The Effect Of Autophagy On The Inflammatory Response Of Macrophages Infected With Toxoplasma Gondii

Objective:In this study,peritoneal macrophages in mice infected with Toxoplasma gondii was established with autophagy regulation.The relationship between autophagy and inflammasome was explored in order to reveal the effect of autophagy on the inflammatory response of macrophages with Toxoplasma gondii infection,which provides scientific basis for the innate immune response of host against Toxoplasma gondii and experimental evidence for the new ideas of prevention and treatment for toxoplasmosis.Methods:1.Peritoneal macrophages in mice infected with Toxoplasma gondii was established which was checked by Giemsa staining,and the cell viability was detected by CCK-8 to determine the optimal time for Toxoplasma gondii and macrophages with the ratio of 1:1;2.The levels of autophagy(LC3)and inflammasome(ASC)of peritoneal macrophages in mice infected with Toxoplasma gondii were detected by Western Blot;3.Peritoneal macrophages in mice were infected with Toxoplasma gondii after regulation with autophagy promoter Rapamycin or autophagy blocker Bafilomycin A1,then the levels of autophagy(LC3,p62)and inflammasome(ASC)were detected by Western Blot,the secretion level of IL-1β by ELISA,the cell viability by CCK-8,and the surface markers of macrophages polarization(iNOS,IL-12,Arg1,IL-10)and the surface antigen 1 of Toxoplasma gondii(SAG1)by Real-time PCR.Results:1.The optimal time for mice peritoneal macrophages infected with Toxoplasma gondii was 4h;2.Compared with the control group,the protein levels of LC3 and ASC of macrophages infected with Toxoplasma gondii were increased dramatically(P<0.01).The protein level of LC3 was increased,while the p62 and ASC were decreased(P<0.05)after Rapamycin treatment;meanwhile the protein levels of LC3,p62 and ASC were increased markedly(P<0.01)after Bafilomycin A1 treatment;3.Compared with the control group,the secretion of IL-1β of macrophages with LPS stimulation or Toxoplasma gondii infection was not increased(P>0.05).However,the secretion of IL-1β was increased significantly(P<0.001)after the pre-stimulation with LPS for 2h and the infection with Toxoplasma gondii for 4h,but the secretion of IL-1β was decreased(P<0.05)after Rapamycin or Bafilomycin A1 treatment;4.Compared with the control group,the cell viability of infected macrophages for 4h was not decreased(P>0.05)with significantly decrease for 6h(P<0.001),but there was no difference of cell viability in infected macrophages for 4h or 6h compared with the groups with Rapamycin,Bafilomycin A1 or DMSO treatment(P>0.05);5.Compared with the control group,the mRNA level of iNOS was slightly increased(P<0.05)with no difference of IL-12(P>0.05),while the mRNA levels of Arg1 and IL-10 were increased dramatically(P<0.01),after the mice peritoneal macrophages were infected with Toxoplasma gondii for 24h;the mRNA level of Arg1 was slightly decreased(P<0.05)without differences of iNOS,IL-12 and IL-10(P>0.05)after Rapamycin treatment;meanwhile the mRNA levels of iNOS and IL-12 were increased markedly(P<0.01)with decrease of Arg1 and IL-10(P<0.05)after Bafilomycin A1 treatment;6.The mRNA level of SAG1 was increased(P<0.05)after Rapamycin or Bafilomycin A1 treatment compared with the infected macrophages.Conclusion:1.The levels of autophagy and inflammasome of mice peritoneal macrophages could be induced by infection of Toxoplasma gondii which may indicate that autophagy plays a negative regulation on inflammation;2.The cell viability of mice peritoneal macrophages infected with Toxoplasma gondii may not modified by autophagy;3.The polarization of mice peritoneal macrophages infected with Toxoplasma gondii were promoted to M1 instead of M2 while the autophagy was blocked;4.The proliferation of Toxoplasma gondii in macrophages was enhanced after the regulation of autophagy and the mechanism needs to be further understood.