Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (1): 216-226.doi: 10.3864/j.issn.0578-1752.2024.01.015

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

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. 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 Online:2024-01-01 Published:2024-01-10

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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

Table 1

Primer sequences"

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

Fig. 1

The detection of inflammatory cytokines and inflammatory damage in the lung after Mhp infection A. Lung lesions; B. Histological examination of lung; C. Quantitative PCR detection of Mhp in lung; D. Detection of inflammatory cytokines in BALF"

Fig. 2

Mhp inhibited the transcription and expression of SPLUNC1 A: IFA detection of SPLUNC1 in lung; B: Relative quantitative PCR detection of splunc1 in lung; C: Western-blotting detection and quantitative analysis of relative protein of SPLUNC1 in PBECs; D: Relative quantitative PCR detection of splunc1 in PBECs"

Fig. 3

splunc1 cloning, construction of recombinant plasmids and the protein expression of SPLUNC1 A: PCR amplification of splunc1 gene, M:Marker;1:splunc1. B: Identification of recombinant plasmids by enzyme digestion, M:Marker;1-2:Identification of pET28a-SPLUNC1 by enzyme digestion;3-4:Identification of pCDNA3.1-SPLUNC1 by enzyme digestion. C: SDS-PAGE identification of SPLUNC1 prokaryotic expression protein, M:Marker;1-2:BSA; 3:Recombinant BL21 expressing bacteria without IPTG induction; 4-5:Recombinant BL21 expressing bacteria with IPTG induction. D. Western-blotting identification of SPLUNC1 prokaryotic expression protein. M: Marker; 1-6: Purified SPLUNC1 protein"

Fig. 4

Antibacterial activity of SPLUNC1 on Mhp A. Antibacterial effect of SPLUNC1 (25 µg·mL-1) on Mhp (103, 105, 107CCU50/mL); B. Antibacterial effect of SPLUNC1 (10, 25, 50 µg·mL-1) on Mhp (105CCU50/mL); C. Effect of SPLUNC1 antibody blocking on the growth of Mhp in the lung of mice"

Fig. 5

The effect of SPLUNC1 on Mhp adhesion in vitro A-B: Western-blotting detection of the effects of over-expression (A) or siRNA interference (B) of SPLUNC1 on Mhp adhesion and quantitative analysis of relative proteins; C: IFA detection of the effects of over-expression or siRNA interference of SPLUNC1 on Mhp adhesion and the integral optical density (IOD) values of the fluorescence staining"

Fig. 6

The effects of SPLUNC1 on the expression of CXCL8 and the activation of ERK pathway induced by Mhp ELISA detection of the effects of over-expression (A) or siRNA interference (B) of SPLUNC1 to the expression of CXCL8 induced by Mhp; Western-blotting detection of the effects of over-expression (C) or siRNA interference (D) of SPLUNC1 to the activation of ERK pathway induced by Mhp and quantitative analysis of relative proteins"

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