Function Study Of UDP And P2Y₆ In Dendritic Cells

As host’s most important antigen-presenting cells, dendritic cells become an important bridge between innate immunity and acquired immunity, which stand in the center of immune responses through uptaking and presenting antigens to effector lymphocytes, subsequently turned on the gate of acquired immunity.At present, many studies have shown that stressed and injured cells will release nucleotides as a danger signal through paracrine or autocrine manners. Then the released nucleotides regulate various important physiological functions by activating P2 receptors on the cell membrane. Our previous study found that P2Y6 can upregulate the expression of MCP-1 thus facilitate recruitment of monocytes to fight against bacterial infection. However, the function of P2Y6 in DC has not been well demonstrated. So, in this paper we want to discover the role of UDP/P2Y6 in DC mediated immune responses.In order to clarify the function of purinergic receptors in DCs, we use QPCR to detect the expression level of three purinergic receptors in dendritic cells. The results showed that the expression of P2Y6 increased during the cell differentiation and development. Then, we used fluorescence polarization to detect the concentration of extracellular UDP from the phenol red-free cell culture supernatant and found that the concentration of UDP continue to increase during the maturation of DC, especially after the cells were maturated with LPS. In addition, we used flow cytometry to detect the expression of costimulatory molecules CD80, CD86 on dendritic cells. Our data showed that under the stimulation of UDP, the expression of CD80, CD86 was significantly increased. Then a significant increasing in inflammatory cytokines TNF-α and IL-12 was also observed in UDP treated DC by QPCR. Accordingly, the lack of P2Y6 receptors can significantly inhibit UDP enhanced maturation of dendritic cells, including reduce the expression of costimulatory molecules and secretion of inflammatory cytokines. Thereafter, the phosphorylation of ERK was also activated obviously by UDP through Western Blotting.Then we used the CHS mouse model to explore the function of UDP in IV hypersensitivity disease. We inject UDP into both P2Y6 knockout and WT CHS mouse model to find the direct role of UDP/P2Y6. Then we injected dendritic cells from knockout mice and WT mice in CHS mouse model to find whether the key role of DCs in UDP mediated immune regulation. Our data showed that the thickness of the ears and cytokine production can be significantly enhanced by injecting UDP and UDP treated DCs, and this kind of enhancement could be eliminated in P2Y6 deficient mice and DCs.In conclusion, our study not only enrich the regulation of UDP/P2Y6 in both innate and acquired immune systems, but also lay the theoretical foundation for the treatment of dendritic cells mediated autoimmune diseases.