| Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens affecting swine industry worldwide. PRRSV infection is characterized by reproductive failure in sows and respiratory disease in all age of pigs, and causes immunosuppression and secondary bacterial infections. It is of significance to explore the mechanism associated with immunosuppression and secondary bacterial infections induced by PRRSV infection, which helping to better understand the pathogenesis of PRRSV. Th17 cells not only participate in anti-bacterial immune responses, but also play important roles in anti-fungal infection, autoimmune disorders and cancer. In the present study, the cytokines related to Th17 cells differentiation were first evaluated on pulmonary alveolar macrophages (PAMs) infected with two strains of PRRSV with different pathogenicity by ELISA, and Thl7 cells responses in SPF piglets that were intranasally inoculated with PRRSV were then analyzed by flow cytometry using prepared anti-porcine IL-17A mAbs, and finally the effect of IL-17A on PRRSV growth and its preliminary mechanism were investigated by using the lentivirus package system and infectious clone technology, in an attempt to provide evidence for elucidating the mechanism of immunosuppression and secondary bacterial infections induced by PRRSV infection.PAMs were infected with highly pathogenic PRRSV (HP-PRRSV) (JXwnO6) and low-virulence PRRSV (HB-1/3.9) respectively and culture supernatants were then collected, and the level of IL-6 and TGF-β were measured by ELISA. It was shown that PRRSV infection suppresses the expression of IL-6 and TGF-P of PAMs, and meanwhile JXwnO6 infection exhibited stronger inhibitory effect on IL-6 expression than HB-1/3.9 infection, suggesting that HP-PRRSV infection can suppress the cytokines related to Th17 cells differentiation of PAMs.To explore the effect of PRRSV infection on Th17 cells response in vivo, SPF pigltes were intranasally inoculated with the PRRSV JXwn06 or HB-1/3.9, and flow cytometry was performed to analyze the changes of the subpopulations of T cells in PBMCs of the infected piglets. The results showed that both the frequency and absolute number of CD3+CD4+ and CD3+CD8+ T cells in JXwn06-infeced group were significantly lower than those of HB-1/3.9-infected group and mock-infected control.Meanwhile, the frequency of CD4+IL-17A+ Th17 cells in JXwn06-infeced group declined compared with HB-1/3.9-infected group and mock-infected control (p<0.05). The Th17 cells in differernt tissues of the inoculated piglets were counted by IHC. The results revealed that JXwn06-infeced group had reduced number of Th17 cells in lungs of the inoculated piglets at different time points compared with HB-1/3.9-infected group, whereas no significant differences were found in Th17 cells number in spleens and inguinal lymph nodes between JXwn06-infeced group and HB-1/3.9-infected group. Bacterial counts in lung of the inoculated piglets showed that the bacterial loads of lung in JXwn06 infected group were significantly higher that those of HB-1/3.9-infected group and mock-infected control.The above data indicated that HP-PRRSV infection not only immunomodulated the Thl7 cells response in peripheral blood of piglets in vivo, but also resulted in the reduction of Th17 cell number in lung of piglets, as well as increased bacterial loads in lung of piglets, implying that secondary bacterial infection caused by HP-PRRSV infection might be closely related to the immunosuppression of Th17 cells response.The infectious cDNA clone of JXwn06 was used to generate four chimeric cDNA clones containing IL-17A gene insertion between its ORFlb and ORF2-coding region. Four chimeric viruses were rescued from the chimeric cDNA clones. The TCID50 titrations of the rescued viruses showed that the viral titers of RvJX-IL17A-R1 had a decreased tendency along with the passaging. However, the decreased virus titers of the other three rescued viruses were only observed at the passage 2. Two nucleotides mutation in IL-17A gene were found at the virus of passage 3 by sequencing. The growth kinetics of the chimeric viruses revealed IL-17A has inhibitory effect on the replication of PRRSV.UV-inactivated culture supernatants of RvJX-IL17A-R1 displayed inhibitory effect on the growth of JXwn06 in MARC-145 cells. MARC-145 cells were transduced with the lentiviruses that were expressing IL-17A, and were then inoculated with JXwn06 at the MOI of 0.1,and the virus titers were measured at different time points. The titers of JXwn06 in the transduced MARC-145 cells were significantly lower than those in the transduced with the lentiviruses expressing GFP and normal cell control (p<0.001), showing that IL-17A can inhibit the replication of HP-PRRSV. The preliminary mechanism was analyzed by luciferase reporter assay. IL-17A was shown to activate ISRE and NF-κB promoter but not IFN-β promoter, and to promote the expression of antiviral protein PKR under the PRRSV infection.In summary, our results indicated that, (i) HP-PRRSV infection not only inhibited Th17 cells response in peripheral blood but also down-regulated the number of Th17 cells in lung of piglets; (ii) HP-PRRSV infection could resulted in the increased bacterial loads in lung of piglets, suggesting the close relationship between secondary bacterial infection and the suppression of Th17 cells response; (iii) IL-17A could inhibit the growth of HP-PRRSV in vitro. |
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