Identifying Host-cell Death Pathways In L929 Cells Induced By Infection Of Chlamydia

Chlamydia is a kind of intracellular pathogen,which infection is one of a globally serious problems for the public health.The reserch between chlamydia infection and ways of cell death is important for infected desease and the clearance of pathogen.Cell death can be divided into three different ways: apoptosis,necrosis and autophagy.In this study,we focus on the host-cell death pathways(apoptosis,autophagy or necrosis)in L929 induced by chlamydia at 4 8 h post infection(h.p.i.).1)For providing experimental materials to subsequent experiments,chlamydia was amplificated,purificated and counted.And the perfect multiplicity of infection was0.85.2)The relationship between apoptosis and infection at 48 h.p.I was assayed by fluorescent DAPI staining,which showed that there weren’t many fragmentation of nucleuses.This suggested apoptosis was not occured in the L929 cells by chlamydia at 48 hours post infection.3)By staining with PI plus Annexin-V and analysising by FACS,we found that most necrosis occurred upon chlamydia infection.Meanwhile,the release of Lactate dehydrogenase was increased after chlamydia infection at 48 h.p.I.These results showed that necrosis was induced in the L929 cells by chlamydia at 48 hours post infection.Then the co-immunoprecipitation of receptor-interacting serine/threonineprotein kinase 1 and receptor-interacting serine/threonine-protein kinase 3 was analysised,but there was no interaction.Next,si RNA of receptor-interacting serine/threonine-protein kinase 1 and Nec-1 were used to identify whether receptorinteracting serine/threonine-protein kinase 1 was participated in infection.The results showed that receptor-interacting serine/threonine-protein kinase 1 was not participated in the infection with chlamydia at 48 h.p.i.4)The autophagy in L929 cells was indicated by a transient transfection of GFP-LC3 previously and detection of the GFP expression post infection by a fluorescent microscopy.Western blotting of LC3 was further used to confirm the occurrence of autophagy.The results showed that fluorescent dots of GFP-LC3 formed upon chlamydia infection,and an increased ratio of LC3 Ⅱ/LC3Ⅰ in the L929 infected by chlamydia was also showed by western blotting,which indicating that autophagy occured during chlamydia infection.In summary,Necrosis and autophagy,but not apoptosis,were induced in the L929 cells infected by chlamydia at 48 hours post infection.However,receptor-interacting serine/threonine-protein kinase 1 was not participated in the infection with chlamydia at48 h.p.i.This study may provide new insights for cell death mechanisms and chlamydia infection,and thus supply novel clues for developing effective prophylactic and therapeutic approaches to chlamydia infection.