猪肺炎支原体通过抑制SPLUNC1功能破坏呼吸道炎性反应平衡

doi:10.3864/j.issn.0578-1752.2024.01.015

中国农业科学 ›› 2024, Vol. 57 ›› Issue (1): 216-226.doi: 10.3864/j.issn.0578-1752.2024.01.015

• 畜牧·兽医 • 上一篇

猪肺炎支原体通过抑制SPLUNC1功能破坏呼吸道炎性反应平衡

王海燕¹^,²(), 张珍珍¹^,², 倪博¹^,², 刘蓓蓓¹^,², 冯志新¹^,²()

¹ 江苏省农业科学院兽医研究所/农业农村部兽用生物制品工程技术重点实验室/国家兽用生物制品工程技术研究中心，南京 210014
² 兽用生物制品（泰州）国泰技术创新中心，江苏泰州 225300

收稿日期:2022-12-23 接受日期:2023-11-30 出版日期:2024-01-01 发布日期:2024-01-10
通信作者:
冯志新，E-mail：fzxjaas@163.com
联系方式: 王海燕，E-mail：wang-haiyan1983@163.com。
基金资助:
国家自然科学基金(31700157); 国家自然科学基金(31900159); 江苏省基础研究计划自然科学基金(BK20221431); 江苏省基础研究计划自然科学基金(CX(21)3140)

Mycoplasma Hyopneumoniae Destroyed the Inflammatory Balance of Respiratory Tract Through Suppressing the Function of SPLUNC1

WANG HaiYan¹^,²(), ZHANG ZhenZhen¹^,², NI Bo¹^,², LIU BeiBei¹^,², FENG ZhiXin¹^,²()

¹ Institute of Veterinary Medicine/Key Laboratory of Veterinary Biological Engineering and Technology of Ministry of Agriculture and Rural Affairs/National Research Center of Engineering and Technology for Veterinary Biologicals, Jiangsu Academy of Agricultural Sciences, Nanjing 210014
² GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, Jiangsu

Received:2022-12-23 Accepted:2023-11-30 Published:2024-01-01 Online:2024-01-10

摘要/Abstract

摘要：

【背景】猪肺炎支原体(Mycoplasma hyopneumoniae，Mhp)通过定植猪呼吸道黏膜层，破坏呼吸道炎性反应平衡，引起炎性损伤和持续性感染。短的上腭、肺及鼻咽上皮克隆1蛋白(short palate lung and nasal epithelial clone1, SPLUNC1)是呼吸道黏膜分泌的具有重要抗菌和抗炎功能的蛋白，被认为是呼吸道黏膜面对危险信号时的“信号传感器”。【目的】从Mhp和SPLUNC1相互作用入手，分析Mhp感染对SPLUNC1表达的影响以及SPLUNC1对Mhp引起的炎性反应的调控作用，揭示Mhp引起炎性损伤的新机制，为解决Mhp持续性感染问题提供参考。【方法】利用猪肺炎支原体对猪支气管上皮细胞（porcine bronchial epithelial cells，PBECs）感染模型和猪体感染模型，通过荧光定量PCR、间接免疫荧光和Western-blotting等方法，分别检测Mhp感染后对肺脏中SPLUNC1以及体外PBECs中SPLUNC1转录和表达的影响。克隆并扩增猪源SPLUNC1基因通过酶切鉴定，构建成功SPLUNC1真核和原核表达重组质粒pCDNA3.1-SPLUNC1以及pET28a-SPLUNC1。与此同时，设计靶向SPLUNC1的siRNA干扰片段。利用体外SPLUNC1蛋白孵育、体内呼吸道黏膜SPLUNC1抗体封闭，通过Mhp CCU50检测明确SPLUNC1对Mhp体内外生长的影响。在猪支气管上皮细胞中，通过过表达或siRNA干扰SPLUNC1基因，后感染Mhp，利用Western-blotting、间接免疫荧光以及酶联免疫吸附方法，明确SPLUNC1对Mhp的黏附作用、诱导炎性因子表达以及MAPK信号通路活化的影响。【结果】Mhp感染猪后可引起肺脏实变，主要组织病理表现为肺脏炎性损伤，同时显著上调肺泡灌洗液中趋化因子CXCL8和炎性因子TNFα和IL-1β分泌。Mhp体内外感染后，体内肺脏和体外猪支气管上皮细胞中SPLUNC1的转录和表达均显著下调。以上研究表明，Mhp可诱导肺脏炎性反应，抑制SPLUNC1表达。另一方面，体外PBECs中过表达SPLUNC1后，Mhp感染引起的CXCL8表达显著减少；siRNA干扰SPLUNC1后，Mhp感染引起的CXCL8表达显著增加，表明SPLUNC1负调控Mhp感染引起的CXCL8表达。基于Mhp感染入侵呼吸道过程，本研究进一步解析SPLUNC1负调控CXCL8表达的机制。结果表明：无论是SPLUNC1和Mhp体外孵育，还是SPLUNC1抗体体内封闭，Mhp的体内外生长均未受影响；SPLUNC1的过表达或siRNA干扰对Mhp体外黏附PBECs的能力也无显著影响；过表达SPLUNC1可抑制pERK和IκBα的激活，相反SPLUNC1 siRNA干扰后，可促进pERK和IκBα的激活。以上研究证明SPLUNC1不是通过调控Mhp的生长和黏附，而是通过负调控MAPK-ERK通路的活化来调控CXCL8的表达。【结论】SPLUNC1可通过MAPK-ERK信号通路来调控炎性因子的过度表达，维护宿主炎性平衡；与此同时，Mhp感染后可通过抑制SPLUNC1的表达来破坏宿主炎性反应的平衡调控，进而引起炎性损伤。本研究为解析Mhp感染损伤机制提供依据。

关键词: 猪肺炎支原体, 支原体-宿主相互作用, 炎性反应, SPLUNC1

Abstract:

【Background】Mycoplasma hyopneumoniae (Mhp) invades the body through respiratory mucosa, then it destroys the inflammatory balance and causes inflammatory damage. Short palate lung and nasal epithelial clone1 (SPLUNC1) is a protein secreted by respiratory mucosa with important antibacterial and anti-inflammatory functions, it is considered to be a “signal sensor” for respiratory mucosa in the face of danger signals. 【Objective】This study focused on the interaction between Mhp and SPLUNC1, and analyzed the influence of Mhp on the expression of SPLUNC1 and the regulatory effect of SPLUNC1 on the inflammatory response caused by Mhp. This study also revealed a new mechanism of inflammatory damage caused by Mhp, and it was of great significance to resolve the problem of persistent infection of Mhp. 【Method】Quantitative PCR, indirect immunofluorescence and Western-blotting methods were used to detect the effects of Mhp on SPLUNC1 transcription and expression in PBECs and piglets. Porcine SPLUNC1 gene was cloned and amplified from the PBECs, and the eukaryotic and prokaryotic expression recombinant plasmids pCDNA3.1- SPLUNC1 and pET28a-SPLUNC1 were constructed successfully. Meanwhile, siRNA interference sequence targeting SPLUNC1 were also designed. The effect of SPLUNC1 on the growth of Mhp was clarified by in vitro incubation experiment and SPLUNC1 blocking experiment in mice. After over-expressing or siRNA interfering of SPLUNC1, Western-blotting, IFA and ELISA were used to detect the effects of SPLUNC1 on the Mhp adhesion, CXCL8 expression and the activation of MAPK pathway. 【Result】Mhp infection induced inflammatory damage, accompanied by CXCL8, TNFα and IL-1β expression in the lung. Meanwhile, the transcription and protein expression of SPLUNC1 was also inhibited in vivo and PBECs after Mhp infection. The above studies indicated that Mhp induced pulmonary inflammatory response and inhibited the expression of SPLUNC1. Over-expression of SPLUNC1 in PBECs significantly decreased CXCL8 expression, on the contrary, the knockdown of SPLUNC1 by siRNA interference significantly increased CXCL8 expression. The result showed that SPLUNC1 negatively regulated CXCL8 expression caused by Mhp infection. The growth of Mhp in vivo and vitro was not significantly inhibited by SPLUNC1. Over-expression or siRNA interference of SPLUNC1 also had no significant effect on the adhesion of Mhp. The over-expression of SPLUNC1 inhibited the activation of pERK and IκBα, whereas siRNA interference of SPLUNC1 upregulated the activation of pERK and IκBα. SPLUNC1 inhibited CXCL8 expression by inhibiting the activation of MAPK-ERK pathway. 【Conclusion】The respiratory mucosa regulated the expression of CXCL8 through SPLUNC1 to maintain the inflammatory balance of the host. However, in the process of infection, Mhp destroyed the inflammatory balance by inhibiting SPLUNC1 expression, thus causing inflammatory damage. Finally, this study provided an important basis for understanding the mechanism of infection and injury of Mhp.

Key words: Mycoplasma hyopneumoniae, interaction between mycoplasma and host, inflammatory reaction, SPLUNC1

王海燕, 张珍珍, 倪博, 刘蓓蓓, 冯志新. 猪肺炎支原体通过抑制SPLUNC1功能破坏呼吸道炎性反应平衡[J]. 中国农业科学, 2024, 57(1): 216-226.

WANG HaiYan, ZHANG ZhenZhen, NI Bo, LIU BeiBei, FENG ZhiXin. Mycoplasma Hyopneumoniae Destroyed the Inflammatory Balance of Respiratory Tract Through Suppressing the Function of SPLUNC1[J]. Scientia Agricultura Sinica, 2024, 57(1): 216-226.

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用途 Classification	引物名称 Primer name	引物序列 Primer sequence
扩增引物 Gene amplification	SPLUNC1-F	ATGTTTCAAGTTGCAGGCCTCATTG
扩增引物 Gene amplification	SPLUNC1-R	TTAGACTTTGATGACAAATTCCTG
真核表达质粒构建 Recombinant plasmids construction	pVAX1-SPLUNC1-F	ATAGAATTCATGTTTCAAGTTGCAGGCCTC
真核表达质粒构建 Recombinant plasmids construction	pVAX1-SPLUNC1-R	ATACTCGAGTTAGACTTTGATGACAAATTCCT
荧光定量PCR Quantitative PCR	SPLUNC1-F	TGAAAGACGAACAGGGGAAGA
	SPLUNC1-R	ATTGACCAGAGGGCACACC
	GAPDH-F	ACATGGCCTCCAAGGAGTAAGA
	GAPDH-R	GATCGAGTTGGGGCTGTGACT

猪肺炎支原体通过抑制SPLUNC1功能破坏呼吸道炎性反应平衡

Mycoplasma Hyopneumoniae Destroyed the Inflammatory Balance of Respiratory Tract Through Suppressing the Function of SPLUNC1

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