Expression And Regulatory Roles Of Chemokine CCL28at Maternal-Fetal Interface In Human Early Pregnancy

The chemokine superfamily includes at least58ligands which bind to19chemokine receptors. Most chemokines are proteins of67to127amino acid, and play a variety of biological roles through binding to the correspondent receptors. A chemokine may have at least one chemokine receptor, and one chemokine receptor may also bind to a plurality of chemokines as ligands. The interactions between chemokines and chemokine receptors form a complicated chemokine-receptor network and present various biological functions. The functions of chemokines and their receptor were studied originally in inflammation. It has been found that chemokines are not only involved in the migration of leukocytes to the inflammatory tissue, but also participate in the body development and tumor progress. Our previous studies have demonstrated the regulatory role of CXCL12/CXCR4, CXCL16/CXCR6and CCL2/CCR2at maternal-fetal interface. The maternal-fetal interface could be considered as specialized mucosa, and thus our present study focused on the role of CCL28and its receptor CCR10at the maternal-fetal interface.Part1. Expression and Regulation of CCL28at Human Maternal-fetal InterfaceObjective To investigate whether CCL28is expressed and its expression regulation at human maternal-fetal interface.Methods Expression of CCL28in decidua and villi from normal early pregnancy was detected by immunohistochemistry, and immunocytochemistry was used to evaluate CCL28expression in primary decidual stromal cells, decidual epithelial glandular cells and trophoblasts. RT-PCR and In-cell western were performed to further confirm the expression of CCL28in decidual stromal cells. Effects of pro-inflammatory cytokines, estrogen and progesterone on the expression of CCL28in decidual stromal cells were investigated in vitro.Results Immunohistochemistry showed that the villous and decidual tissues from normal pregnancy were positively stained with anti-CCL28antibody. The primary DSCs, DECs and trophoblasts were also positively stained for CCL28by immunocytochemistry. RT-PCR and In-cell western further confirmed the expression of CCL28in DSCs. IL-1β, TNF-α, IFN-γ and IL-17A up-regulated the CCL28expression in DSCs. Estrogen and progesterone had no regulatory effect on the expression of CCL28in DSCs.Conclusions Maternal-fetal interface constitutively expresses CCL28. The production of CCL28in DSCs is up-regulated by pro-inflammatory cytokines.Part2. CCL28induces apoptosis of DSCs via binding to CCR3and enhances the invasion of DSCs through binding to CCR10and CCR3Objective To probe into the effect of CCL28on the biological behavior of human first-trimester decidual stromal cells.Methods Flow cytometry was used to analyze expression of CCR10and CCR3on decidual stromal cells either from normal pregnancy or from unexplained miscarriage. The effect of rhCCL28on the viability of human first-trimester deciduaf stromal cells was evaluated by CCK8assay. The invasion of decidual stromal cells was observed by matrigel invasion assay after various concentrations of rhCCL28treatments and anti-CCR10/3neutralized antibody. DSC apoptosis was investigated by Annexin V/PI staining and flow cytometry. CCR10and CCR3expression on decidual stromal cells following different pro-inflammatory cytokines treatments was analyzed by flow cytometry. The secretion of pro-inflammatory cytokines of DSCs from normal or unexplained miscarriage was determined by ELSIA.Results Increased CCR3/CCR10expression was observed in DSCs from miscarriage than that of the normal pregnancy. The rhCCL28had no effects on the viability of DSCs, but promoted DSC apoptosis, which was eliminated by pretreatment with neutralizing antibodies against CCR3/CCR10or CCL28. The rhCCL28enhanced the invasiveness of DSCs through CCR10and CCR3. Recombinant human CCL28and IL-1β up-regulated expression of CCR10in DSCs, and CCR3expression was increased after IL-1β or IL-17A treatment. DSCs from unexplained miscarriage secreted higher level of IL-1β, TNF-α and IL-17A.Conclusions CCL28promotes apoptosis of DSCs mainly through CCR3, and CCL28-CCR10/3interaction is involved in invasion of DSCs. Up-regulation of CCR10and CCR3on DSCs is found in the unexplained miscarriage. IL-1β promotes the expression of CCR10and CCR3in DSCs while IL-17A does only up-regulate the CCR3expression. DSCs from unexplained miscarriage secrete more IL-1β、TNF-α and IL-17A.Part3. Characterization of human first-trimester decidual CCR10+TregsObjective To determine the phenotype of CCR10+decidual Treg.Methods The presence of peripheral and decidual CCR10+Treg was analyzed by flow cytometry. Expression of a series of cell-surface molecules including chemokine receptors and integrins in CCR10+decidual Tregs was analyzed by using flow cytometry. Phenotypic difference between CCR10+and CCR10-Treg was further analyzed.Results10%CCR10+Treg was observed in periphery or decidual Tregs. Increased CCR10+decidual Tregs was observed in unexplained miscarriage. Compared to CCR10Treg, CCR10+decidual Tregs expressed higher level of CTLA4, TGF-β1, IL-10, CD45RA, CCR7, CXCR3, CXCR6, CD103, CD29and αVβ5. Intriguingly, CCR10+decidual Tregs expressed lower level of CXCR4and Foxp3.Conclusions Decidual Treg contains a subset of CCR10+Treg which is increased in the unexplained miscarriage. Decidual CCR10+Treg has an unique phenotype.Part4CCL28and SDF-1recruit Tregs and regulate the expression of CCR10/3and Foxp3in TregsObjective To investigate the recruitment of Treg by CCL28and SDF-1and to analyze the expression regulation of CCR10, CCR3and Foxp3in Treg.Methods We first analyzed the chemokine expression profile of Tregs from the periphery and decidua. Chemotaxis assay was used to evaluate the migration of Treg to CCL28and the CCR10+Treg migration to SDF-1. We also isolated and expanded Tregs in vitro which were treated by rhCCL28, and then Foxp3, CCR10, CCR3, CD39and CTLA4expression was detected by flow cytometry.Results Tregs from periphery and decidua expressed CXCR4, CXCR6. Decidual Tregs expressed higher level of CCR5, CCR6, CCR9, CXCR1, CXCR2and CXCR3compared to periphery Tregs. Either CCL28or SDF-1could recruit Tregs, but SDF-1mainly recruited CCR10Tregs. CCL28decreased the Foxp3expression while increased CCR10and CCR3expression in Tregs. No effect of CCL28was observed on the CD39and CTLA4expression in Tregs.Conclusions Decidual Tregs are a heterogeneous population and express a variety of chemokine receptors. SDF-1mainly recruits CCR10Tregs, and CCL28recruits Tregs and down-regulates their Foxp3expression. CCR10and CCR3expression is also up-regulated by CCL28. CCL28is a good complement of SDF-1in terms of recruitment of CCR10+Treg.