Chlamydia Psittaci Plasmid-encoded CPSIT＿P7 Induces M1 Polarization To Promote Clearance Of Chlamydia Through TLR4-mediated MAPK And NF-κB Pathways

Objective:The zoonotic pathogen Chlamydia psittaci(C.psittaci)causes a respiratory illness called psittacosis,which could lead to a pandemic.Due to its high infectivity and pathogenicity,a preventive vaccine may be one way to prevent infection,but there is currently no effective vaccine.Thus,the antibacterial response of the host is critical for the control of chlamydial infection.As a fundamental part of innate immunity,macrophages undergo M1 activation to acquire phagocytic and microbicidal competence,which promotes the host antibacterial response.This article explores the effect and mechanism of the C.psittaci plasmid-encoded protein CPSIT＿P7 on macrophages polarization to provide new insights into new prevention and treatment strategies for chlamydial infection.Method:1.Expression and purification of the recombinant protein CPSIT＿P7 were achieved as described previously under nondeformed conditions.2.The variable amounts of CPSIT＿P7 were used to stimulate the RAW264.7 cells for different times,and then the transcriptional levels of the macrophage markers(CD86,CD206,iNOS,Arg1,TNF-α,and IL-6)were analyzed by q RT–PCR.Western Blot was used to analyze the expression of the M1 marker iNOS and the phosphorylation of the transcription factor STAT1.The results of q RT-PCR and Western Blot were combined to determine the optimal concentration and time of the plasmid protein CPSIT＿P7 to induce macrophage polarization.3.RAW264.7 cells were stimulated with 10μg/m L CPSIT＿P7 for 24 h,the proportion of M1 macrophages(F4/80~+CD11b~+CD86~+)and M2macrophages(F4/80~+CD11b~+CD206~+)was measured using flow cytometric.4.After the plasmid protein CPSIT＿P7 incubated with RAW264.7 cells,the activation of the macrophage polarization-related pathways(TLR4,ERK,JNK,p38,and NF-κB)were detected by Western Blot,and the inhibitors were used for the further verification.5.BMDMs isolated and induced macrophages from mouse bone marrows.After the plasmid protein CPSIT＿P7 incubation,the proportion of M1macrophages and M2 macrophages was measured using flow cytometric,ELISA were used to detect the expression of macrophage polarization-related cytokines TNF-α,IL-6,IL-1βand IL-10.6.After CPSIT＿P7 intranasal administration,mice lung macrophages were harvested to analyze the proportion of M1/M2,and the supernatant of lung homogenates were collected for the cytokine(IL-6,TNF-α,IL-1β,and IL-10)analysis using ELISA kits.7.After CPSIT＿P7 intranasal administration,mice were challenged with C.psittaci and sacrificed at Day 1,2,3 or 7.Immunofluorescence was performed to observe the C.psittaci and macrophages in mouse lung tissue at 1 dpi,2 dpi,and 3 dpi.The supernatant of lung homogenates were harvested at 7 dpi,and fluorescence microscope was used to count chlamydial inclusion forming units.H&E was used to assess the degree of pathological changes in mouse lung tissue.Results:1.The expression of M1 macrophage markers CD86,iNOS TNF-α,and IL-6 were upregulated in RAW264.7 cells after CPSIT＿P7 treatment,but the gene transcription of CD206 and Arg-1 were inhibited.2.A remarkable elevation of iNOS was observed after 10μg/m L CPSIT＿P7 incubation for 24 h in RAW264.7 cells.The phosphorylation of STAT1 was evident 120 min after CPSIT＿P7 stimulation.In addition,the proportion of M1 macrophages increased after CPSIT＿P7 stimulation.3.Western blot analysis revealed that incubation with CPSIT＿P7 resulted in the significant phosphorylation of ERK,JNK,and p38 in RAW264.7cells.Compared to cells preincubated with DMSO,pretreatment of macrophages with SP600125 and BAY117082 significantly restrained the CPSIT＿P7-induced production of iNOS,and a slight decrease in iNOS production was observed after preincubation with SB203580.However,the PD98059 inhibitor did not affect the expression of iNOS.Furthermore,inhibition of JNK attenuated the blots of IκB phosphorylation and inhibition of NF-κB also led to inactivation of JNK MAPK pathway,which indicated that there is crosstalk between the two pathways.4.The expression of TLR4 was upregulated by CPSIT＿P7.TAK-242 effectively suppressed the phosphorylation of JNK and IκB and the expression of iNOS.5.Plasmid protein CPSIT＿P7 induced the secretion of inflammatory factors TNF-α,IL-6 and IL-1βin BMDMs cells,and the expression of C86 surface molecule on the cell surface also increased.6.Compared to PBS-treated mice,the levels of M1 macrophage polarization marker CD86 were markedly increased,but M2 macrophage polarization marker CD206 was slightly decreased in CPSIT＿P7-treated mice pulmonary macrophages.And the release of IL-6,TNF-α,and IL-1βfollowed the same upregulation as the CD86 expression in the CPSIT＿P7-treated mice.7.During C.psittaci infection,the lungs of mice exposed to CPSIT＿P7 always had lower chlamydial burden than mice exposed to PBS and the volume of inclusions was also smaller than the PBS group.H&E analysis showed alveolar loss,diffuse inflammatory cell infiltration and other serious lesions in PBS-treated mice lung tissue.Conclusion:C.psittaci plasmid-encoded CPSIT＿P7 induces M1polarization to promote chlamydial clearance through TLR4-mediated MAPK and NF-κB pathways.