Pathogenesis Of Aggravation Of Avian Influenza Virus H9N2 With Primary Infection Of Chlamydia Psittaci In Chickens

Chlamydia psittaci(C.psittaci)is one of zoonotic agents and it is able to transfer to the target organs using macrophages as a delivery vehicle.In light of zoonotic implication,C.psittaci is listed as a bioweapon both in USA and European Unions.Currently,avian respiratory diseases are prevalent and seasonal outbreak in Chinese poultry farms.Mortality amounts to 30-40%,resulting in antibiotic abuse and food safety concerns.Clinically,avian H9N2,C.psittaci and E.coli have been identified from the diseased birds while highly seroprevalence of C.psitaci,avian metapeumonia and ORT are detected from the recovered poultry flock.Therefore,understanding the pathogenesis of C.psittaci infection and its con-infection will shed a light on the strategic control of avian airsacculitis in future.Polymorphic membrane proteins(Pmps)are unique protein families located on the outer membranes across all Chlamydial species.In a previous report,PmpD is confirmed to be a virulent factor of Chlamydia trachomatis(C.trachomatis).However,what is a potential role of PmpD of C.psittaci?Does it contribute to immune evasion during C.psittaci infection?The answer is not clear due to lack of future investigation.Our hypothesis is that dysfunction of macrophage might be downregulated directly by C.psittaci-specific PmpD.Alternatively,dysfunction of macrophage might be associated with the activation of TLRs/MyD88/NF-KB and imbalance of Thl/Th2 immune response,leading to innate immune failure and widely infection.In current study,the experiments were carried out to illustrate our above rationales as the followings:1).To understand the immune suppression induced by C.psittaci and secondary infection by avian influenza virus H9N2 using C.psittaci+H9N2,C.psittaci/H9N2,H9N2/C.psittaci,H9N2 alone and C.psittaci alone.2)To elucidate macrophage and expression of toll-like receptors during con-infection with C.psittaci and H9N2.3)To explore the activation of TLRs/MyD88/NF-KB signaling pathways and Thl/Th2 cytokines post inoculation with HD11 and PmpD-N.Meanwhile,both siRNA and inhibitor were to block the signaling pathways for warranting the speculation.Effects of C.psittaci and H9N2 mixed infection on SPF chickens:SPF chickens infected with highly virulent C.psittaci were found to be poor immune organ index and lower antibody levels against Newcastle disease as compared to the birds with lower virulence strains.In other experiment,Lower body weight,immune organ index and cytokine expression were found in the birds inoculated C.psittaci and H9N2 simultaneously and the C.psittaci/H9N2 group.More importantly,33%mortality,highly lesions in immune organs and H9N2 virus loads were determined in he C.psittaci/H9N2 group.Chickens inoculated with C.psittaci/H9N2 is confirmed to be a better animal models for replicating avian respiratory diseases.Also,C.psittaci infection triggers immune suppression and facilities secondary H9N2 infection,characterized by lower humoral response.Effects of con-infection-of C.psittaci/H9N2 on dysfunction of HD11 cells:In the present study,chicken macrophage HD11 cell line were used as a target cells during combination or inoculation with C.psittaci or H9N2 agent,12h interval during experiment.Subsequently,HD11 were suffered from cell death post infection with C.psittaci and then H9N2 infection.Meanwhile,inactivation of HD11 and iNOS were obviously observed as compared to C.psittaci alone or H9N2 alone.Moreover,Thl cytokines were reduced significantly in C.psittaci/H9N2 group.Effect of C.psittaci-specific PmpD-N on HD11 function:Firstly,pEGFP-N1-pmpD-N plasmids were constructed and transfected into HD11 cell lines,then expression of PmpD-N was identified using RT-PCR and Western-blotting assay.Afterwards,HD11 cell lines were stimulated with rPmpD-N,or pEGFP-NI-pmpD-N plasmids or LPS as the control post administration at 6,12,24 and 48h.Chlamydia loads were detected using direct immunofluorescence assay,cell phagocytosis was determined using fluorescence microsphere method and concentration of NO2-in cell culture supernatants were measured by Griess method.Results showed that PmpD-N could significantly decrease C.psittaci infection and HD11 cell phagocytic ability,but did not increase the secretion of NO,and increase expression of Th2 type cytokine,such as IL-6 and IL-10.Moreover,expression of TLR2,TLR4 and TLR5 were upregulated and TLR2 expression was significant increase.Furthermore,upregulating MyD88 and transcription factor NF-κB mRNA expression were also observed in the study,suggesting the activation of TLR2/MyD88 signaling pathway and Th2 innate immunity.Effect of C.psittaci-specific PmpD-N on HD11 and signaling pathway:HD11 cell lines were stimulated with rPmpD-N,pEGFP-N1-pmpD-N plasmids or LPS as the control post administration at 6,12,24 and 48h.Toll like receptor,adaptor protein MyD88 and transcription factor NF-κB in NF-κB signaling pathway were detected by Western-blotting assay.The migration of NF-κB subunit p65 to the nucleus was detected by laser confocal microscopy while EMSA technique was used to detect the binding of DNA to the target protein after the transfer of p65 into the nucleus.Transcription of NF-κB and the transcription of the target gene after it was combined with the corresponding target protein using Dual luciferase reporter gene analysis.Finally,RNA interference and the NF-κB inhibitor PDTC were used to verify above results.The results showed that both upregulating TLR2 and MyD88 were found obviously.NF-κB subunit p65 was activated,migrated to the nucleus and then combined with the corresponding promoter gene.Post blocking with TLR2,MyD88 and NF-κB signaling pathway,IL-6 and IL-10 levels were significantly reduced while macrophage function recovered later.Based on all above data,chickens infected with C.psittaci/H9N2 not only replicate typical avian respiratory diseases as a good model,but also C.psittaci and C.psittaci-specific-PmpD-N are able to induce immune suppression and facility H9N2 infection by downregulating macrophage function.Also,macrophage dysfunction is dependent on an indirect way via TLR2/MyD88/NF-KB signaling pathway and Thl/Th2 imbalance,which contributes to C.psittaci survive in macrophage and immune evasion.