| Objective:Polyunsaturated fatty acids (PUFA) are indispensable parts of metabolism of our bodies. They are not only important parts of the cell membrane, but also have different effects to mediate the pro-inflammatory or anti-inflammatory effects. However, their molecular mechanism in the pro-inflammatory or anti-inflammatory procedure had not been well elucidated. Our study focused on the regulation of prostaglandin on the dendritic cells and its possible molecular pathway.Methods:1. Regulation of prostaglandin E2 and E3 on bone marrow stem cells differentiate to dendritic cells.(1) Bone marrow (BM) cells were flushed out from femur and tibia of C57BL/6 mice. Erythrocytes were lysed by ACK Lysis buffer. Bone marrow cells were collected to count the total number of cells, and then single BM cell suspension was prepared.(2) Bone marrow cells were plated at 6×105 per well into 24 plate, and were all cultured in the serum-free medium supplement with 10ng/ml GM-CSF and 1ng/ml IL-4, and with or without 50ng/ml PGE2 or PGE3.50ng/ml PGE2 while 50ng/ml PGE3 was supplement. Half of the medium was replaced every day. After being cultured for six days later, cells were harvested and used for the further experiments.(3) Total cell numbers were counted. The morphology of differentiated cells were observed under microscopy by using Wright staining.(4) The percentage of CD11b+CD11c+ dendritic cells and Ly6G+CDllb+myeloid MDSCs were analyzed by the flow cytometry to explore the regulatory effects of prostaglandins in DC differentiation.(5) The mRNA levels of IL-12, IDO, IL-10 Arg-land COX-2 in total differentiated BM cells and purified CD11c+ dendritic cells were determined by Q-PCR.(6) Considering the important role of PPARy in the regulation of lipid metabolism, we examined the PPARy expression in GM-CSF and IL-4 treated bone marrow cells at Oh, 2h,6h,12h,24h which with or without prostaglandins.2. The effects of prostaglandin E2 and E3 on mature dendritic cells.(1) Mouse bone marrow cells were cultured with GM-CSF and IL-4 for 7 days, and then, differentiated dendritic cells were collected. Cells were plated into wells of the 24 plate at 6×105 cells per well with different concentrations of prostaglandin. After being incubated for 24h, cells were collected and used for the further experiments.(2) The expression of CD80, CD86, MHC-Ⅱ on dendritic cells were detected by flow cytometry.(3) The mRNA level of IL-12 and IL-10 was detected by Q-PCR.(4) To analyze its capability in priming the antigen specific T cells proliferation, dendritic cells were firstly incubated with OVA for 6h, and then cultured with CFSE labeled CD4+T cells, which purified from the F1 generation of C57BL/6 and DO 11.10 mice. Being cultured for 3 days, cells were collected, labeled with anti-CD4-PE and 7-AAD, and cell divisions were detected by using flow cytometric analysis.(5) To analyze the phagocytosis of dendritic cells, prostaglandin treated dendritic cells were incubated with OVA-Alexa488 at 37℃ for 2h, and the phagocytic function of DC was determined by flow cytometry.Results:(1) In the procedure of bone marrow stem cells differentiating into dendritic cells, prostaglandin addition lead the total cell number increase, espeacially granulocyte, which confirmed by Wright’s staining. Compared with the control group, the percentages of CDllb+CDllc+DC cells were decreased and CDllb+Ly6G+ cell population were increased in PGs treated cells. The levels of anti-inflammatory mediators Arg-1, IDO and IL-10 were higher in PGE2-DC than PGE3-DC. The Q-PCR analysis data of CDllc+ dendritic cells showed that immune suppression factor and COX-2 genes were up-regulated in prostaglandin treated DC, especially in PGE2-DC.(2) The western blot data showed bone marrow stem cells treated with PGE2 expressed the highest of PPARy at 2h, but cells treated with PGE3 were at 12h and indicated that PGE2 has a strong effect than PGE3 in PPARy regulation.(3) The data showed that low concentration (50ng/ml) of PGE2 promoted mature DCs highly express CD86 and MHC-Ⅱ, but high concentration (1000ng/ml) PGE2 inhibited its expression. Consistent with the flow cytometry results, the expression of inflammatory cytokines IL-12 was increased, while the anti-inflammatory cytokine IL-10 expression was decreased in low concentration PGE2 treated DC.(4) PGE2-DC had stronger capability in priming OVA specific CD4+ T cell proliferation than PGE3-DC. On the contrary, PGE3-DC expressed enhanced DC capacity in antigen phagocytosis.Conclusion:Prostaglandins had regulatory functions in bone marrow derived dendritic cells differentiation in vitro, which cultured with GM-CSF and IL-4. Actually, both PGE2 and PGE3 inhibited CD11c+ dendritic cells differentiation, but promoted Ly6G+ immature myeloid cells production, which showed MDSC cells like immunsuppressive characteristics. This prostaglandin mediated regulation may be depended on the up-regulation of PPARy and COX-2. Interestingly, low concentrations of PGE2 up-regulated the expression of costimulatory molecules, MHC-Ⅱ and cytokines secretion of DCs. To the contrary, high concentration of PGE2 (500ng/ml) showed suppression on DCs. Comparing the effects of PGE2 or PGE3 at concentration of 50ng/ml on the function of DCs, we found that PGE2 treated DCs (PGE2-DC) showed higher OVA antigen presenting ability to CD4+T cells than the PGE3 treated DCs (PGE3-DC), and PGE2-DC primed more OVA antigen specific CD4+T cells into proliferation. Contrarily, the OVA phagocytosis ability of PGE3-DC was stronger than PGE2-DC. These data demonstrated that PGE2 had higher efficiency in promoting the immune functions of DCs than PGE3 at low concentration. As prostaglandins work as major inflammatory mediators, our study suggested that different prostaglandins had different regulatory functions in immune cells, and then indicated its potential usage in inflammatory diseases. |
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