Establishment Of Porcine Alveolar Macrophage(PAM) Polarization Model And Its Dynamic Change During Prrsv Infection

In order to estabish different subtypes of porcine alveolar macrophages (PAMs) and resarch its dynamic change during PRRSV infection, in this study, porcine alveolar macrophages was induced in vitro by IFN-y/LPS to M1subtype of porcine aveolar macrophages and induced by IL-4to M2subtype of porcine aveolar macrophages. We observed morphological changes in different cells by calcein-AM staining and electron microscopy. The M1-type macrophages were successed by related marks moleculesNO, IL-12. The M2-type macrophages were successed by related marks molecules Arg-1, IL-10. Normal porcine alveolar macrophages were infected with PRRSV, related marks molecules of Ml and M2macrophages were measured, to study polarzation type for porcine alveolar macrophages infected with PRRSV. Ml and M2macrophages were cultured with PRRSV, the impact of PRRSV on two subtypes of macrophages was studied by measuring the related molecules.In order to detect the expression level of NO, the supernatant of different groups of cells was ertracted after24h, NO expression of each group was measured by Griess assay. It showed that porcine alveolar macrophages were stimulated by IFN-y/LPS, compared to the unstimulated group and IL-4group, showing very significant difference (P<0.01), thus to be compared with stimulated with IL-4and unstimulated group, the porcine alveolar macrophages stimulated by IFN-y/LPS showed significant difference(P<0.01). The protein of diffrent groups of macrophages were extracted after cultured24-72h, western results showed that compared with the group of porcine alveolar macrophages stimulated by IFN-y/LPS and unstimulated, the group of porcine alveolar macrophages stimulated by IL-4showed a significant difference at Iconic element Arg-1of M2macrophages (P<0.01). Meanwhile, compared with the group of porcine alveolar macrophages stimulated by IL-4and unstimulated, the group of porcine alveolar macrophages stimulated by IFN-y/LPS showed a significant difference at related mark IL-12of M2macrophages (P<0.01). Compared with the group of porcine alveolar macrophages stimulated by IFN-y/LPS and unstimulated, the group of porcine alveolar macrophages stimulated by IL-4showed a significant difference at related mark IL-10of M2macrophages (P<0.01). Through M1-type molecules related marks NO, IL-12, M2-type molecules related marks Arg-1, IL-10expressed differently,suggesting that M1-type and M2-type of swine alveolar macrophages were successfully induced in vitro.To study polarization type after the porcine alveolar macrophages infected with PRRSV, the different groups of porcine alveolar macrophages were infected with100TCID50,500TCID50,1000TCID50,5000TCID50virus titer. According to the above method to detect NO, Arg-1, IL-12, IL-10. The measurement results showed after porcine alveolar macrophages infected by different doses of PRRSV, the Arg-1, IL-10which were related molecules on M2macrophages showed significant differences(P<0.01or P<0.05), the NO, IL-12which were related molecules on M1macrophages expressed sightly, suggesting that porcine alveolar macrophages infected with PRRSV, porcine alveolar macrophages were to M2-type polarization.To study the effects on M1-type and M2-type porcine alveolar macrophages which were cultured with PRRSV, firstly, the porcine alveolar macrophages were induced to two subtypes of macrophages, two differernt type macrophages were cultured with PRRSV12～72h, NO content was measured by Griess assay, Arg-1, IL-10, IL-12were measured by western. The results showed that Ml-type porcine alveolar macrophages infected with PRRSV transformed to M2-type porcine aveolar macrophages, M2-type porcine alveolar macrophages infected with PRRSV changed little.In conclusion:The M1-type porcine alveolar macrophages were successfully induced by IFN-γ/LPS in vitro, the M2-type porcine alveolar macrophages were successfully induced by IL-4in vitro.After infected by PRRSV, porcine alveolar macrophages were polarized to M2-type macrophages. The M1-type porcine alveolar macrophages infected with PRRSV could transform to M2-type porcine alveolar macrophages. M2-type porcine alveolar macrophages infected with PRRSV changed little.