Chlamydia Psittaci Plasmid Protein CPSIT_P7 Elicits Inflammtory Cytokines From Human Monocytes Via TLR4 And TLR6 Signaling Pathways

Objective: Chlamydia psittaci(C.psittaci)is an intracellular pathogen of atypical pneumonia.The Chlamydial plasmid is an important virulence factor and encodes plasmid proteins that play important roles in pathogen invasion and host inflammatory response.So far,the virulence factors and the molecular mechanisms of C.psittaci are still unclear.The aim of this study investigated the inflammatory ability induced by C.psittaci plasmid protein CPSIT_P7 and the signal pathways of THP-1 cells by which induce inflammatory cytokines interleukin-6(IL-6),interleukin-8(IL-8),and monocyte chemoattractant protein-1(MCP-1)in THP-1 cells,and to provide new ideas and basis for elucidating the pathogenesis of C.psittaci.Methods: CPSIT_p7 protein induced by IPTG was overexpressed,and purified by Ni-nitrilotriacetic acid(NTA)beads.Subsequently,the fusion proteins were concentrated and passed through Toxin Eraser? Endotoxin Removal Kit for removing of its contaminating endotoxin.Cytokine arrays assay was used to detect the inflammatory cytokines of THP-1 cells stimulated by 10 ?g/m L CPSIT_P7.THP-1 cells were stimulated with CPSIT_P7(0.1,1,5,or 10 ?g/m L)for 24 h or treated with 10 ?g/m L CPSIT_P7 for different time points(0,6,12,24,36,or 48 h).We uses dominant negative plasmids p ZERO-h TLR2,p ZERO-h TLR6,p De Ny-h Mal,p De Ny-h My D88,or si RNA of psi RNA-h TLR4 to inhibit the function of TLR2,TLR6,Mal,My D88,and TLR4.THP-1 cells stimulated with 10 ?g/m L CPSIT_P7.RT-q PCR and ELISA were used to detect the levels of IL-6,IL-8,and MCP-1 m RNA transcriptions and the protein expressions,respectively.THP-1 cells were treated with CPSIT_P7 for various times(0,15,30,60,120,and 180 min)and pretreated with NF-?B inhibitor BAY11-7082 to examine intracellular I?B? and ?-actin levels by western blotting.Then we used immunofluorescence approach to further investigate whether NF-?B was involved in CPSIT_P7 induced IL-6,IL-8,and MCP-1 productions.After incubation with 10 ?g/m L CPSIT_P7,RT-q PCR was used to detect the levels of IL-6,IL-8,and MCP-1 m RNA transcriptions,and ELISA was used to detect the levels of IL-6,IL-8,and MCP-1 protein expressions.Western blotting was used to detect the levels of signaling molecule I?B? phosphorylation.Immunofluorescence approach was used to detect the NF-?B subunit p65 nuclear translocation.Results: After Ni-NTA beads purification and concentration,Western blotting result showed the CPSIT_P7 molecular weight was 28 k Da.(1)After tested 42 cytokines,the granulocyte-monocyte colony-stimulating factor(GM-CSF),IL-6,IL-8,and MCP-1 were most abundantly secreted by THP-1 cells;(2)CPSIT_P7 induced IL-6,IL-8,and MCP-1 expressions in a concentration-and time-dependent fashion;(3)CPSIT_P7 induced IL-6,IL-8,and MCP-1 productions through TLR4,TLR6,Mal,and My D88;(4)CPSIT_P7 induced I?B? degradation,I?B? were increased from 15 min,and reached peak levels at 30 min.The phosphorylation of I?B? and inflammatory cytokines were found to be inhibited when the cells were pretreated with NF-?B inhibitor BAY11-7082.(5)Treatment with CPSIT_P7 result in significant increase of the NF-?B immunofluorescence signal in the nucleus,When the cells were pretreated with NF-?B inhibitor BAY11-7082,NF-?B subunit p65 nuclear translocation of NF-?B was significantly abolished.Conclusions:(1)Chlamydia psittaci plasmid protein CPSIT_P7 induces THP-1 cells to express IL-6,IL-8,and MCP-1;(2)TLR4,TLR6,Mal,My D88 and NF-?B are involved in the IL-6,IL-8,and MCP-1 expression in THP-1 cells under the stimulation of CPSIT_P7.