerance is a process that involves complex interactions of the immune system,and myeloid-derived suppressor cells(MDSCs)have emerged as one of the novel immuno-modulator in the maintenance of maternal-fetal immune tolerance.MDSCs are myeloid progenitor cells with immunosuppressive activities on both innate and adaptive cells through various mechanisms.Emerging evidence demonstrates the accumulation of MDSCs during healthy pregnancy to establish maternal-fetal immune tolerance,placentation,and fetal-growth process.In contrast,the absence or decreased MDSCs in pregnancy complications like preeclampsia,preterm birth,stillbirth,and recurrent spontaneous abortion has been reported.T cell immunoglobulin and mucin-containing protein-3(Tim-3)/Galectin-9(Gal-9)pathway regulates the function of various immune cells in the maternal-fetal interface.Nonetheless,the regulatory effects of Tim-3/Gal-9 signaling on MDSCs and its role in preeclampsia(PE)remain elusive.PE is a syndrome that is characterized by an inability of the trophoblast to adequately invade the decidual arteries leading to alterations in placental development,placental perfusion,and an insufficient transport of nutrients.It predominantly affects the brain,liver,and kidneys,and also causes aberrations in the clotting mechanism.Therefore,it represents a potential risk for hypertension and associated cardiovascular and cerebrovascular disease.It is a major cause of perinatal and maternal morbidity and mortality,and affects 2-8%of pregnancies,particularly in middle-and low-income countries.The incidence of PE shows remarkable variations among countries and ethnicities,with increased morbidity in developing countries such as Africa and Latin America.According to published data,morbidity due to PE declined from 2004 to 2016 in China.Therefore,to alleviate the burden of PE globally we need to diagnose it and treat it as early as possible.Here we made an effort to demonstrate that targeting MDSCs functions could positively influence the outcome of immunological pregnancy complications.Moreover,regulating the expression of Tim-3 provides a unique and novel target for clinical interventional immunotherapy for PE.Methods:To investigate the role of the Tim-3/Gal-9 pathway on MDSCs in pregnancy and PE,we used peripheral blood samples and placenta at term labor of healthy pregnancy(HP)and PE.The samples were immediately collected for the isolation of PBMCs.(1)We measured the frequencies of T cells(CD4~+T cells and CD8~+T cells),NK cells and their subsets,subsets of CD4~+T cells,CD8~+T cells,and myeloid cells in peripheral blood mononuclear cells of pre-eclamptic patients and healthy controls by flow cytometry.Then,Tim-3 molecules expression on the surface of T cells,NK cells,and myeloid cells was detected by flow cytometry.(2)We detected MDSC,G-MDSC,and M-MDSC cells by CD11b,HLA-DR,CD33,CD66b,and CD14 peripheral blood mononuclear cells of pre-eclamptic patients and healthy controls by flow cytometry.Then the expression of Tim-3 on each cell was also assessed.After that,placental tissues were used to detect Gal-9 protein expression by immunohistochemistry and Western blot analysis.Next,RNA isolation and q RT-PCR were done to detect the m RNA levels of Tim-3 in M-MDSCs and G-MDSCs.IFN-γand TGF-βcytokines were measured from serum samples.Proliferation assay was done on PBMC with 2.5μmol/L CFSE for 20 min in a 37?C water bath.For inhibition studies,Tim-3 antibodies were added to the proliferation assays at concentrations of 10μg/ml,5μg/ml,and 2.5μg/ml.Cells were stained with CD45 antibodies,CD4antibodies,and CD8 antibodies for 30 min,then they were washed and analyzed by flow cytometer.(3)Finally,we select MDSCs for further analysis to investigate their role in the pathogenesis of PE.We performed RNA sequencing of MDSC cells between HP and PE and find out which signal pathways were altered,and identify the gene expression which was in accordance with RNA sequencing.Differentially expressed genes(DEGs)were identified by using the DESeq algorithm.Then a further functional analysis of DEGs was done by GO function,KEGG pathway,Reactome,DO function,and Dis Ge NET enrichment analysis.Results:(1)Compared to healthy pregnant controls,in the peripheral blood of pre-eclamptic patients,there is an increase in the frequency of T cell subsets and myeloid cells but it didn’t reach the level of significance(p>0.05).The frequency of NK cells is decreased in PE but these results were not statistically significant.Tim-3 expression by the CD3~+T cells is higher in pre-eclamptic pregnancy and it reaches the level of statistical significance while Tim-3 expression by NK cells and myeloid cells was statistically non-significant.(2)There was no statistically significant differences in the proportions of CD4~+T and CD8~+T subsets:na?ve T cells,effector T cells,effector memory cells,and central memory cells(p-value>0.05)in the peripheral blood of HP and PE.The differences in CD158A~+CD56~+NK,NKG2A~+CD56~+NK,LILRB1~+CD56~+NK,NKp46~+CD56~+NK,and NKp44~+CD56~+NK are statistically insignificant(p>0.05).(3)The percentage of Tim-3~+M-MDSC cells in PE patients was significantly increased(p<0.01)than that in HP controls.The expression of Gal-9 proteins was found to be upregulated in the placenta of pre-eclamptic women.(4)The m RNA levels of Tim-3 in the M-MDSC of PE patients were significantly higher compared to that in HP controls.The concentrations of serum IFN-γin PE patients were raised,although there were no statistical differences between the two groups.While TGF-βdecreased significantly.(5)The proliferation of CD4~+T cells was strongly suppressed by MDSC,while MDSC adding anti-Tim-3 m Ab displayed dose-dependent suppression on the proliferation of T cells.Our data suggest that blocking Tim-3 could attenuate the inhibitory function of MDSC.(6)Of the total 23,122 genes expressed in PE and HP,12001 genes are DEGs in PE and 11121 genes are DEGs in HP.10,338 DEGs are found to be common in both PE and HP.292 DEGs were upregulated in PE while 45 DEGs were seen to be down-regulated in PE.Amongst the top 20 upregulated and downregulated DEGs;HLA-DRB1,HEY1,ANGPT1,HLA-DQA1,GCGR,MAS 1,and CYP19A1,etc.,genes are appeared to be associated with hypertensive disease.(7)GO analysis showed an inflammatory response,humoral immune response,and association with the basement membrane in PE MDSC cells.KEGG pathway enrichment analysis showed activation of the JAK-STAT signaling pathway in MDSCs of PE.Additionally,the involvement of ECM-receptor interaction and cytokine-cytokine receptor interaction suggest an immune response pathway associated with the MDSCs of PE.Reactome analysis showed signaling by interleukins,decreased expression of PD-1 signaling,and interferon-gamma signaling,etc.which also suggests an involvement of inflammatory response in the MDSCs of PE.Disease enrichment analysis indicated that the key upregulated PE-related genes might be involved in respiratory system disease,lung disease,lower respiratory tract disease,integumentary system disease,skin disease,and prostate cancer.Dis Ge NET enrichment pathway analysis suggests inflammatory response and defective artery remodeling in the MDSCs of PE.Conclusion:(1)Innate and adaptive immune responses play an important role in maintaining maternal-fetal immuno-tolerance.The dysregulation of the maternal immune system is a significant feature of symptomatic PE.For a successful pregnancy,a fine balance of memory T cells is imperative to induce tolerance toward fetal antigens.(2)Overexpression of Tim-3 results in a hyperimmune response leading to PE.Therefore,regulating the expression of Tim-3 provides a unique and novel target for clinical interventional immunotherapy for PE.In our study,there was an abnormal expression of Tim-3 on M-MDSC in PE patients.Hence,the immune tolerance at the maternal-fetal interface of PE patients was disrupted,and pro-inflammatory and anti-inflammatory cytokine get imbalanced.Moreover,blocking Tim-3 could affect the inhibitory function of MDSC in vitro assay.The relationship between Tim-3 and M-MDSC provides a new pathway to study the immunological pathogenesis of PE.(3)Analysis of DEGs in combination with the use of bioinformatics analysis divulged a variety of biological pathways that seem to be coordinately dysregulated in PE.The functional analysis showed an inflammatory response,humoral immune response,and activation of the JAK-STAT signaling pathway in MDSCs of PE.Moreover,this research implies that the development of non-invasive methods focused on the maternal PBMC transcriptome and/or proteome can be a productive future research area.