Effects And Mechanism Of NMU On Function Of Porcine Dendritic Cells In Vitro

Neuromedin U(NMU)is a neuropeptide,first separated from pig spinal cord in 1985,because it can cause the uterine contraction of rat in vitro,it was named NMU.NMU is usually present as peptides 8 or 25 amino acids long(NMU-8 and NMU-25,respectively),which have similar biological activities.There are two NMU receptors,NMU1R(FM-3/GPR66)and NMU2R(FM-4/TGR-1)respectively,which belong to the typical G protein coupled receptor(GPCR),and have seven transmembrane domains.NMU plays biological roles through these two receptors.As NMU and its receptors widely distribute in human and animal bodies,they are involved in a variety of physiological functions,including stimulating smooth muscle contraction,regulation of feeding,energy balance,blood pressure,stress,gastrointestinal motility,endocrine and immunity etc.NMU mRNA has been detected in dendritic cells,and NMUR1 mRNA has been detected in immune effector cells,particularly in T and natural killer cells,suggesting an importance role of NMU in the immune response.Dendritic cell(DC),one of the most powerful antigen presenting cells,is famous for its abundant antigen presenting molecules on its surface,which can effectively activate the initial T cells.DC can also regulate memory B cells and plasma cells by secreting IL-6 and IFN-y,thus affecting the humoral immunity.NMU can induce synthesis and release of several different cytokines from Th2 cells in vitro.In vivo studies were shown that NMU is required for IL-6 production by macrophages,which have the same antigen presenting function as DC.At present,the studies on the immunity regulation of NMU have been reported,and mainly concentrated in mast cells,neutrophils and immunologic effector cells.Because very few DC exists alone within animal body,the study on effect of NMU function of dendritic cells has not been reported.Therefore,in this study,we isolated from peripheral blood of the wholesome pigs,and then transformed into immature dendritic cells by GM-CSF and IL-4 in vitro.These cells were later matured by LPS.First,we studied the distribution of NMU,NMUR1 and NMUR2 in dendritic cells.Next,we investigated the effect of NMU on phagocytosis,proliferation and apoptosis of pig DC.Finally,we studied the effect and mechanism of NMU on secretion of cytokines of pig DC.These studies provide the experimental evidence for the role of NMU in pig immune regulation.1 The effect of NMU on the proliferation and apoptosis of dendritic cells in vitroThis experiment detected the expression of NMU and its receptors mRNA in pig DC by RT-PCR and the distribution of their proteins in the DC with immunocytochemistry,and studied the effect of different doses of(0.01,0.1,1.0,10,100 and 1 000 nM)NMU on the proliferation and apoptosis of the DC by CCK-8 method and FACS respectively.The results are as follows:(1)NMU and NMUR1 mRNA expressed in pig DC,and their protein were localized in cellular membrane and cytoplasm of the DC.(2)0.1?1 000 nM NMU significantly promoted the proliferation of pig immature DC(P<0.01),with the maximum proliferation effect by 1 000 nM NMU(1.33±0.060).0.1?10 nM NMU also significantly stimulated the proliferation of porcine mature DC(P<0.01),but 1 000 nM NMU significantly inhibited the proliferation of porcine mature DC.(3)0.1,1.0,10 and 100 nM NMU significantly reduced the apoptosis of porcine iDC(P<0.01),with the minimum apoptosis ratio by 10 nM NMU(2.360±0.3113%).1?100 nM NMU and LPS acted synergistically in inhibition of the apoptosis of the DC(P<0.01).2 The effect of NMU on function of porcine dendritic cells in vitroVaried doses of NMU(0.01,0.1,1,10,100,1 000 nM)were added into pig DC in culture medium for 2,4,8,12 and 24 h,then the culture supernatant of mDC was collected to study the secretion of IL-1?,IL-4,IL-5,IL-6,IL-8,TNF-a,IFN-?,IL-10 and IL-13 in mDC and the mechanism of NMU-evoked cytokin secretion by RIA or ELISA Kit.Finally,the phagocytosis of DC and the capacity of DC to induce T cell proliferation were investigated by FACS and CCK-8 Kit.The results are as follows:(1)Low and medium doses(1?100 nM)NMU promoted the phagocytic capacity of pig immature dendritic cells.However,but high dose(1 000 nM)NMU decreased the phagocytosis of the iDC.(2)Both iDC and mDC can promote the proliferation of T lymphocytes after incubation with NMU in a dose-dependent manner.(3)1.0 nM and 10 nM NMU up-regulated the secretion of IL-1? in porcine dendritic cells.(4)All doses of NMU increased the secretion of IL-8,TNF-?,IFN-?,IL-5 and IL-6,and decreased the secretion of IL-4 and IL-10 in porcine dendritic cells,except 1 000 nM NMU,which down-regulated IFN-y secretion at 4 h.However,the secretion of IL-13 appeared diversity trend.It turned out that 0.01?0.1 nM NMU significantly inhibited the expression of IL-13.1?10 nM NMU inhibited the IL-13 release in the early stage,while it increased the IL-13 secretion of the DC after 2 h.100?1 000 nM NMU mainly increased the expression of IL-13.(5)After pig DCs were pretreated with PLC inhibitor,JAK inhibitor,PI3K inhibitor and MEK1/2 inhibitor,NMU-evoked IL-6,TNF-a and IL-10 secretion in the DC was detected by RIA.The results showed that NMU stimulated the secretion of IL-6 and TNF-a in the DC by PLC,JAK,PI3K and MEK signaling pathway,while inhibited the secretion of IL-10 only through PLC,JAK and PI3K pathway,not through the MEK signaling pathway.In summary,certain doses of NMU can promote the proliferation of pig DC,inhibit their apoptosis,improve their endocytic activity,stimulate the T lymphocytes proliferation induced by the DC,and affect the secretion of cytokines in dendritic cells.The results suggested that NMU can involve in the regulation of pig immune function.