Study On The Immunoprotection And Proinflammatory Function Of Galt Protein Of Actinobacillus Pleuropneumoniae

Actinobacillus pleuropneumoniae?APP?is pathogenic agent of porcine contagious pleuropneumonia?PCP?,which causes fibrinous pleurisy and hemorrhagic pneumonia in pigs,causing a huge economic loss in world pig industry.Studies have shown that APP has a variety of virulence factors,including Apx exotoxin,lipopolysaccharide,capsular polysaccharide and so on.These virulence factors are expressed in vitro,and there are not many studies on the induction of antigen in APP.In vivo induced antigens are antigens that specifically expressed in the process of infection,and these antigens play an important role in the pathogenesis.The identification of A.pleuropneumoniae genes,specially expressed in vivo,is a useful tool to reveal the mechanism of infection.In vivo induced antigen technology?IVIAT?was used to screen in vivo induced antigens of APP.It's demonstrated that these antigens are preferentially expressed during infection.Immunity experiments showed that GalT can induce effective humoral and cellular immune response.Secretion of pro-inflammatory cytokines of macrophages were promoted after stimulated with GalT,and reveals the signal transduction pathway in this process.The content of our word are summarized as follows.1.IVIAT was used in this work to identify antigens expressed in vivo during A.pleuropneumoniae infection,using sera from individuals with chronic porcine pleuropneumonia.Sequencing of DNA inserts from positive clones showed 11 open reading frames with high homology to A.pleuropneumoniae genes.Based on sequence analysis,proteins encoded by these genes were involved in metabolism,replication,transcription regulation,and signal transduction.Moreover,three function-unknown proteins were also indentified in this work.Expression analysis using quantitative real-time PCR showed that most of the genes tested were up-regulated in vivo relative to their expression levels in vitro.IVI?in vivoinduced?genes that were amplified by PCR in different A.pleuropneumoniae strains showed that these genes could be detected in almost all of the strains.It is demonstrated that the identified IVI antigen may have important roles in the infection of A.pleuropneumoniae.2.Six in vivo-induced?IVI?antigens—RnhB,GalU,GalT,Apl₁061,Apl₁166,and HflX were selected for a vaccine trial in a mouse model.The results showed that the IgG levels in each immune group were significantly higher than that of the negative control?P < 0.001?.Except rRnhB group,proliferation of splenocytes was observed in all immunized groups and a relatively higher proliferation activity was observed in rGalU and rGalT groups?P < 0.05?.In the rGalT vaccinated group,the proportion of CD4 C T cells in spleen was significant higher than that of negative control?P < 0.05?.Moreover,proportions of CD4 C T cells in other vaccinated groups were all up-regulated to varying degrees.Up-regulation of both Th1?IFN-g,IL-2?and Th2?IL-4?cytokines were detected.A survival rate of 87.5,62.5,and 62.5% were obtained among rGalT,rAPL₁166,and rHflX group,respectively while the remaining three groups was only 25%.Histopathological analyses of lungs indicated that surviving animals from the vaccinated groups showed relatively normal pulmonary structure alveoli.These findings confirm that IVI antigens used as vaccine candidates provide partial protection against Actinobacillus pleuropneumoniae infection in a mouse model,which could be used as potential vaccine candidates in piglets.3.GalT is an important antigen of Actinobacillus pleuropneumoniae?APP?,which was shown to provide partial protection against APP infection in a previous study in our lab.The main purpose of the present study is to investigate GalT induced cross-protection between different APP serotypes and elucidate key mechanisms of the immune response to GalT antigenic stimulation.Bioinformatic analysis demonstrated that GalT is a highly conserved gene in APP,widely distributed across multiple pathogenic strains.Homologies between any two strains ranges from 78.9% to 100% regarding the GalT locus.Indirect enzyme-linked immunosorbent assay?ELISA?confirmed that GalT specific antibodies could not be induced by inactivated APP L20 or MS71 whole cell bacterin preparations.A recombinant fusion GalT protein derived from APP L20,however has proven to be an effective cross-protective antigen against APP sevorar 1 MS71?50%,4/8?and APP sevorar 5b L20?75%,6/8?.Histopathological examinations have confirmed that recombinant GalT vaccinated animals showed less severe pathological signs in lung tissues than negative controls after APP challenge.Immunohistochemical?IHC?analysis indicated that the infiltration of neutrophils in the negative group is significantly increased compared with that in the normal control?P<0.001?and that in surviving animals is decreased compared to the negative group.Anti-GalT antibodies were shown to mediate phagocytosis of neutrophils.After interaction with anti-GalT antibodies,survival rate of APP challenged vaccinated animals was significantly reduced?P<0.001?.This study demonstrated that GalT is an effective cross-protective antigen,which could be used as a potential vaccine candidate against multiple APP serotypes.4.GalT is a highly conserved antigen in gram-negative bacteria,and has been shown to play a crucial role in the pathogenesis of many zoonoses.Actinobacillus pleuropneumoniae?APP?is a widespread respiratory system pathogen belonging to Pasteuriaceae.The functional mechanisms of GalT in the process of infection remain unclear.The aim of this study is to analyze roles of GalT in the pathogenesis of APP infection.In this study,recombinant GalT was expressed in E.coli,purified,and was used to treat a RAW264.7 macrophage line.Stimulation of RAW264.7 macrophages with recombinant GalT protein induced the expression of pro-inflammatory cytokines?TNF-?,IL-1? and IL-6?.Compared with negative control,GalT led to increased production of pro-inflammatory cytokines in treated cells.Furthermore,specific inhibitors of the extracellular signal-regulated P38 and JNK MAPKs pathways significantly decreased GalT-induced pro-inflammatory cytokine production,and a western blot assay showed that GalT stimulation induced the activation of the MAPKs pathway.This process included cell-signaling pathways like P38 and JNK MAPKs and NF-?B.Both TLR2 and TLR4 were receptors of GalT antigens,whereas they played negative and positive roles?respectively?in the process of induction and expression of pro-inflammatory cytokines.Taken together,our data indicate that GalT is a novel pro-inflammatory mediator and induces TLR2 and TLR4-dependent pro-inflammatory activity in RAW264.7 macrophages through P38 and JNK MAPKs pathways.