S100A12在胸膜肺炎放线杆菌诱导猪肺泡巨噬细胞炎性细胞因子表达及吞噬中的作用

doi:10.16431/j.cnki.1671-7236.2021.11.036

摘要/Abstract

摘要： 试验旨在探究S100A12在胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae，APP）诱导猪肺泡巨噬细胞（porcine alveolar macrophages，PAM）炎性细胞因子表达及吞噬中的作用。PAM细胞接种APP菌株后，用实时荧光定量PCR检测感染后不同时间点炎性细胞因子和S100A12的mRNA表达水平；构建S100A12重组蛋白表达载体并进行重组蛋白的表达纯化，用MTT法检测其对PAM的细胞毒性，实时荧光定量PCR检测不同浓度（0、5、50和500 ng/mL） S100A12重组蛋白对PAM炎性细胞因子mRNA表达水平的影响；构建S100A12干扰质粒，用Western blotting、实时荧光定量PCR及流式细胞术检测其干扰效率及其对APP感染PAM后炎性细胞因子mRNA表达水平和细胞凋亡的影响；用平板计数法检测S100A12表达对APP的黏附侵袭及其在PAM细胞内存活的影响。结果表明，APP感染促进PAM中IL-6、IL-8、IL-18、IL-1β和TNF-α等炎性细胞因子表达的同时也促进S100A12的表达，且该表达随APP感染剂量增大及时间的延长均显著或极显著增加（P<0.05；P<0.01）。5、50和500 ng/mL重组蛋白S100A12对PAM均没有毒性。5和50 ng/mL的重组蛋白S100A12均可显著促进PAM中IL-6、IL-8、TNF-α、IL-1β、IL-21和IL-5等炎性细胞因子的表达（P<0.05），而高浓度的S100A12重组蛋白则对上述炎性细胞因子表达无影响（P>0.05）。干扰质粒可成功阻断S100A12蛋白的表达，与转染shNC的对照组相比，在APP诱导下转染shS100A12干扰质粒的PAM中细胞因子IL-6、IL-8、IL-1β和IL-5的转录水平显著或极显著降低，且细胞凋亡率也显著或极显著增加（P<0.05；P<0.01）。进一步研究发现，干扰PAM中S100A12蛋白的表达可显著增强APP对PAM的黏附侵袭能力及其在PAM内的存活能力（P<0.05），相反，体外添加S100A12重组蛋白则显著抑制APP对PAM细胞的黏附侵袭及其在PAM内的存活（P<0.05）。以上研究表明，S100A12具有增强APP感染后PAM炎性细胞因子的表达、抑制APP诱导的PAM凋亡和降低APP对PAM的黏附侵袭及其在PAM内存活的作用，为进一步揭示APP感染过程中S100A12对PAM调控作用及机制奠定了基础。

关键词: 胸膜肺炎放线杆菌(APP); S100A12; 猪肺泡巨噬细胞(PAM); 细胞因子

Abstract: The purpose of this study was to explore the role of S100A12 in the expression of inflammatory cytokines and phagocytosis in porcine alveolar macrophages (PAM) induced by Actinobacillus pleuropneumoniae (APP). After PAM cells were inoculated with APP strain, Real-time quantitative PCR was used to detect the mRNA expression levels of inflammatory cytokines and S100A12 at different time points after infection. S100A12 recombinant protein expression vector was constructed and purified, and its cytotoxicity to PAM was detected by MTT method. The effects of different concentrations (0, 5, 50 and 500 ng/mL) of S100A12 recombinant protein on the expression level of inflammatory cytokine mRNA in PAM were detected by Real-time quantitative PCR. S100A12 interference plasmid was constructed, and its interference efficiency and effects on inflammatory cytokine mRNA expression and apoptosis of PAM after APP infection were detected by Western blotting, Real-time quantitative PCR and flow cytometry. Plate counting method was used to detect the influence of S100A12 expression on APP adhesion and invasion and survival in PAM cells. The results showed that APP infection promoted the expression of IL-6, IL-8, IL-18, IL-1β and TNF-α as well as the expression of S100A12 in PAM, and the expression increased significantly or extremely significantly with the increase of APP infection dose and time (P<0.05;P<0.01). 5, 50 and 500 ng/mL recombinant protein S100A12 had no toxicity to PAM. The low concentration of S100A12 recombinant protein significantly promoted the expression of inflammatory cytokines IL-6, IL-8, TNF-α, IL-1β, IL-21 and IL-5 in PAM (P<0.05), while the high concentration of S100A12 recombinant protein had no effect on the expression of the above-mentioned inflammatory cytokines (P>0.05). The interference plasmid could successfully block the expression of S100A12 protein. Compared with control group transfected with shNC, the transcriptional level of the cytokines IL-6, IL-8, IL-1β and IL-5 in PAM transfected with shS100A12 interference plasmid under APP induction were significantly or extremely significantly reduced, and apoptosis rate was also significantly or extremely significantly increased (P<0.05;P<0.01). Further research found that interfering with the expression of S100A12 protein in PAM could significantly enhance the adhesion and invasion ability of APP to PAM and its survival ability in PAM (P<0.05). On the contrary, adding S100A12 recombinant protein in vitro significantly inhibited the adhesion, invasion and survival of APP to PAM cells (P<0.05). The above results showed that S100A12 could enhance the expression of inflammatory cytokines after APP infection in PAM cells, inhibit APP-induced PAM apoptosis, and reduce APP's adhesion and invasion to PAM and its survival in PAM cells, which laid a foundation for further revealing the regulatory effect and mechanism of S100A12 on PAM in the process of APP infection.

Key words: Actinobacillus pleuropneumoniae (APP); S100A12; porcine alveolar macrophages(PAM); cytokines

中图分类号:

S859.7

刘海瑶, 李娜, 张晓光, 李子亨, 鲍春彤, 姜合祥, 刘柏君, 肖佳孟, 王俊, 李丰阳, 雷连成. S100A12在胸膜肺炎放线杆菌诱导猪肺泡巨噬细胞炎性细胞因子表达及吞噬中的作用[J]. 中国畜牧兽医, 2021, 48(11): 4262-4273.

LIU Haiyao, LI Na, ZHANG Xiaoguang, LI Ziheng, BAO Chuntong, JIANG Hexiang, LIU Baijun, XIAO Jiameng, WANG Jun, LI Fengyang, LEI Liancheng. Role of S100A12 in the Expression of Inflammatory Cytokines and Phagocytosis in Porcine Alveolar Macrophages Induced by Actinobacillus pleuropneumoniae[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(11): 4262-4273.

参考文献

[1] HATHROUBI S, HANCOCK M A, BOSSE J T, et al. Surface polysaccharide mutants reveal that absence of O antigen reduces biofilm formation of Actinobacillus pleuropneumoniae[J]. Infection and Immunity, 2016, 84(1):127-137.
[2] PATTISON I H, HOWELL D G, ELLIOT J.A haemophilus-like organism isolated from pig lung and the associated pneumonic lesions[J]. Journal of Comparative Pathology, 1957, 67(4):320-330.
[3] GALLE L, MOREAU M, BALLAIRE R, et al. Targeted invalidation of SR-B1 in macrophages reduces macrophage apoptosis and accelerates atherosclerosis[J]. Cardiovascular Research, 2020, 116:554-565.
[4] WANG Y J, WEI S, SONG H, et al. Macrophage migration inhibitory factor derived from spinal cord is involved in activation of macrophages following gecko tail amputation[J]. FASEB Journal, 2019, 33:14798-14810.
[5] MARTIN S, GRAEME J K, KATHYYN J M.Connecting transcriptional and functional macrophage heterogeneity in atherosclerosis[J]. Circulation Research, 2019, 125(12):1052.
[6] CHIOU J W, FU B, CHOU R, et al. Blocking the interactions between calcium-bound S100A12 protein and the V domain of RAGE using tranilast[J]. PLoS One, 2016, 11:e0162000.
[7] MINTS M, LANDIN D, NASMAN A, et al. Tumour inflammation signature and expression of S100A12 and HLA class Ⅰ improve survival in HPV-negative hypopharyngeal cancer[J]. Scientific Reports, 2021, 11(1):1782.
[8] WANG Q, ALESHINTSEV A, JOSE A N, et al. Calcium regulates S100A12 zinc sequestration by limiting structural variations[J]. Chembiochem, 2020, 21(9):1372.
[9] ZHANG Z, HAN N N, SHEN Y.S100A12 promotes inflammation and cell apoptosis in sepsis-induced ARDS via activation of NLRP3 inflammasome signaling[J]. Molecular Immunology, 2020, 122:38-48.
[10] DEMIRCI S G, ZAMANI A, YOSUNKAYA S, et al. Serum S100A12 and S100B proteins are independent predictors of the presence and severity of obstructive sleep apnea[J]. Turkish Journal of Medical Sciences, 2019, 49:746-754.
[11] JIANG H, ZHU R, LIU H, et al. Transcriptomic analysis of porcine PBMCs in response to Actinobacillus pleuropneumoniae reveals the dynamic changes of differentially expressed genes related to immuno-inflammatory responses[J]. Antonie Van Leeuwenhoek, 2018, 111(12):2371-2384.
[12] HOU F, WANG L, WANG H, et al. Elevated gene expression of S100A12 is correlated with the predominant clinical inflammatory factors in patients with bacterial pneumonia[J]. Molecular Medicine Reports, 2015, 11(6):4345-4352.
[13] 陈旋, 熊旭东.RAGE及其配体钙粒蛋白S100A12在急性肺损伤中研究进展[J]. 临床急诊杂志, 2017, 18(11):867-869. CHEN X, XIONG X D.Review of RAGE and ligand S100A12 in the research of acute lung injury[J]. Journal of Clinical Emergency, 2017, 18(11):867-869.(in Chinese)
[14] KOVAČIĆ M, MITROVIĆ-AJTIĆ O, BELESLIN-ČOKIĆ B, et al. TLR4 and RAGE conversely mediate pro-inflammatory S100A8/9-mediated inhibition of proliferation-linked signaling in myeloproliferative neoplasms[J]. Cell, 2018, 41(5):541-553.
[15] SKRONSKA W W, DURLANIK S, GARNETT J P, et al. Polarized cytokine release from airway epithelium differentially influences macrophage phenotype[J]. Molecular Immunology, 2021, 132:142-149.
[16] HOFMANN M A, DRURY S, FU C, et al. RAGE mediates anovel proinflammatory axis:A central cell surface receptor for S100/calgranulin polypeptides[J]. Cell, 1999, 97(7):889-901.
[17] GOYETTE J, YAN W X, YAMEN E, et al. Pleiotropic roles of S100A12 in coronary atherosclerotic plaque formation and rupture[J]. Journal of Immunology, 2009, 183(1):593-603.
[18] JANSSEN W J, BARTHEL L, MULDROW A, et al. Fas determines differential fates of resident and recruited macrophages during resolution of acute lung injury[J]. American Journal of Respiratory and Critical Care Medicine, 2011, 184:547-60.
[19] 叶钧, 宋丽丽, 刘韵, 等.肠分化细胞黏蛋白O-型糖链合成抑制导致MUC2表达减低以及对细菌侵袭易感[J]. 第三军医大学学报, 2013, 19:2056-2059. YE J, SONG L L, LIU Y, et al. Inhibition of O-glycan chains synthesis in intestinal differentiated epithelial cells induces lower MUC2 expression and susceptibility to bacterial invasion[J]. Journal of Third Military Medical University, 2013, 19:2056-2059.(in Chinese)
[20] 陈雪燕, JOHN M D, 江玲丽, 等.重组质粒在减毒沙门氏菌中的稳定性及其对细菌侵袭力的影响[J]. 微生物学报, 2005, 5:744-747. CHEN X Y, JOHN M D, JIANG L L, et al. Stability of recombinant plasmids in attenuated Salmonella typhimurium and their effects on bacterial invasiveness and multiplication[J]. Acta Microbiologica Sinica, 2005, 5:744-747.(in Chinese)