TFPR1通过激活树突状细胞并促进其成熟发挥佐剂功能
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摘要
目的研究树突状细胞(DC)在重组蛋白TFPR1发挥佐剂功能中的作用。方法在无菌条件下取4~5周龄雄性BALB/c小鼠的骨髓细胞,小鼠重组粒细胞-巨噬细胞集落刺激因子(rm GM-CSF)与重组白介素4(rm IL-4)诱导分化6 d后,加入TFPR1共孵育24 h,并以LPS处理为阳性对照、PBS为阴性对照,普通光学显微镜观察DC形态变化,罗丹明标记鬼笔环肽(TRITC Phalloidin)和DAPI共染色后,激光共聚焦显微镜观察细胞肌动蛋白和细胞核的分布情况,流式细胞术检测细胞表面CD40、CD80、CD86、MHCⅡ的表达水平,ELISA检测细胞培养上清中的细胞因子。结果在普通光镜和共聚焦显微镜下,加入TFPR1的DC与加入PBS的阴性对照DC相比具有显著的不同,而和加入LPS的阳性对照相似:光镜下发现大多数TFPR1处理的DC伪足变短直至消失,并变成圆形、悬浮于培养基中;激光共聚焦显微镜观察发现TFPR1处理的DC肌动蛋白由分布于细胞两极转至均匀分布于细胞膜上,从形态学上表明TFPR1可促进DC成熟;流式细胞术检测到TFPR1处理的DC表面CD40、CD80、CD86、MHCⅡ的表达量上调,进一步证明TFPR1具有促进DC成熟的作用。ELISA检测发现TFPR1处理的DC可产生高水平的IL-6、IL-8、TNF-α等细胞因子。结论 TFPR1不但能促进DC成熟,且能激活DC产生细胞因子,说明DC在TFPR1的佐剂功能中发挥重要作用。
Objective To study the role of dendritic cells in the function of a recombinant protein TFPR1 as an adjuvant. Methods Bone marrow cells were collected from four-to five-week-old male BALB/c mice under aseptic conditions,and cultured with complete RPMI 1640 containing rm GM-CSF and rm IL-4 for six days. TFPR1 was added on day 6,and cells were incubated for another 24 hours. LPS was used as positive control,while PBS as negative. The morphology of dendritic cells was observed under an optical microscope and laser confocal microscope,cell surface makers( CD40,CD80,CD86 and MHCⅡ) were detected with flow cytometry,and the cytokines in the supernatant were detected with ELISA. Results Compared with negative control,dendritic cells incubated with TFPR1 for 24 hours were significantly different in morphology as was observed by optical and laser confocal microscopes,but were similar to positive control. Most of the dendritic cells treated with TFPR1 showed less adherence and became round,whose podosomes became shorter,and even disappeared. Actin distribution changed from two poles of the cell to the membrane. CD40,CD80,CD86 and MHCⅡon the cell surface were up-regulated on stimulation by TFPR1,as was detected by FACS. These results showed that TFPR1 was capable of promoting dendritic cell maturation. ELISA showed dendritic cells treated with TFPR1 secreted high levels of cytokines( IL-6,IL-8 and TNF-α). Conclusion TFPR1 is capable of promoting dendritic cell maturation,and activating cells to produce cytokines,indicating that dendritic cells can play an important role in the function of TFPR1 as a novel adjuvant.
Objective To study the role of dendritic cells in the function of a recombinant protein TFPR1 as an adjuvant. Methods Bone marrow cells were collected from four-to five-week-old male BALB/c mice under aseptic conditions,and cultured with complete RPMI 1640 containing rm GM-CSF and rm IL-4 for six days. TFPR1 was added on day 6,and cells were incubated for another 24 hours. LPS was used as positive control,while PBS as negative. The morphology of dendritic cells was observed under an optical microscope and laser confocal microscope,cell surface makers( CD40,CD80,CD86 and MHCⅡ) were detected with flow cytometry,and the cytokines in the supernatant were detected with ELISA. Results Compared with negative control,dendritic cells incubated with TFPR1 for 24 hours were significantly different in morphology as was observed by optical and laser confocal microscopes,but were similar to positive control. Most of the dendritic cells treated with TFPR1 showed less adherence and became round,whose podosomes became shorter,and even disappeared. Actin distribution changed from two poles of the cell to the membrane. CD40,CD80,CD86 and MHCⅡon the cell surface were up-regulated on stimulation by TFPR1,as was detected by FACS. These results showed that TFPR1 was capable of promoting dendritic cell maturation. ELISA showed dendritic cells treated with TFPR1 secreted high levels of cytokines( IL-6,IL-8 and TNF-α). Conclusion TFPR1 is capable of promoting dendritic cell maturation,and activating cells to produce cytokines,indicating that dendritic cells can play an important role in the function of TFPR1 as a novel adjuvant.
引文
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[18]Brahmakshatriya V,Kuang Y,Devarajan P,et al.IL-6 production by TLR-activated APC broadly enhances aged cognate CD4helper and B cell antibody responses in vivo[J].J Immunol,2017,198(7):2819-2833.
[19]Russo RC,Garcia CC,Teixeira MM,et al.The CXCL8/IL-8chemokine family and its receptors in inflammatory diseases[J].Exp Rev Clin Immunol,2014,10(5):593-619.
[20]Kalliolias GD,Ivashkiv LB.TNF biology,pathogenic mechanisms and emerging therapeutic strategies[J].Nat Rev Rheumatol,2016,12(1):49-62.
[2]Gar9on N,Di Pasquale A.From discovery to licensure,the adjuvant system story[J].Hum Vaccin Immunother,2017,13(1):19-33.
[3]López-Yglesias AH,Zhao X,Quarles EK,et al.Flagellin induces antibody responses through a TLR5-and inflammasome-independent pathway[J].J Immunol,2014,192(4):1587-1596.
[4]Guo J,Yang Y,Xiao W,et al.A truncated fragment of Ov-ASP-1 consisting of the core pathogenesis-related-1(PR-1)domain maintains adjuvanticity as the full-length protein[J].Vaccine,2015,33(16):1974-1980.
[5]Wang J,Tricoche N,Du L,et al.The adjuvanticity of an O.volvulus-derived r Ov-ASP-1 protein in mice using sequential vaccinations and in non-human primates[J].Plo S One,2012,7(5):e37019.
[6]寇志华,周育森,孙维来,等.TFPR1蛋白及其编码基因与其在免疫调节中的应用:中国,2015100276641[P].2015-05-06.
[7]孙维来,杨裔,宁秀哲,等.蛇毒蛋白triflin功能区TFPR1在大肠杆菌中的表达及其功能初步研究[J].生物技术通讯,2015,26(3):329-333.
[8]宁秀哲,寇志华,孙维来,等.新型佐剂蛋白TFPR1在毕赤酵母系统中的表达及鉴定[J].中华微生物学和免疫学杂志,2016,36(4):294-299.
[9]Worbs T,Hammerschmidt SI,F9rster R.Dendritic cell migration in health and disease[J].Nat Rev Immunol,2017,17(1):30-48.
[10]Olguín-Alor R,de la Fuente-Granada M,Bonifaz LC,et al.A key role for inhibins in dendritic cell maturation and function[J].Plo S One,2016,11(12):e0167813.
[11]Burns S,Hardy SJ,Buddle J,et al.Maturation of DC is associated with changes in motile characteristics and adherence[J].Cytoskeleton,2004,57(2):118-132.
[12]Schulz AM,Stutte S,Hogl S,et al.Cdc42-dependent actin dynamics controls maturation and secretory activity of dendritic cells[J].J Cell Biol,2015,211(3):553.
[13]Vargas P,Maiuri P,Bretou M,et al.Innate control of actin nucleation determines two distinct migration behaviours in dendritic cells[J].Nat Cell Biol,2016,18(1):43-53.
[14]Roche PA,Furuta K.The ins and outs of MHC classⅡ-mediated antigen processing and presentation[J].Nat Rev Immunol,2015,15(4):203-216.
[15]Zaccard CR,Watkins SC,Kalinski P,et al.CD40L induces functional tunneling nanotube networks exclusively in dendritic cells programmed by mediators of type 1 immunity[J].J Immunol,2015,194(3):1047-1056.
[16]Pulendran B.The varieties of immunological experience:of pathogens,stress,and dendritic cells[J].Ann Rev Immunol,2015,33:563-606.
[17]Benko S,Magyarics Z,SzabóA,et al.Dendritic cell subtypes as primary targets of vaccines:the emerging role and cross-talk of pattern recognition receptors[J].Biol Chem,2008,389(5):469.
[18]Brahmakshatriya V,Kuang Y,Devarajan P,et al.IL-6 production by TLR-activated APC broadly enhances aged cognate CD4helper and B cell antibody responses in vivo[J].J Immunol,2017,198(7):2819-2833.
[19]Russo RC,Garcia CC,Teixeira MM,et al.The CXCL8/IL-8chemokine family and its receptors in inflammatory diseases[J].Exp Rev Clin Immunol,2014,10(5):593-619.
[20]Kalliolias GD,Ivashkiv LB.TNF biology,pathogenic mechanisms and emerging therapeutic strategies[J].Nat Rev Rheumatol,2016,12(1):49-62.