Influence Of Porcine Circovirus Type 2 On TLRS In Porcine Alveolar Macrophages In Vitro

Porcine circovirus type 2 (PCV2) is the causative agent of post weaning multisystemic wasting syndrome (PMWS). It has been widely disseminated around the world since the first emergence in western Canada in 1991 and has caused largely negative effects on pig industry. PCV2 causes immunosuppression in the infected piglets. Toll-like receptors (TLRs), identifying one or more specific pathogen-associated molecular patterns (PAMP), play an important role in the connection between the innate and adaptive immune responses. Porcine alveolar macrophages (PAMs) are the main cell tropism of PCV2 in piglets, and are the key cells involved in virus proliferation and dissemination. PAMs also play an important role in innate immunity due to their strong phagocytic activity. Lung is the major damaged organ after PCV2 infection. The study of the changes in TLRs caused by PCV2 infection can help to reveal the mechanism of PCV2-induced immune suppression.Twelve conventional piglets were used in this study, which were free of PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV) as verified by ELISA and fluorescence quantitative PCR. PAMs were isolated aseptically and divided into two groups, i.e., control group and PCV2 infection group (PCV2 group). and then cultured in vitro. The cells and the culture supernantants of PAMs were collected after 0 h,6 h,12 h,24 h and 48 h, respectively. The amount of PCV2-infected cells was determined by indirect immunofluorescence assay (IFA), the expression of TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9 mRNAs was detected by real-time PCR, and the expression of TLR3, TLR4 and TLR9 proteins were detected by western blot. The results show that in comprison with control group, the expression level of TLR2 mRNA increased dramatically in PCV2 group after 6 h, but decreased markedly after 12 h. At this time point, the expression level of TLR2 mRNA in PCV2 group remained significantly higher than that in control group (P<0.01 or P<0.05), however after 48 h, the TLR2 mRNA levels in the two groups were of equal values. The expression of TLR3 mRNA in PCV2 group was significantly elevated after 6 h and 12 h (P<0.05), but decreased to a level similar to that of control after 24 h and 48 h. In addition, PCV2 infection led to a significant increase in the expression of TLR4 mRNA in PAMs after 12 h (P<0.05) and a significant decrease in the TLR4 mRNA expression after 24 h and 48 h as compared to control group (P<0.05). It also led to significant increases in the expression levels of TLR7, TLR8 and TLR9 mRNAs in PAMs after 6 h and 12 h (P<0.01 or P<0.05), however after 24 h and 48 h, these mRNA valueswere similar to their counterparts in control group.These results indicated that PCV2 significantly affected the expression of TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9 mRNAs in PAMs cultured in vitro.In PCV2 group, the TLR3 protein content in PAMs after 12 h significantly increased as compared to control group (P<0.05), but decreased to a level similar to that of control after 24 h. The increase in the TLR4 protein expression in PCV2 group declined modestly after 6 h, however it gradually increased after 12 h and was significantly higher than that of control after 24 h and 48 h (P<0.05). And the expression level of TLR9 protein after 48 h pf PCV2 infection significantly increased as compared to control group (P<0.05). The results demonstrated that PCV2 infection could upregulate the expression of TLR3, TLR4 and TLR9 proteins at different times, i.e., the expression content of TLR3 protein might increase in the middle stage of PCV2 infection, while the increase in the expression of TLR4 and TLR9 proteins might occur during the later stage of PCV2 infection.