| Streptococcus suis(S.suis)is an important bacterial pathogen that causes significant economic losses on pig farms in the world and is also an emerging zoonotic pathogen.S.suis infection causes clinical symptoms such as meningitis,septicemia,arthritis,endocarditis and pneumonia.Macrophages are important part of the innate immune system can exhibit considerable heterogeneity in different microenvironments.Macrophages can be divided into two main phenotypes:proinflammatory M1 type and anti-inflammatory M2 type.As the primary immune cells,porcine alveolar macrophages(PAMs)form the first line of defense against S.suis infection in the lungs.However,the preparation of primary PAMs is costly and complicated.PAM-Tang is an immortalized cell line by introducing SV40 large T antigen to primary PAMs and can be continuously passaged in vitro with ordinary medium,but it is unknown whether immortalized PAM-Tang cells can replace primary PAMs as a better cell model for studing the interaction between S.suis and macrophages.Therefore,in the present study,the anti-infective properties of PAM-Tang cell line against S.suis were studied by molecular biology experiments.To evaluate the phagocytosis of PAM-Tang,the phagocytosis of PAM-Tang was monitored by imaging live cells.PAM-Tang could effectively engulfs S.suis at about 15 hpi,and the number of lysosomes significant increased during the phagocytic process(P<0.05).The phagocytic vesicles were observed in PAM-Tang cell by electron microscopy,and bacteria were observed in the phagocytic vesicles,indicating that PAM-Tang could phagocytose S.suis effectively.To determine the effect of S.suis infection on PAM-Tang polarization,the polarization of macrophages after infection was analyzed with antibodies for M1 type and M2type macrophages.The results showed that S.suis induced M1 polarization of PAM-Tang.To determine if PAM-Tang exposed to S.suis have the ability to secrete pro-inflammatory cytokines,the levels of porcine IL-18,IL-1βand TNF-αwere detected using ELISA,and the m RNA expressions of IL-6 and IL-8 were detected by RT-q PCR.The results showed that S.suis could induce the secretion of pro-inflammatory cytokines in PAM-Tang.In order to investigate the inflammation signaling pathway induced by S.suis in PAM-Tang,the expression of inflammation-related proteins was detected by Western blot.The results showed the expression of NLRP1 and Caspase-1 increased significantly after infection that indicated NLRP1 inflammasome signaling pathway is activated,which may result in the release of numerous proinflammatory cytokines.What’s more,the increased expression of phosphorylated ERK1/2 and p38 proteins indicated that the MAPK pathway was also involved in the inflammatory response during S.suis infection.To explore the interaction between S.suis and PAM-Tang with different virulence,PAM-Tang cells were inoculated with S.suis of different virulence in this study.The results showed that the S.suis serotype 9 avirulent strain W7119 had higher levels of adhesion in PAM-Tang cells(2.69×10~6)than the S.suis serotype 2 virulent strain 700794(3.4×10~4).Cellular cytotoxicity of different virulent strains of S.suis was assessed using LDH assay,and the results demonstrated a positive correlation between incubation time of S.suis and its cytotoxicity,and the virulent strain induced a higher level of cytotoxicity(OD492=2.42)than the avirulent strain(OD492=1.2).The apoptosis of PAM-Tang induced by S.suis was analyzed by TUNEL assay and the results showed that the virulent strain induced higher levels of apoptosis in PAM-Tang(207 cells/mm~2)than the avirulent strain(112 cells/mm~2).To determine the apoptosis signaling pathway of PAMs induced by S.suis,the expression levels of apoptosis signaling pathway-related proteins were detected by Western blot.The results showed S.suis-induced apoptosis of PAM-Tang and primary PAM cells depended on Caspase and AIF/p53 signaling pathways.In conclusion,this study shows the phagocytic ability of PAM-Tang to S.suis and its anti-infective properties against S.suis,and verified PAM-Tang could be used as a cell model for study the apoptosis mechanism of S.suis in vitro. |
