The Role Of Cholecystokinin Octapeptide In The Regulation Of Treg Function By CDC

n Treg cells mainly take part in suppressing the proliferation of T cells and inflammatory response, and occupy about 5-10% of peripheral CD4+T cells and play important roles in the maintenance of immune homeostasis. Treg dysfunction often leads to severe autoimmune disease and even death. It is important to investigate the regulatory mechanism of Treg function for discussing the related pathogenesis of autoimmunity.Myeloid dendritic cells is the conventional classic DC(c DC),and mainly participate in the induction and irritate of immune response. It is confirmed that c DC play an critical role in n Treg response. According to the ability of stimulating T cells, DC has two states of immature(immature DC, i DC) and mature(mature DC, m DC). i DCs are characterized by a high capacity for antigen uptake, but they can not induce primary immune responses. In the presence of inflammatory signals, they rapidly take up foreign antigens and undergo a differentiation/maturation process that down-regulate further antigen- processing capacity, but enhance their expression of major histocompa-tibility complex class I and II and co-stimulatory molecules, and the production of inflammatory cytokines important for successful antigen presentation. They then migrate to the lymphoid organs where they gain a high capacity to present antigens and consequently become m DCs. It has confirmed that c DC can indunce Treg proliferation and then abolish Treg anergy. These results showed that the regulatory of Treg cells by DC is related to the maturity states of DC. Therefore, the regulatory mechanism research of phenotypic and function of DCs will provide new ideas for the prevention and treatment of autoimmunity that related with Treg function imbalance.Cholecystokinin(cholecystokinin, CCK) is atypical brain gut peptide andimmune regulatory peptide. Cholecystokinin octapeptide(CCK-8) is thebiologically predominant active form of CCK. As an immune regulator, CCK-8 exerts immune regulatory effects through activating its CCK receptors(CCKR) on cell surface. CCKR conclude CCK1 R and CCK2 R, which belong to G protein-coupled receptor(GPCR) superfamily. CCK1 R mainly expresses in the gastrointestinal system and CCK2 R expresses in the stomach and the central nervous system. CCKR has a variety of signaling pathways, including CCKR and Gs protein-coupled c AMP-PKA pathway, coupled with Gα DAG-PKC pathway, the interaction between the various signaling pathways and ultimately exert biological effects. Domestic and international researches and our previous studies have shown that CCK-8 has anti-inflammatory and can regulate immune cell differentiation, chemotaxis and immune killing and so on, however, there are still lots of questions about immune regulation and mechanisms. Our recent study have shown that CCK-8 can inhibite the expression of CD80 and CD86 on human peripheral blood p DC induced by CD40 L and Cp G ODN and allogeneic CD4+T cell proliferations. Otherwise human peripheral blood c DCs also express CCKR. Therefore, we speculated that CCK-8 might regulate Treg function by modulating peripheral blood c DC.According to the results of above researches, in this study, we used puried CD14+ monocytes from human peripheral blood and in vitro differentiated the cells under i DC and m DC-plolarizing conditions, and investigated the effect of CCK-8 on the regulatory of phenotype of different states DCs and the effect of CCK-8 treated DC with different states on Treg function, and further discussed the molecular mechanism and receptor mechanism in this actions of CCK-8. In addition, we explored the modulation of CCK-8 on the differentiation and suppressor function of Tregs affected by PGE2 and thereby further observed immunomodulatory effects of CCK-8.Part 1 Effect of CCK-8 on the phenotypic of human peripheral blood c DCObjective: In this part, we differentiated CD14+ monocytes under i DC and m DC of c DC polarizing conditions in vitro, and determined the effects of CCK-8 on the phenotype of i DC and m DC.Methods:1 CD14+ monocytes were isolated by MACS selection from healthy human peripheral blood, and the purity of the sorted cells was determined by flow cytometry analysis. CD14+ monocytes were cultured with GM-CSF and IL-4 for 6d after MACS selection, and the cells were i DCs. The positive cells of CD14-CD1a+ cells was determined by flow cytometry analysis. Then the cells were stimulated with LPS for 48 h, and we obtained m DCs.2 Effects of CCK-8 on the expression of DC-sign CD209 and CD83 on i DCs and m DCs: CD14+ cells were cultured under i DC or m DC polarizing conditions and were divide into control group, CCK-8 group(10-6,10-7, 10-8, 10-9, 10-10 M). Using flow cytometry analysis, we evaluated the expression of DC-sign CD209 and CD83 and using RT-PCR, we measured the CD209 and CD83 m RNA expression.3 Effects of CCK-8 on the expression of CD80, CD86, HLA-DR and GITR-L on i DC and m DC: CD14+ cells were cultured under i DC or m DC polarizing conditions and were divide into control group, CCK-8 group(10-6,10-7, 10-8, 10-9, 10-10 M). Using flow cytometry analysis, we evaluated the expression of CD80, CD86, HLA-DR and GITR-L.Results:1 The purity of the sorted cells was > 90%, and the positive cells of CD1a+CD14- cells induced by GM-CSF(100ng/ml) and IL-4(50ng/ml) from CD14+ monocytes were reached(62.45±10.63)%. It is proved that we successfully induced DC generation.2 The expression of DC-SIGN CD209 and CD83 on i DC was lower and the expression by m DC was higher than i DC(P<0.05). CCK-8 dose-dependently inhibited the expression of DC-SIGN CD209 and CD83 and the m RNA expression, peaking at 10-6M. 10-6M and 10-7M of CCK-8 groups had significant difference compared with control group(P<0.05). 3 The expression of CD80, CD86, HLA-DR and GITR-L on i DC was lower, compared with m DC. And the expression of CD80, CD86, HLA-DR and GITR-L on m DC was higher(P<0.05). CCK-8 dose-dependently inhibited the expression of CD80, CD86, HLA-DR and GITR-L, peaking at 10-6M.10-6M and 10-7M of CCK-8 groups had significant difference compared with control group(P<0.05).Summery: These results indicated that CCK-8 can suppress the expression of DC-SIGN CD209, CD83, CD80, CD86, HLA-DR and GITR-L and showed that CCK-8 has inhibition on i DC and m DC phenotype. There is another state DC that semi-mature DC which is tolerogenic DC(t DC) in addition to i DC and m DC. It expressed lower levels of phenotype molecules than m DC. The above results suggested that CCK-8 can induced semi-mature DC or t DC.Part 2 Effect of CCK-8 on the regulation of Treg function by c DCObjective: The results of the first part indicated that CCK-8 can inhibit m DC phenotype, but induce t DC generation and suggested that it is complicated that CCK-8 in the regulation of c DC phenotype and function.As CD80/CD86 and GITR-L are costimulatory molecules of modulating Treg cells,so we speculated that CCK-8 might affect Treg function by regulating DC. But how CCK-8 impact Treg function by c DC, we observed the role of CCK-8 educated DC in Treg function by coculturing DC and Treg, in order to further reveal the immune regulatory effect of CCK-8.Methods:1 CD4+CD25+Treg cells and CD8+T cells from human peripheral blood were isolated by MACS selection, and the purity of the sorted cells was determined by flow cytometry analysis.2 The effect of CCK-8 educated i DC/m DC on the expression of Surface molecules of Treg cells and the production of cytokines secreted by Treg cells: The culture method of DC was the same as the first part. The cultured i DC/m DC were collected and treated with 50μg/ml MMC for 0.5 hour at 37°C, then added to purified Treg cells in 24-wells for 48 h with the proportion 1:5(1×105:5×105), the expression of CTLA-4 and GITR on Treg cells were determined by folw cytometry analysis and the production of TGF-β and IL-10 were detected by ELISA.3 The effect of CCK-8 educated i DC/m DC on the proliferation of Tregcells: The cultured iDC/mDC were collected and treated with 50μg/ml MMC for 0.5 hour at 37°C, then added to CFSE labeled-Treg cells with the proportion 1:5(1×105:5×105), 5d later, the proliferation of Treg cells were determined by folw cytometry analysis.4 The effect of CCK-8 educated m DC on inhibitory of Teff proliferation by Treg cells: The cultured i DC/m DC were collected and treated with 50μg/ml MMC for 0.5 hour at 37°C, then added to Treg cells and Teff with the proportion 1:5:5(1×105:5×105:5×105), 4d later, the proliferation of Teff were determined by Brd U incorporation.Results:1 The purity of the sorted CD4+CD25+ Treg cells and CD8+T cells were both > 90%.2 CCK-8 educated m DC upregulated the expression of CTLA-4 and GITR and the production of IL-10 and TGF-β by Treg cell and the difference was statistically significant(P<0.05), however, CCK-8 educated i DC had no effect on the expression of CTLA-4 and GITR and the production of IL-10 and TGF-β by Treg cell.3 CCK-8 educated m DC upregulated the proliferation of Treg cells and the difference was statistically significant(P<0.05), however, CCK-8 educated i DC had no effect on the proliferation of Treg cells.4 CCK-8-educated m DC play a positive role in the inhibitory of Teff proliferation by Treg cells. The difference was statistically significant between CCK-8 group and control group(P<0.05).Summary: These results indicated that CCK-8 educated m DC can upregulate Treg suppressor function and Treg proliferation. Because CCK-8 can inhibit m DC phenotype, we speculated that the effects of CCK-8 were related to the generation of t DC. However, i DC or CCK-8 educated i DC had no effect on Treg suppressor function and proliferation.Part 3 The mechanism of CCK-8 educated c DC in the Treg functionObjective: Since the first part and the second part of the study has confirmed that CCK-8 inhibited m DC phenotype, but upregulated Tregfunction affected by DC and this actions might be related to the generation of t DC. CD80/86 and GITR-L were the costimulatory molecules associated with Treg function, so we used anti-CD80/CD86 and anti-GITR-L to investigate the effect of Treg function of CCK-8 induced t DC, and further confirmed that whether the regulation of Treg function by CCK-8 induced t DC achieved by mudulating the expression of CD80/CD86 and GITR-L on DC. And we also investigated the receptor role of CCK-8 in the regulation of Treg function by m DC.Methods:1 Effect of anti-CD80, anti-CD86 and anti-GITR-L on the regulation of Treg function by CCK-8 induced t DC: CTLA-4 groups: control group, CCK-8 group, anti-CD80+CCK-8 group, anti-CD86+CCK-8 group and anti-CD80+anti-CD86+CCK-8 group. GITR groups: control group, CCK-8 group, anti-GITR-L+CCK-8 group. Cells were cocultured for 48 h and the expression of CTLA-4 and GITR were detected by folw cytometry analysis and the production of TGF-β and IL-10 were detected by ELISA.2 Effect of anti-CD80, anti-CD86 and anti-GITR-L on the regulation of Treg proliferation by CCK-8 induced t DC: groups: control group, CCK-8 group, anti-CD80+CCK-8 group, anti-CD86+CCK-8 group, anti-CD80+ anti-CD86+CCK-8 group, anti-GITR-L+CCK-8 group. Cells were cocultured for 5d and the proliferations of Tregs were detected by CFSE.3 Effect of anti-CD80, anti-CD86 and anti-GITR-L on the regulation of Treg suppressing Teff proliferation by CCK-8 induced t DC: groups: DC+Treg+Teff group;CCK-8+DC+Treg+Teff group;anti-CD80+CCK-8+ DC+Treg+Teff group;anti-CD86+CCK-8+DC+Treg+Teff group;anti-CD80/ anti-CD86+CCK-8+DC+Treg+Teff group;anti-GITR-L+CCK-8+ DC+Treg+ Teff group. Cells were cocultured for 4d and the proliferations of Teff were detected by Brd U incorporation.4 The role of CCKR antagonists in the regulation of CD80, CD86 and GITR-L on m DC: groups: control group, CCK-8 group, CCK1 R antagonist(Devazepid) group and CCK2 R antagonist group(L 365260). Cocultured cellswere collected and detected the expression of CD80, CD86 and GITR-L by folw cytometry analysis.5 Effect of CCKR antagonists on the regulation of Treg function by CCK-8 induced t DC: cells were cultured under the above conditions, and treated with 50μg/ml MMC for 0.5h at 37°C and cocultured with Tregs for 48 h. The expression of CTLA-4 and GITR were detected by folw cytometry analysis and the production of TGF-β and IL-10 were detected by ELISA.6 Effect of CCKR antagonists on the regulation of Treg proliferation by CCK-8 induced t DC: DC and CFSE labeled Tregs were cocuultured under the above conditions for 5d. The proliferations of Tregs were detected by folw cytometry analysis.7 Effect of CCKR antagonists on the regulation of Treg suppressing Teff proliferation by CCK-8 induced t DC: DC, Tregs and Teffs were cocultured with the proportion 1:5:5(1×104:5×104: 5×104) for 4d and the proliferations of Teff were detected by Brd U incorporation.8 Effect of CCK-8 on PKA/PKC activity in m DC during its mature and activation: monocytes were collected and cultured under mature and activity culture conditions and divided into: control group, CCK-8 group, Devazepid+CCK-8 group, L 365 260+CCK-8 group and Devazepid+ L 365 260+CCK-8 group. Cultured cells were collected and extracted cellular proteins. PKA/PKC activity in m DC were dectected by a Protein Kinase A Activity Assay Kit and a Protein Kinase C Activity Assay Kit.Results:1 anti-CD80 and anti-CD86 alone or both can partly block the effect of CCK-8 induced t DC on the expression of CTLA-4 on Treg cells. Anti-GITR-L reversed the effect of CCK-8 induced t DC on the expression of GITR. Every anti-body alone or all couldn’t regulated the effect of CCK-8 induced t DC on the production of TGF-β and only anti-CD80 and anti-CD86 act together can downregulate the production of IL-10 modulated by CCK-8 induced t DC(P<0.05).2 Both anti-CD80 and anti-CD86 had no effect on Treg proliferationmodulated by CCK-8 induced t DC, and the two together can inhibit the effect of CCK-8 induced t DC, and anti-GITR-L also can downregulate the effect of CCK-8 induced t DC on Treg proliferation(P<0.05).3 anti-CD80 and anti-CD86 had no effect on the inhibitory of Teff of Treg cells modulated by CCK-8 induced t DC, but anti-CD80/CD86 together downregulated the inhibitory of Treg cells by CCK-8 induced t DC, so is anti-GITR-L(P<0.05).4 L 365260 or L 365260 and Devazepid can reversed the downregulated the expression of CD80, CD86 and GITR-L on m DC by CCK-8(P<0.05), but not Devazepid(P>0.05).5 L 365260 or L 365260 and Devazepid can block the effect of CCK-8 induced t DC on the expression of CTLA-4 and GITR and the production of TGF-β and IL-10 by Treg cells(P<0.05), but not Devazepid(P>0.05).6 L 365260 or L 365260 and Devazepid can block the effect of CCK-8 induced t DC on Treg differentiation(P<0.05), but not Devazepid(P>0.05).7 L 365260 or L 365260 and Devazepid can reverse the inhibitory of Teff proliferation by Treg cells moudulated by CCK-8 induced t DC(P<0.05), but not Devazepid(P>0.05).8 CCK-8 can increase the activity of PKA but decrease the activity of PKC by CCK2R(P<0.05), but not CCK1R(P>0.05).Summery:These results indicated that the application of anti-CD80, anti-CD86 and anti-GITR-L further determined the role of CCK-8 modulated DC in Treg function. CCK-8 affected Treg function by the expression of CD80/CD86 and GITR-L. CCK-8 modulated the expression of CD80, CD86 and GITR-L on DC by CCK-2R, and then regulated Treg function by this receptor on DC, and PKA and PKC involved in this actions of CCK-8 on DC.Part 4 The role of CCK-8 in the regulatory of Treg differentiation of PGE2Objective: DC can secreted a large numbe of inflammatory mediators PGE2 in the stimulation of viruses or other pathogens and CCK-8 hasanti-inflammatory effects, so we give the exogenous PGE2 and investigated PGE2 on the differentiation of human peripheral blood Treg cells differentiation from na?ve CD4+T cells and the related mechanism and the role of CCK-8 in the regulatory of Treg differentiation and function affected by PGE2 and in order to reveal the anti-inflammatory of CCK-8 in immune system.Methods:1 Involvement of EP2 and EP4 receptors in the actions of PGE2: na?ve T cells were cultured under Treg differention. We first investigated the involvement of EP agonists, the experiments were divided into control group, Butaprost group, Sulprostone group and L-902,688. We then used AH6809, an EP2 antagonist, and GW627368 X, an EP4 antagonist, to verify the above results. The experiments were divided into control group, PGE2 group, AH6809+PGE2 group, GW627368X+PGE2 group. The cells were cultured for 7 days, the proportion of CD25+Foxp3+ cells were analyzed by flow cytometry and the expression of Foxp3 m RNA were measured by RT-PCR.2 Effects of PGE2 on the production of c AMP and the activity of PKA in Treg differentiation: Seven-day ploarised Treg cells were assayed for c AMP accumulation using a LANCETM c AMP Kit and PKA activity using a Protein Kinase A Activity Assay Kit.3 The role of c AMP/PKA signaling in the actions of PGE2: we used db-c AMP and H-89 to investigated the patyway in the differentiation and function of Treg cells affected by PGE2. Relative expression of Foxp3 m RNA were measured by RT-PCR and CD25+Foxp3+ cells, the expression of CLTA-4 and GITR were analyzed by flow cytometry and the production of IL-10 in the culture supernatants were detected by ELISA.4 The role of CCK-8 in the inhibitory of PGE2 on the differention and function of Treg cells: na?ve T cells were cultured under Treg differention and divieded into control group, PGE2 group, CCK-8 group and CCK-8+PGE2 group. 7-day later, Relative expression of Foxp3 m RNA were measured by RT-PCR and CD25+Foxp3+ cells, the expression of CLTA-4 and GITR wereanalyzed by flow cytometry and the production of IL-10 in the culture supernatants were detected by ELISA.Results:1 Butaprost and L-902,688 decreased the quantities of CD25+Foxp3+ cells and the expression of Foxp3 m RNA, but Sulprostone had no effect. This finding indicated that PGE2 suppressed Treg differentiation mainly via the EP2 and EP4 receptors. We then used AH6809, an EP2 antagonist, and GW627368 X, an EP4 antagonist, to verify the above results. AH6809 and GW627368 X could reverse the decreases in the number of Tregs and Foxp3 m RNA expression caused by PGE2. Moreover, AH6809 and GW627368 X did not affect the differentiation of Treg cells in the absence of PGE2. These results further proved that PGE2 suppressed human Treg differentiation via the EP2 and EP4 receptors.2 PGE2 could markedly induce the production of intracellular c AMP compared with the control group. The effects of Butaprost and L-902,688 were comparable to that of PGE2 and induced the production of c AMP. db-c AMP served as a positive control and exerted a similar effect. These effects were rescued by the EP2 and EP4 antagonists. AH6809 and GW627368 X blocked the effect of PGE2 on the c AMP levels. Similar to the intracellular c AMP levels, the PGE2-mediated PKA activity was significantly higher than that of the control group. The administration of Butaprost and L-902,688 increased this activity. Nevertheless, AH6809 and GW627368 X abrogated these effects.3 The effect of db-c AMP on Treg differentiation and was similar to that of PGE2; it decreased the quantity of Tregs and expression of Foxp3 m RNA. However, H-89 abolished the inhibitory effect of PGE2 on Treg differentiation. Similarly, H-89 blocked the effects of Butaprost and L-902,688 on the percentage of Tregs and the expression of Foxp3 m RNA. PGE2 decreased the percentage of Foxp3+CTLA-4+ cells and Foxp3+GITR+ cells and suppressed the production of IL-10. The effect of db-c AMP was similar to that of PGE2, but H-89 reversed the inhibition of CTLA-4 and GITR expression and IL-10secretion by PGE2. Butaprost and L-902,688 partly simulated the suppression of Foxp3+CTLA-4+ and Foxp3+GITR+ cells and the secretion of IL-10 by PGE2. However, H-89 blocked the actions of Butaprost and L-902,688.4 CCK-8 increased the quantity of Tregs and has a significant upregulation of Foxp3 m RNA, and increased the percentage of Foxp3+CTLA-4+ cells and Foxp3+GITR+ cells and the production of IL-10, however, CCK-8 can only partly reversed the inhibitory effect of PGE2 on Treg differentiation and the expression of CTLA-4 and GITR expression and IL-10 secretion, and couldn’t completely reversed it.Summery: In summary, PGE2 suppressed the differentiation of human na?ve CD4+ T cells into Treg cells via EP2/EP4 receptors and the c AMP/PKA signaling pathway. By this signaling, PGE2 inhibited the function of Treg cells. CCK-8 can upregulate the expression of Foxp3 m RNA and the function of Treg cells, and can partly reversed the inhibitory of PGE2 on the Treg differentiation and function.Conclusions:In present study, we used purified CD14+ monocytes from human peripheral blood and in vitro dfferentiated the cells under i DCs and m DCs. We determined the effects of CCK-8 on the phenotypic of i DC and m DC, and examined the influence of CCK-8 educated i DC/m DC on Treg functions, and detected the related receptor mechanism and the role of CD80, CD86 and GITR-L in Treg function regulated by DC.1 CCK-8 can inhibited m DC phenotype and induce the generation of semi-mature DC, which is t DC. CCK-8 can upregulated Treg suppressor function and Treg proliferation by m DC, which may be related to the generation of t DC.2 CCK-8 may regulate Treg function by modulated the expression of CD80/CD86 and GITR-L on DC. CCK-8 modulated the expression of CD80, CD86 and GITR-L on DC by CCK-2R, and then regulated Treg function by this receptor, and PKA and PKC involved in this actions of CCK-8 on DC. In addition, anti-CD80, anti-CD86 and anti-GITR-L further determined the roleof CCK-8 modulated DC in Treg function.4 PGE2 suppressed the differentiation of human na?ve CD4+ T cells into Treg cells via EP2/EP4 receptors and the c AMP/PKA signaling pathway. By this signaling, PGE2 inhibited the function of Treg cells. CCK-8 can upregulate the expression of Foxp3 m RNA and the function of Treg cells, and can partly reversed the inhibitory of PGE2 on the Treg differentiation and function.