Chlamydia Psittaci Modulates Autophagy Of Human Bronchial Epithelial Cells Through UPR-related Signaling Pathways

Objective:To investigate the effects of Chylamydia psittaci(C.psittaci)on human bronchial epithelial cells(HBE)autophagy and explore the regulatory mechanism of unfolded protein response(UPR)on this prosess,so as to further clarify the pathogenic mechanism of C.psittaci.Methods:HBE cells were infected with C.psittaci with a multiplicity of infection(MOI)of 0,0.5,1,1.5,and 2 for 24 hours,and western blot was used to detect the expression levels of the autophagy-associated protein“LC3-II,Beclin1,p62”,and UPR and its signal pathway-associated protein“Bip,PERK,phosphorylated PERK,e IF2α,IRE1α,phosphorylated IRE1αand ATF6”.The m RNA expression of XBP1 in the downstream of the IRE1αpathway was detected by Real-time Quantitative PCR(q RT-PCR).The autophagosomes and autophagosomes were observed by transmission electron microscopy(TEM),the formation of LC3 spots were detected by immunofluorescence analysis(IFA).To investigate whether C.psittaci-induced autophagy is associated with UPR and its signaling pathways,si RNA was used to knock down the expression of Bip in HBE cells,or HBE cells were treated with specific IRE1αinhibitor-4μ8C and specific PERK inhibitor-GSK26026414 for 2 hours separately before the C.psittaci infection.Then,the expression of LC3 II,Beclin1 and p62were detected by Western blot,the formation of LC3 spots was detected by IFA.Finally,the numbers of C.psittaci inclusion bodies were counted in Bip si RNA-treated and inhibitors-treated infected cells to detect the relationship between UPR and C.psittaci development.Results:The expression of LC3-II,Beclin1,Bip,PERK,phosphorylated PERK,e IF2α,IRE1α,XBP1 and phosphorylated IRE1αin HBE cells infected with different MOI of C.psittaci for 24 hours were increased and reached a peak at MOI of 1.5.The expression levels of ATF6 and p62 proteins were down-regulated in a dose-dependent manner after infection.When HBE cells were infected with C.psittaci at MOI of1.5 for 24 hours,green LC3 spots were observed around the nucleus of HBE cells,and appeared many autophagosomes and some autophagosolysosomes in HBE cells.After interfered with 50 n M Bip si RNA for 48 hours,the expression of Bip was significantly dreased in HBE cells,and the expression level of LC3-II and Beclin1 were decreased in C.psittaci infected cells,while the expression level of p62increased compared with the control group.Immunofluorescence analysis showed that the intensity of the LC3 spots were reduced comparing with the control group.After treated with 30μM 4μ8C or 40μM GSK26026414 inhibitors for 2 hours separately,the expression level of LC3-II and Beclin1 were decreased and the expression level of p62 was increased similarly.Moreover,the inhibitors treatment significantly inhibited the production of LC3 spots around the nucleus.After interfered the expression of Bip,the number of intracellular C.psittaci inclusion bodies were significantly decreased(1.13×10~7 IFU/m L),compared with the infected group(2.33×10~7 IFU/m L)or NC si RNA-infected group(2.50×10~7 IFU/m L).And when the IRE1αor PERK signaling pathway was inhibited with 4μ8C or GSK26026414,the formation of C.psittaci inclusion bodies was also significantly decreased.Conclusion:These data suggest that C.psittaci infection induces autophagy and endoplasmic reticulum stress in HBE cells,simultaneously initiates UPR and induces activation of PERK and IRE1αsignaling pathways,and this autophagy process is regulated by UPR and its PERK and IRE1αsignaling pathways.Furthermore,UPR and its PERK and IRE1αsignaling pathways are involved in C.psittaci reproduction and development in HBE cells.These observations provide clues on the pathogenesis of C.psittaci infection.