| Salmonella spp.is one of the main zoonotic pathogens that seriously threatens the health of humans and animals.Salmonella Enteritidis(SE)is one of the most important serotypes to cause human infections,so its prevention and control have become an urgent need.Salmonella can survive in host macrophages to escape immune clearance,and bring to a persistent infection,which is mainly caused by Salmonella-induced M2 macrophages polarization,it is one of the main pathogenicity.However,few studies have been conducted on Salmonella-induced M2 macrophages polarization,which seriously restricts the development of effective vaccines and drugs,and also hinders the prevention and control of Salmonella in the livestock and poultry industry.Macrophage polarization refers to the functional and phenotypic change of macrophages,which includes typical activation(M1)and alternative activation(M2).M1 macrophages can promote an inflammatory response and Th1 type immune response.M2 macrophages can inhibit the inflammatory response and promote Th2 type immune response,it plays an important role in the immune escape of pathogens to promote persistent infection.Salmonella can not only induce M1 macrophages to eliminate intracellular bacteria,but also regulate M2 macrophages through heterogeneous gene expression to promote Salmonella persistence in vivo.In previous studies,it was found that the SE spiC mutant(SEΔspiC)had a stronger proliferation ability and induced higher levels of IL-10 in macrophages than SE.This suggested that SEΔspiC-infected macrophages might belong to M2 macrophages,but it had not been reported that the spiC gene mediated the M1/M2 shift in SE-infected macrophages.Therefore,this study aimed to elaborate the mechanism of SpiC-mediated M1/M2 shift of SE-infected macrophages.1.SEΔspiC induces RAW264.7 macrophage M2 polarization in vitroSEΔspiC and SECoΔspiC were constructed,and their growth and biochemical characteristics were consistent with SE,but the proliferation ability of SEΔspiC enhanced in macrophages.qPCR analysis showed that mRNA levels of M1 indicators CXCL9,TNF-α,iNOS,and CD86 decreased,while the M2 indicators IL-10,TGF-β,Arg1,and CD206 increased in SEΔspiC-infected RAW264.7 cells,compared with that of SE.Similarly,in the culture supernatants of SEΔspiC-infected RAW264.7 cells,IL-1β and IFN-γ levels decreased while IL-10 level increased by ELISA,and the TNF-α level decreased while IL-10 level increased by FACS.the levels of intracellular NO,ROS and CD86 decreased,while the level of CD206 increased by FACS.iNOS level was decreased and Argl level was increased in SEΔspiC-infected RAW264.7 cells by Western blot and qPCR.The cytotoxicity of SEΔspiC to RAW264.7 cells based on LDH release was decreased,and the activity of intracellular arginase increased in SEΔspiC-infected RAW264.7 cells.Further analysis showed that the intracellular proliferation ability and IL-10 levels in the culture supernatants of the spiC promoter mutant were consistent with those of SEΔspiC.SpiC protein could be induced for high expression by three media LPM1,LPM2,and LPM3,and SpiC level in LPM2 was the highest.Laser confocal,qPCR and Western blot analysis showed that the expression of SpiC protein can be inhibited by NO in a concentration-dependent manner in LPM2 media.After SE-infected RAW264.7 cells being treated with NO,the mRNA level of intracellular Salmonella SpiC was significantly reduced,the proliferation ability of SE was stronger,and the IL-10 level was higher.These results were consistent with the polarization effect of M2 macrophages induced by SEΔspiC.2.SEΔspiC induces M2 macrophages via the PKM2-JAK2-STAT3-IL10 axisIn SEΔspiC-infected RAW264.7 cells compared to SE,IL-10 levels in the culture supernatants were significantly increased by ELISA,the ability of p-STAT3 nuclear transfer was enhanced by Laser confocal method,and the phosphorylation levels of STAT3 and JAK2 were increased,as well as the expression level of PKM2 by Western blot.Meanwhile,compared to the untreated control,the proliferation ability of SEΔspiC in macrophages treated with STAT3,JAK2 and PKM2 inhibitors was significantly reduced,and IL-10 level in the culture supernatants was significantly decreased in a concentration-dependent manner.Western blot analysis showed that compared to the untreated group,the phosphorylation level of STAT3 in SEΔspiC infected RAW264.7 cells treated with JAK2 inhibitor decreased in a concentration-dependent manner.This indicates that STAT3 is the downstream substrate of JAK2.After PKM2 inhibitor treatment,the phosphorylated JAK2 and STAT3 levels in SEΔspiC-infected RAW264.7 cells decreased in a concentration-dependent manner,indicating that the functions of JAK2 and STAT3 were both in the downstream of PKM2.In conclusion,these results indicated that PKM2 could regulate SEΔspiC-indnced M2 macrophages.3.SEΔspiC induces M2 macrophage in vivoCompared to SEΔspiC-infected C57BL/6 mice,the bacterial loads in the organs of the SE-infected mice were significantly higher,and serum IL-10 levels were significantly increased;qPCR analysis showed that the mRNA levels of the M1 factors TNF-α,ROS,and CD86 decreased,while the mRNA levels of IL-10,Argl,and CD206 increased in the spleen of SEΔspiC-infected mice.The numbers of M1 macrophages(CD86+)and M2 macrophages(CD206+)in murine spleen were analyzed by FACS.The results showed that the number of M1 macrophages(CD86+)decreased gradually and M2 macrophages(CD206+)increased slightly in the spleen of mice infected with SE and SEΔspiC,but there was a difference in the number between the two groups.Compared with SE group,the number of M1 macrophages(CD86+)in spleen of mice infected with SEΔspiC was lower,and M2 macrophages(CD206+)in spleen of mice infected with SEΔspiC continued to increase.These results indicated that SEΔspiC was more inclined to induce M2 macrophages.4.SpiC protein induces Ml macrophages by stimulating NF-κB pathway and interacting with PKM2SpiC was expressed in macrophage cells by laser confocal microscopy and Western blot analysis.The TEM-1 report system proved that SpiC protein could be secreted into the cytoplasm of host cells infected by SE and functioned as an effector of type Ⅲ secrection system.NF-κB pathway was analyzed as SpiC target because the NF-κB pathway is classic in M1 macrophage polarization.The results showed that SpiC could mediate SE to promote NF-κB p65 promoter activity by luciferase reporting system,intracellular IκBα degradation by Western blot and the nuclear transfer of p65 by laser confocal,indicating that SpiC might activate the NF-κB pathway in HeLa and RAW264.7 cells.SpiC expressed by eukaryotic vector also could promote NF-κB p65 promoter activity,intracellular IκBα degradation and nuclear transfer of p65 in HeLa cells and RAW264.7 cells.Therefore,SpiC may directly participate in the M1 polarization;SEΔspiC-induced M2 macrophages were positively regulated by PKM2,suggesting that SpiC might interact with PKM2 to mediate M1 macrophages.Pull-down and laser confocal assays confirmed that SpiC interacted with PKM2 and co-located in the cytoplasm.In conclusion,SpiC protein induced SE-infected macrophage M1 polarization by activating NF-κB pathway and interacting with PKM2.As a control,when spiC being knocked out or inhibitied by NO,the SE strain would induce macrophage M2 polarization by promoting PKM2-JAK2-STAT3-IL10 axis.The discovery of this mechanism broadened the theoretical mechanism of Salmonella infection and immunity,and also laid the theoretical base for the development of Salmonella vaccines and new drugs,as well as the prevention and control of Salmonella disease in livestock and poultry. |
