Modulation Of Human Peripheral Blood Plasmacytoid Dendritic Cells Maturation And Activation By Cholecystokinin Octapeptide

Plasmacytoid dendritic cells (pDC) are specialized in rapid and massive secretion of type I interferon (IFN-α/β) in response to foreign nuclei acids. Combined with their antigen presentation capacity, this powerful functionality enables pDC to orchestrate innate and adaptive immune responses. This function of pDC is linked to their expression of Toll-like receptor7(TLR7) and TLR9, which sense nucleic acids within the early endosomes, and pDC may be more specialized in presenting viral antigens and endogenous self-antigens. As mentioned previously, pDC not only play a role in the initiation of immunity but are also indispensable for the preservation of tolerance.pDC rapidly produce large amounts of type1IFN, which not only has direct inhibitory effects on viral replication but also contribute to the activation of NK cells, B cells, T cells, and classical dendritic cells (cDC), leading to the induction and expansion of an antiviral immune response, or breaking immune tolerance. One of the most remarkable features of pDC is their constitutive expression of high levels of IRF-7, a possible molecular component underlying the ability to rapidly produce huge amounts of type I IFN upon signaling TLR-7/TLR-9upon viral infection. TLR7/TLR9-mediated IFN-a production by pDC depends on the forming and activation of Myd88-IRAK4-TRAF6-IRF-7complexes.Upon exposure to environmental (i.e. viruses) and/or endogenous (i.e. nucleic acid-containing immune complexes) triggers, pDC from patients produce IFN-a in a sustained fashion. IFN-a activates classical DC, and expresses costimulatory molecules and triggers the expansion and differentiation of autoreactive CD4+and CD8+T cells, and possibly mature B cells, into autoreactive effectors. Cytotoxic T cells kill tissue targets thereby generating nucleosomes and autoantigen fragments, which further feed the autoimmune process. Type I IFN, together with other products of activated pDC such as IL-6, drive these autoreactive B cells to differentiate into plasma cells that secrete autoantibodies. Immune complexes derived from DNA and RNA-containing antibodies can further activate pDC to release IFN-a, amplifying this pathogenic loop. IFN-a also directly promotes abnormal vasculogenesis, which might contribute to the development of premature pathological changes.Cholecystokinin (CCK) is wellknown as a typical brain-gut peptide and immunomodulating peptide, and it is identified as several different size of the peptide. Cholecystokinin octapeptide (CCK-8) is the smallest molecular owed full biological function of CCK. For recent years, a series of studies focused on the effect of CCK-8against endotoxin shock and inflammation have being performed in our laboratory. Our previous studies showed that CCK-8can regulate the immune function of several immunocyte.It is well known that CCK exerts a variety of physiological actions through its cell surface receptors, which have been pharmacologically classified into two subtypes CCK-1R and CCK-2R according to their affinity to the peptide agonists CCK-8and gastrin. CCK receptors belong to G protein-coupled receptor (GPCR) super family and distribute extensively in mammalian body.With the emergence of elevated IFN levels as a pathogenesis factor in several autoimmune diseases, the potentially important role of pDC in autoimmunity has been recognized. Modulation of the immune response by pDC might provide novel immune-based therapies in autoimmunity and transplantation. CCK-8is a potent immunomodulator, whose role in pDC function is unknown. The present study was designed to investigate whether human pDC express CCK receptors, effects of CCK-8on pDC maturation&activation, and the related signal transduction pathway.1Expression of CCK1R and CCK2R in human peripheral blood plasmacytoid dendritic cells Objective:The present study was designed to investigate whether human pDC, cDC, PBMC express CCK receptors, and influence of pDC maturation and activation on the expression of CCK receptors.Methods:Approval for all experiments with human blood was provided by the Blood Center of Hebei Province. PBMCs from venous blood of healthy volunteers were separated by Ficoll-Hypaque gradient density centrifugation. CD303+pDC precursors were isolated from PBMC with anti-BDCA-4/CD304magnetic beads or the pDC isolation kit (Miltenyi Biotec). BDCA-1/CD1c+cDC were induced by monocytes treated with100ng/mL GM-CSF and10ng/mL IL-4. Expression of CCK1R/CCK2R was detected in human peripheral blood dendritic cells, including BDCA-2/CD303+pDC or BDCA-1/CDlc+cDC, and peripheral blood mononuclear cell (PBMC), by means of RT-PCR and immunofluorescence stain.Results:(1) CCK1R and CCK2R mRNA were detected in human peripheral blood DC, including BDCA-2/CD303+pDC or BDCA-1/CD1c+cDC, and PBMC.(2) Effect of CD40L or CpG ODN on CCK1R/CCK2R mRNA expression in human pDC:The relative expressed quantity of CCK1R was (100±0)%in PBMC and (56.7±3.38)%,(49.2±4.41)%,(42.7±5.84)%, in r-pDC, m-pDC, a-pDC respectively and a little bit decreases, but it was not statistically significant (P>0.05vs r-pDC group). For CCK2R mRNA, their relative expressed quantity was (100±0)%in PBMC and (23.7±4.38)%,(29.2±3.41)%,(59.7±4.84)%in r-pDC, m-pDC, and a-pDC respectively, and significant increases in its expression were observed at24h after incubating human pDC with CpG ODN (P<0.01).(3) Effect of CD40L or CpG ODN on CCK1R and CCK2R expression in human pDC:CCK-1R and CCK-2R protein were all co-expressed on their membrane and cytoplasm of human pDC, and significant increases in CCK-1R and CCK-2R protein expression were observed after incubating human pDC with CD40L(P<0.05) or CpG ODN(P<0.01).Conclusion:CCK1R and CCK2R were coexpressed in human peripheral blood pDC/IPC and cDC/mDC. The expression of CCKR was obviously up-regulated after maturation or activation of pDC2Effect of cholecystokinin octapeptide on human peripheral blood plasmacytoid dendritic cells maturation&activationObjective: CCK-8is a potent immunomodulator, whose role in pDC function is unknown. In this study, up-regulation of CCK1R and CCK2R expression was observed after maturation or activation of human peripheral blood pDC. We then investigated whether CCK-8could modulate the maturation&activation of pDC. DCs undergo the change in phenotype and function during the maturation. In this study, we investigated whether CCK-8could modulate the phenotype and cytokine secretion of pDC and the potential of pDC to induce adaptive immune response of CD4+T cells.Methods:Using flow cytometry analysis, we evaluated pDC expression profiles of surface molecules with significance for T cell-pDC interactions including the costimulatory molecules CD80, CD83, CD86, HLA-DR ligand, and the DC maturation marker CD208. FITC-, PE-, and PECy5-labeled antibody to BDCA-2/CD303, BDCA-1/CDlc and CD123were used to stain pDC or cDC. Cell culture supernatants were collected and the content of IFN-a was determined by enzyme-linked immunosorbent assay (ELISA) kits. T cell/pDC/cDC-cocultures supernatants were collected and the content of IFN-y/IL-17was also determined. Semi-quantitative Realtime RT-PCR was used to analyze the expression of IFN-α mRNA. Mixed lymphocyte reaction (MLR) was performed using purified allogene T cells as responder cells and pDC as stimulator cells. T-cell proliferation was measured by MTT assay with a microplate reader at490nm. Each experiment was performed in duplicate and repeated at least five times. Data were expressed as the mean±standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA) with SPSS software version11.5. A level of P<0.05was considered statistically significant.Results:(1) CCK-8inhibits the maturation of pDC induced by CD40L The CD303+CDlc-CD123high r-pDC was the cells we used to complete the next study. We analyzed the effects of CCK-8(various concentrations as follow:10-10,10-9,10-8,10-7,10-6M) on the CD40L-induced expression of DC maturation-marker CD208. The expression of CD208in CD40L group was higher than that of control (IL-3) group (P<0.05). Compared to CD40L group, the expression of CD208was decreased in CD40L+CCK-8(10-8,10-7M) group (P<0.05). CD40L-stimulated (with or without CCK-8/PGE2) m-pDC were harvested, and mean fluorescence intensities of CD80,CD83,CD86,CD208and HLA-DR was determined by FACS analysis. The expression of CD80, CD86,CD208and HLA-DR in CD40L group was higher than that of control group (P<0.01). CD83was not seriously influenced by CD40L (P>0.05). Compared to CD40L group, the expression of CD80, CD86, CD208and HLA-DR was decreased in CD40L+CCK-8(10-8M) group, increased in CD40L+PGE2(10-8M) group (P<0.05). But compared to CD40L+PGE2group, the expression of CD80, CD86, CD208and HLA-DR was not impacted by the treatment with CCK-8(P>0.05). Compared to CD40L group, the expression of CD80, CD86, CD208and HLA-DR was also not impacted by the treatment with CCK-8(10-8M)+PGE2(10-7M)(P>0.05)(2) CCK-8inhibits the activation of pDC induced by CpG ODN:pDC were isolated as described and cultured in the presence of CpG ODN (5μg/ml) and with or without CCK-8(10-8M) for48h. After2days, cocultures were harvested, and the expression of CD208and HLA-DR was determined by FACS analysis, the IFN-a mRNA was measured by RT-PCR. Cell culture supernatants were collected and the content of IFN-a was determined by enzyme-linked immunosorbent assay (ELISA) kits. Allogene CD4+T cells from healthy donors were isolated, and coincubated with precultured pDC at different ratios as indicated, then the percentage of proliferated T cells was determined by MTT assay. Resting pDC, following CpG ODN treatment, express high levels of CD208and HLA-DR, while CCK-8treatment inhibited CD208and HLA-DR expression (P<0.05, vs CpG ODN group). These results indicated that CCK-8could inhibit the CpG ODN-induced phenotypic maturation of pDC. The r-pDC produced too little IFN-a, and after activated following CpG ODN treatment, a-pDC produced much higher level of IFN-α. CCK-8(10-8M) treatment decreased the production of IFN-a, compared to CpG ODN cocktail group (P<0.05). After activated by CpG ODN pDC obtained the antigen presenting capacity and induced T cell proliferation, however, treatment with CCK-8(10-8M) reduced the stimulatory capacity (P<0.05, vs CpG ODN group).(3) CCK-8inhibits the countervailing actions of cDC on the activation of pDC induced by LPS: pDC were isolated as described and cultured in the presence of LPS (1μg/ml) and with or without CCK-8(10-8M) for48h. CD4+T cells from healthy donors were isolated via positive selection (Miltenyi Biotec) and resuspended in PBS (1x107cells/ml). cDC were induced with GM-CSF(100ng/ml) and IL-4(10ng/mL) from PBMC. pDC were then coincubated with allogeneic CD4+T cells or/and homogenic cDC at different ratios as indicated. After48hours, cocultures were harvested, and the expression of CD208and HLA-DR was determined by FACS analysis, the IFN-α mRNA was measured by RT-PCR, the percentage of proliferated T cells was determined by MTT assay. Cell coculture supernatants were collected and the content of IFN-α was determined by enzyme-linked immunosorbent assay (ELISA) kits. pDC expressed low levels of CD208or HLA-DR, but cDC, expressed high levels of CD208or HLA-DR, treated by LPS (P<0.05, vs cDC control group). While adding cDC (pDC:cDC=1:1) into pDC cocultures, concomitant with LPS, the expression of CD208and HLA-DR increased (P<0.05, vs cDC LPS group), and the value of gate%was more than50%. So, pDC was also activated in this coculture, concomitant with LPS and cDC. These results indicated that CCK-8could inhibit the LPS and cDC-induced phenotypic maturation of pDC. The r-pDC and LPS-treated pDC produced too little IFN-α, but r-pDC were activated by LPS+cDC treatment, produced much higher level of IFN-α. Meanwhile CCK-8(10-8M) treatment decreased the expression of IFN-α, compared to LPS+cDC+pDC cocktail group (P<0.05). Cocktail stimuli (pDC+LPS+cDC) can induce T cell proliferation, however, treatment with CCK-8(10-8M) reduced the stimulatory capacity, compared to pDC+LPS+cDC cocktail group (P<0.05).(4) CCK-8inhibits the potential of pDC with or without the presence of PGE2: CCK-8inhibits the potential of pDC, induced by cocktail stimuli (IL-3+CD40L+CpG ODN class A/B), to induce adaptive immune response of allogene CD4+T cells, with or without the presence of PGE2. pDC were isolated as described and cultured in the presence of IL-3(1μg/mL), CD40L (1μg/mL), CpG ODN class A/B (5μg/mL), and with or without the presence of CCK-8(10-8M) and PGE2(10-7M) for48h. CD4+T cells from healthy donors were isolated via positive selection (Miltenyi Biotec) and resuspended in PBS (1x107cells/mL). pDC were then coincubated with allogene CD4+T cells (pDC:CD4+T=1:50/100/150), different stimuli pretreated, at different ratios as indicated. After48hours, cocultures were harvested, and the expression of CD208and HLA-DR was determined by FACS analysis, the IFN-a mRNA was measured by RT-PCR, the percentage of proliferated T cells was determined by MTT assay. Cell coculture supernatants were collected and the content of IFN-α was determined by enzyme-linked immunosorbent assay (ELISA) kits. Isolated pDC, following cocktail stimuli (IL-3+CD40L+CpG ODN class A/B) treatment, pDC expressed high levels of CD208and HLA-DR, and CCK-8(10-8M) treatment decreased, PGE2(10-7M) treatment increased the expression of CD208and HLA-DR, compared to cocktail group (P<0.05). These results indicated that CCK-8could inhibit the cocktail stimuli (IL-3+CD40L+CpG ODN class A/B)-induced phenotypic maturation of pDC, with or without the presence of PGE2. The cocktail stimuli-treated pDC produced high level of IFN-a, and level of IFN-a increased following PGE2treatment, decreased following CCK-8treatment. Purified pDC can activated by cocktail stimuli (IL-3+CD40L+CpG ODN class A/B) as APC to induced T cell proliferation, however, treatment with CCK-8(10-8M) reduced, treatment with PGE2(10-7M) increased the stimulatory capacity, compared to cocktail stimuli group (P<0.05). Supernatants of different group were harvested and assayed for IFN-y, IL-17by ELISA. CD4+T cells exposed to cocktail stimuli-treated pDC, plus PGE2treatment, produced high levels of IFN-y and IL-17. If the pDC were treated with cocktail stimuli, with or without the expression of PGE2, plus CCK-8treatment, IFN-γ and IL-17production was significantly decreased(P<0.05).Conclusion:CCK-8modulated the maturation and activation of pDC, including the phenotypic maturation, IFN-a synthesis and secretion, and the potential to induce adaptive immune response of allogene T cells, with or without the presence of PGE2.3Lentivirus-mediated CCK2R silencing reversed the modulation of TLR9-mediated activation of human peripheral blood plasmacytoid dendritic cells by CCK-8Objective:CCK receptor is an important member of the G-protein coupled receptors and is present in pulmonary vascular endothelial cells, macrophages, bronchial epithelial cells and alveolar epithelial cells, which play an important role in mediating the regulatory actions of CCK-8on these cells.[13-16] Our data proved that CCK-8, as an immunomodulatory cytokine, modulated the CD40L-induced phenotypic maturation of pDC. And CCK-8inhibited synthesis and secretion of IFN-a, and the potential of pDC to induce adaptive immune response of allogene CD4+T cells. Interestingly, we found that CCKIR and CCK2R co-expressed on their membrane and cytoplasm of human peripheral blood pDC, and the expression of CCK2R mRNA/protein could be up-regulated after maturation or activation of human pDC, induced by CD40L or CpG ODN. Why CCKIR and CCK2R co-expressed on pDC membrane, and the role of them in the modulation of pDC maturation and activation by CCK-8is still unknown.Methods:(1) Screening effective sequence of CCKIR miRNA and CCK2R miRNA and constructing the LV-miR-CCKIR and LV-miR-CCK2R: According to Human CCKIR, NM₀00730.2and Human CCK2R, NM₁76875.2two target gene sequences, using Invitrogen's RNAi Designer, we designed and synthesized four pairs for each gene, a total of eight pairs. miR single-stranded DNA oligonucleotides, and conduct recombinant plasmid DNA for CCKIR and CCK2R. Each transformation plate was picked up four clones to verify by sequencing. Before we detected effective sequence of CCKIR and CCK2R for each transfection sample (including of miRNA-control (no load), CCK-1R-mil, CCK-1R-mi2, CCK-1R-mi3, CCK-1R-mi4, CCK-2R-mil, CCK-2R-mi2, CCK-2R-mi3, CCK-2R-mi4) in human derived cell line SH-SY5Y, plates were checked for transfection by examining fluorescence intensity, using fluorescent microscopy(plasmid carrying the EGFP fluorescence marker). RT-PCR and Western blot were used to detect the silencing efficiency of eight recombinant plasmid DNA of pcDNA6.2TM-GW/EmGFP-CK1R/2R-miRNA in SH-SY5Y cells. Then, Invitrogen corporation helped to construct a large number of LV-miR-CCK1R/LV-miR-CCK2R in293T cells with our selected target sequence of miRNA.(2) Transducing the lentiviral construct into pDC and determining the optimal MOI:The CD303+CD1c-CD123high r-pDC is the cells we used to complete the next study. CD303+CD1c-CD123high r-pDC were transfected by Lenti6.3-negtive and examined fluorescence intensity, using fluorescent microscopy (plasmid carrying the EGFP fluorescence marker). Silencing efficiency of CCKIR and CCK2R gene RT-PCR and In-Cell Western blot were used to detect the silencing efficiency of CCKIR and CCK2R gene by recombinant LV-miR-CCKIR and LV-miR-CCK2R on human peripheral blood pDC.(3) Effect of CCKIR or CCK2R silencing on the CpG ODN-induced activation of pDC with the presence of CCK-8:Isolated pDC, CCK1R-knockdown pDC, and CCK2R-knockdown pDC were cultured in fat-bottom24-well plates at5x105cells per well in a final volume of1mL RPMI1640medium together with5μg/mL CpG ODN with or without the presence of CCK-8(10-8M). Using flow cytometry analysis, we evaluated the expression of DC maturation marker CD208. Cell culture supernatants were collected and the content of IFN-a was determined by enzyme-linked immunosorbent assay (ELISA) kits. Semi-quantitative Realtime RT-PCR analyzed the IFN-a/CCR7mRNA expression. T cell/pDC-cocultures supernatants were collected and the content of IFN-y/IL-17was also determined. T-cell proliferation was measured by MTT assay with a VERSA max Tunable microplate reader at490nm.(4) Isolated pDC, CCK1R-knockdown pDC, and CCK2R-knockdown pDC culture supernatants were collected and the protein kinase A activity was determined by Protein Kinase A Activity Assay Kit(Enzo Life Sciences, EKS-390A), the protein kinase C activity was determined by Protein Kinase C Activity Assay Kit(Enzo Life Sciences, EKS-420A). Expression of TRAF6and IRF7were analyzed by hi-cell western blot. Each experiment was performed in duplicate and repeated at least five times. Data were expressed as the mean±standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA) with SPSS software version11.5. A level of P<0.05was considered statistically significant.Results:(1) Sequencing analysis revealed that eight plasmid insert fragments are consistent with the sequences we designed, which have been inserted correctly in the predetermined position. Many of the cells (>70%) should be strongly GFP positive after14days transfection and cultured with selection pressure (10ug/ml blasticidin). The decline of CCK1R mRNA was most marked (>60%) by CCK-1R-mi2, and CCK2R mRNA was most marked (>60%) by CCK-2R-mi2. This result was consistent with that of Western blot.(2) Much of the pDC (>75%) should be strongly GFP positive after72hours transfection (MOI:100). Silencing efficiency of CCK1R and CCK2R gene RT-PCR and In-Cell Western blot were used to detect the silencing efficiency of CCK1R and CCK2R gene by recombinant LV-miR-CCKIR and LV-miR-CCK2R on human peripferiral pDC. The decline of CCK1R mRNA was most marked (>65%), and CCK2R mRNA was most marked (>65%). This result was identified by in-cell western blot.(3) Effect of CCK1R or CCK2R silencing on the CpG ODN-induced activation of pDC with the presence of CCK-8:CD303+CD1c-CD123high r-pDC, following cocktail stimuli (IL-3/CD40L/CpG ODN class A/B) treatment, expressed high levels of CD208. LV-negtive, transfected pDC, could increase a little bit of the CD208expression, but CCK-8(10-8M) decreased the CD208expression compared to cocktail group (P>0.05). In contrast to lentivirus-mediated RNA interference (MOI:100) of CCK2R reversed the effect induced by CCK-8treatment(P<0.05). The cocktail stimuli-treated pDC produced high level of IFN-a, including IFN-a mRNA, and level of CCR7mRNA increased following CpG ODN treatment, all this decreased following CCK-8treatment. The decrease of IFN-α and CCR7mRNA (compared to cocktail group) following CCK-8(10-8M) treatment, could be reversed by lentivirus-mediated CCK2R knockdown (MOI:100, P<0.01). Purified pDC can activated by cocktail stimuli (IL-3/CD40L/CpG ODN class A/B) as APC to induced T cell proliferation, however, treatment with CCK-8(10-8M) reduced the capacity, and treatment with LV-miR-CCK2R (MOI:100), in the presence of CCK-8, reversed the costimulating capacity compared to cocktail+CCK-8group (P<0.05). Supernatants of different group were harvested and assayed for IFN-γ, IL-17by ELISA. The cocktail stimuli-treated pDC produced high level of IFN-γ, IL-17, and CCK-8could decrease the secretion of IFN-γ and IL-17, which could be reversed by LV-miR-CCK2R (MOI:100)(P<0.01).(4) Effect of CCK1R or CCK2R knockdown on PKA and PKC kinase activity in human peripferiral pDC activated by CpG ODN:The present study was performed to investigate the effect of CCK1R or CCK2R knockdown on PKA and PKC kinase activity in human peripferiral blood pDC stimulated by CpG ODN. pDC was isolated and stimulated with CpG ODN in the absence or presence of CCK-8for indicated time. PKC kinase activity was detected by non-radioactive enzyme assay of all forms PKA or PKC. Stimulating pDC with CpG ODN resulted in an obvious increase in PKA and PKC kinase activity. Treating pDC with CCK-8lead to an inhibition of PKC kinase activity induced by CpG ODN, and promotion of PKA kinase activity. Silencing CCK2R was so potent that had an obvious effect on CCK-8-resulted inhibition of CpG ODN induced PKC kinase activity, and promotion of PKA kinase activity (P<0.01vs cocktail+CCK-8group).(5) Effect of CCK1R or CCK2R knockdown on the expression of TRAF6and IRF7:Relative protein levels of TRAF6, IRF7and p-IRF7analyzed by hi-Cell Western blot and Odyssey System software. Stimulating pDC with CpG ODN resulted in an obvious increase in TRAF6, IRF7and p-IRF7expression. Treating pDC with CCK-8lead to an inhibition of TRAF6, IRF7and p-IRF7expression induced by CpG ODN. Silencing CCK2R was so potent that had an obvious effect on CCK-8-resulted inhibition of CpG ODN induced TRAF6, IRF7and p-IRF7expression (P<0.05vs cocktail+LV-negative+CCK-8group).Conclusion:These results indicated that CCK-8modulated the maturation and activation of pDC, including the phenotypic maturation, IFN-a synthesis and secretion, and the potential to induce adaptive immune response of allogene T cells, with or without the presence of PGE2. Silencing CCK2R, not CCK1R, obviously reversed the effect resulted by CCK-8, as an immunomodulatory cytokine. CCK-8modulated the PKC/PKA kinase activity and the TRAF6and IRF7protein expression or modification, which might be involved in the effect of CCK-8on pDC maturation&activation.