胸膜肺炎放线杆菌Adh通过活化NLRP3炎症小体诱导小鼠体内外炎症反应OA

To investigate the role and mechanism of the important virulence factor trimeric autotransporter adhesin(TAA)Adh of Actinobacillus pleuropneumoniae(APP)in APP-induced inflammatory responses,porcine alveolar macrophages(PAMs)and BALB/c mice were infected with the APP serotype 5b wild-type strain L20(5bWT)and its TAA-deficient mutant(5bΔAdh),respectively.In addition,the nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)specific inhibitor CRID3 sodium salt(MCC950)was used for treatment.Western blotting(WB),enzyme-linked immunosorbent assay(ELISA),real-time fluorescent quantitative reverse transcription polymerase chain reaction(RT-qPCR),and hematoxylin-eosin(H.E.)staining were performed to detect relevant indicators.The results showed that,compared with the Control group,the relative protein expression levels of cleaved gasdermin D(Cle-GSDMD)and cleaved cysteinyl aspartate specific proteinase-1(Cle-Caspase-1)in the 5bWT group were significantly increased(P<0.001).The levels of interleukin-1β(IL-1β),interleukin-18(IL-18),and interleukin-17 A(IL-17A)were significantly upregulated(P<0.05 or P<0.01),and the relative mRNA expression levels of monocyte chemoattractant protein-1(MCP-1),Caspase-1,interleukin-6(IL-6),and IL-18 were significantly increased(P<0.05).Compared with the 5bWT group,the protein expression levels and contents of the above inflammatory factors in the 5bΔAdh group were significantly reduced to varying degrees(P<0.05,P<0.01,or P<0.001).Compared with the DMSO+PBS group,the relative mRNA expression levels of IL-1β and IL-18 in lung tissues of mice in the DMSO+5bWT group were significantly increased(P<0.05),accompanied by obvious inflammatory pathological changes.Compared with the MCC950+5bWT group,the relative mRNA expression levels of IL-1β and IL-18 in lung tissues of mice in the MCC950+5bΔAdh group were significantly decreased(P<0.05),with reduced inflammatory cell infiltration in lung tissues.These results indicate that Adh is an important virulence factor of APP that activates the NLRP3 inflammasome and induces inflammatory responses both in vivo and in vitro.This study provides a scientific basis for elucidating the pathogenic mechanism of APP.