Involvement Of RORα In The Regulation Of Dendritic Cells Maturation By α-Melanocyte Stimulating Hormone And Its Analogue STY39

Retinoic acid receptor-related orphan receptorα(RORa), a transcription factor belonging to the nuclear receptor superfamily, plays an important role in the physiopathological processes, such as morphological development, cell proliferation and differentiation, metabolic homeostasis, neural function control and immunoregulation. The staggerer (Rorasg/sg) mutant mouse carries a deletion in the Rora gene that leads to extensive and seriously functional disorders. An important aspect of the staggerer phenotype is abnormal immunity characterized by an increased susceptibility to autoimmune or hypersensitivity diseases. These abnormal reactions may suggest that RORa is closely related with immune system. The anti-inflammatory actions of RORa have been proved in lots of researches on animal models.α-Melanocyte stimulating hormone (α-MSH) is a small molecular neuropeptide with potent anti-inflammatory and immunomodulatory effects which had been confirmed by means of animal models of inflammation.α-MSH is able to up-regulate the release of IL-10 by monocytes and down-regulate the production of nitric oxide and pro-inflammatory cytokines by macrophages.α-MSH also significantly suppresses the expression of CD86 and CD40 on monocytes and dendritic cells (DCs).α-MSH prolongs allograft survival and improves the histopathologic alteration of the allograft in experimental heart transplantation.α-MSH functions specifically via melanocortin receptor (MC-R) which include five subtypes (MC-1R to MC-5R). Recent studies showed that melanocortin 1 receptor (MC-1R) and melanocortin 5 receptor (MC-5R) were expressed on the surface of immune and inflammatory cells, such as monocytes, macrophages, DCs and lymphocytes. The actions of anti-inflammation and immunoregulation ofα-MSH are mainly transmitted through MC-1R and MC-5R. However,α-MSH has MC1 and MC3 receptor-selective and low affinities for MC4 and MC5 receptors. It can also affect the neuroendocrine functions besides melanin pigmentation. Considering these disadvantages ofα-MSH, we have already designed a new peptide, named STY39, by InsightⅡsoftware, which is aα-MSH analogue and has MC1 and MC5 receptor-selective. The effects of STY39 were appraised in the mouse model for endotoxic shock, pulmonary fibrosis and ARDS. Thus,α-MSH is regarded as an important factor involved in the regulation of inflammation and immune responses. However, its mechanism of action remains unclear.Our previous work has demonstrated thatα-MSH inhibited tumor necrosis factor-alpha (TNF-α)-induced maturation of human peripheral monocyte-derived DCs (MoDCs), both phenotypically and functionally. Moreover, we found that it may be connected with high expression of RORα. DCs are crucial regulators of immunity, including the abilities to initiate or activate immune response and induce immunotolerance. The functions of DCs are closely related with their mature state. So we hypothesized that the transcription factor RORαmay take part in the signal pathways which underlying the regulatory effects ofα-MSH in DC maturation. Although RORαhas been reported to function as an anti-infalmmatory factor, its role on DC maturation has not been examined. The primary aim of this study was to investigate the role and mechanism of RORαin the reversion of DC maturation byα-MSH or STY39.PartⅠ. Effects ofα-MSH and its analogue STY39 on the expression of RORa in DCs.DCs isolated from mouse bone marrow were investigated. DCs were stimulated with 50 ng/ml TNF-α(TNF-a-DCs) at first, and treated with different concentrations ofα-MSH or STY39 treatment (10-6 mol/L,10-8 mol/L,10-10 mol/L,10-12 mol/L). Then the expression levels of RORαprotein and mRNA were detected by Western blotting and Real-time PCR. We confirmed that the protein and mRNA expression of RORαwas decreased in TNF-α-DCs, while increased during the reversion of TNF-α-DC maturation byα-MSH (10-8 mol/L and 10-10 mol/L) or STY39 (10-10 mol/L).PartⅡ. Regulatory effects of RORαon DC maturation induced by TNF-α.The amplified RORαcDNAs were linked and inserted into AAV-IRES-hrGFP plasmid. Then the recombinant expression vector, AAV-RORα-hrGFP, was constructed and transfected into TNF-α-DCs (RORa-TNF-α-DCs) to complement the decreased expression of RORα. To determine the effects of RORαon the DCs maturation, we compared different groups of DCs in terms of surface marekers, phagocytic capacity, cytokine production and the ability to stimulate antigenic specific T cells. The expression of Iab, CD86 and CD54 on DCs and FITC-BSA phagocytic function of DCs were estimated by FCM. The proliferation of antigenic specific T cells stimulated by different groups of DCs was detected in vitro by [3H]-thymidine incorporation assay. The concentrations of IL-6, IL-12p40 and IL-10 in the supernatants of co-cultured cells were determined using ELISA. Compared with IRES-TNF-α-DCs (empty vector control), some characteristics of RORα-TNF-α-DCs were found as follows:①The expression of Iab, CD86 and CD54 on DCs was decreased.②The phagocytic capability of DCs was significantly increased.③The ability to stimulate antigenic specific T cell was inhibited.④Down-regulated IL-6 and IL-12 secretion and up-regulated IL-10 production were also observed. The results are consistent with our hypothesis that over-expression of RORαmay reverse the maturation of TNF-α-stimulated DCs.PartⅢ. Role of RORαin the suppression of DC maturation byα-MSH and its analogue STY39.RORα-siRNA duplexes were obtained and transfected into TNF-α-DCs with treatment of 10-10 mol/Lα-MSH or STY39 [α-MSH (or STY39)-ROR-si-TNF-α-DCs]. To determine whether the transfection of RORα-siRNA could affect the maturation of DCs, we compared different groups of DCs in terms of surface marekers, phagocytic capacity, cytokine production and the ability to stimulate antigenic specific T cells. The experimental methods were similar with Part Two. Compared withα-MSH (or STY39)-non-si-TNF-α-DCs, some characteristics ofα-MSH(or STY39)-ROR-si-TNF-α-DCs were found as follows:①The expression of Iab, CD86 and CD54 was increased.②The phagocytic capability was significantly reduced.③The ability to stimulate antigenic specific T cell was enhanced.④Up-regulated IL-6 and IL-12 secretion and down-regulated IL-10 production were also observed. It can be concluded that RORαtakes part in the reversal procedure of DC maturation byα-MSH or STY39.PartⅣ. The molecular mechanisms underlying the involvement of RORa in the suppressive effects ofα-MSH or STY39 on TNF-αinduced DC maturationFirstly, DCs cultured in vitro were divided into imDCs group, TNFα-DCs group,10-10 mol/Lα-MSH treated group,10-10 mol/L STY39 treated group, RORα-TNF-α-DCs group and IRES-TNF-α-DCs group. The expression levels of MC-1R to MC-5R mRNA were detected by real-time quantitative PCR, respectively. The dual-luciferase reporter assay was used to analyze the transcriptional activity of nuclear factor-kappa B (NF-κB), and the expression of NF-κB p65 and IκBαprotein was detected by Western blot in different groups. Secondly, TNF-α-DCs were treated with 10-10 mol/Lα-MSH or STY39 in the presence of a number of specific signaling protein inhibitors, including SB203580 (p38/MAPK),GF10933X (PKC), H89 (PKA), PD98059 (ERK1/ERK2) and PDTC (NF-κB). Then the expression of RORαmRNA was analyzed and determined by real-time quantitative RT-PCR.Some significant phenomena were found as follows:①MC-1R to MC-5R mRNA were all expressed on DCs.α-MSH or STY39 could both up-regulate MC1-R expression on imDCs and TNF-α-DCs.②High expressed RORαalso enhanced the MC-1R expression on TNF-α-DCs.③α-MSH or STY39 mediated induction of RORαwas observed in control group, while attenuation of this response was observed to varying degrees with the inhibitor compounds, such as SB203580, GF10933X or H89.④IκBαprotein were increased, NF-κB p65 nuclear translocation were decreased, and the transcription activity of NF-κB was also inhibited in RORα-TNF-α-DCs, compared with IRES-TNF-α-DCs.⑤The transcription activity of NF-κB in STY39-RORα-siRNA-TNF-α-DCs was enhanced, compared with STY39-non-siRNA-TNF-α-DCs. Our results indicate thatα-MSH or STY39 increases expression of ROR a through multiply signal pathways such as p38/MAPK, PKC and PKA.α-MSH or STY39 inhibits the maturation of DCs through RORa at least partially. RORαmay not only inhibit NF-κB signal transduction but also increase the expression of MCR1 on DCs.In a word, we found that RORαtakes part in the modulation of DC maturation, and the suppressive effects ofα-MSH and STY39 on DC maturation may be through MCRs-mediated multiple signal transduction pathways. RORαtakes a very important role in this process and could be used as a new drug target for DC-based measures treating autoimmune diseases, cancer, infection diseases and transplant rejection. The present research provides a new evidence for exploring the roles of neuroendocrine-immune regulation network in the pathogenesis, prevention and treatment of the diseases.