The Novel Function Of Cyclooxygenase-2 (COX-2) In Anti-mycobacterial Immune Response

Due to Mycobacterium tuberculosis(MTB)is hard to be eradicated and persists in macrophages,tuberculosis,caused by MTB infection,is still a significant threat to global health.In MTB-infected macrophages,cyclooxygenase-2(COX-2)expressed increase and acted as an important influencing factor for bactericidal activity.It has been reported that,when compared with wild type macrophages,the growth of MTB was enhanced in Ptges-/-macrophages(which cannot produce COX-2).Furthermore,COX-2 also promotes bacterial elimination by activating CD8+ T cells through dendritic cell-dependent cross-priming and repairing plasma membrane disruption.COX-2 is also known as an important rate-limiting step limited enzymes to transform arachidonic acid into prostaglandins(PG)(including PGD2,PGE2,PGF2?,PGI2,and PGJ2).Each PG interacts with its receptor and activates different signaling pathways to regulate the immune response.The bactericidal mechanism of COX-2 reported before were via PGE2 production,and the function in bacterial elimination of PG,like PGD2,PGF2?,PGI2 and PGJ2 need to be explored.In cancer researches,downstream products of COX-2,prostaglandins,have been reported to enhance autophagy,a confirmed bactericidal process during MTB infection.However,the role of COX-2 in regulation of autophagy induced by M.tuberculosis infection has not been explored.In this study,we found that silencing COX-2 expression in the murine macrophage cell line RAW264.7 or murine bone marrow-derived macrophages reduced the autophagy level and bactericidal activity against intracellular mycobacterium,while COX-2 overexpression reversed the above effects.However,enhancement of bactericidal activity was suppressed by inhibition of autophagy in COX-2-overexpressing cells,indicating that COX-2 accelerated mycobacterial elimination via promoting autophagy.Furthermore,the inhibition of autophagy and bactericidal activity caused by COX-2 silencing were remarkably reversed by catalysis products of COX-2,prostaglandins,suggesting that the effects of COX-2 were mediated by its catalytic products.Importantly,the function of COX-2 and prostaglandins on the immune response to infection was through inhibiting the AKT/mTOR pathway which negatively regulates autophagy,thus accelerated the process of autophagy and in turn,mycobacterial elimination.In conclusion,we firstly demonstrated a novel role of COX-2 in the immunity against mycobacterial infection:accelerating mycobacterial elimination by promoting autophagy.Methods1.The expression levels of COX-2 in mycobacterium-infected macrophages1)Murine bone marrow-derived macrophages(mBMDMs)were isolated from 4 to 6 weeks old C57BL/6 mice,and cultured in DMEM with 10%FBS and 100 ng/ml GM-CSF for 7 days to induce BMDMs maturation.After BMDMs maturated,experiments were performed with those cells.2)mBMDMs and RAW264.7 cells were infected with H37Rv or BCG as different multiplicity of infection(MOI)for 24 h.Then mRNA and protein levels of COX-2 were examined by real-time RT-PCR and western blot.3)H37Rv or BCG was used to challenge mBMDMs and RAW264.7 cells as MOI of 2 for different time points(0 h,6 h,12 h,24 h).Then mRNA and protein levels of COX-2 were examined by real-time RT-PCR and western blot.2.The changes of bactericidal activity in COX-2-slicenced or-overexpressed macrophages1)mBMDMs and RAW264.7 cells were transfected with COX-2 siRNA and followed by H37Rv or BCG infection,and then mRNA and protein levels of COX-2 were detected by real-time RT-PCR and western blot.Moreover,COX-2-overexpressing cell line(RAW264.7-COX-2 cells)was constructed and the protein of COX-2 was detected by western blot.2)The viability of BCG or H37Rv in COX-2 inhibiting or overexpressing macrophages was confirmed by colony-forming unit(CFU)assay.3.The mechanism of COX-2 promoting mycobacterial elimination1)After BCG were used to infect COX-2-inhibited macrophages,the levels of cytokines,including IFN-?,IL-1(3,TNF-? and IL-6,were detected by real-time RT-PCR and ELISA.2)COX-2-slienced macrophages were infected with BCG,and then the levels of iNOS and NO were detected by real-time RT-PCR and ELISA.3)In BCG-infected COX-2-knockdown macrophages,the production of ROS was detected by flow cytometry.4)After BCG were used to infect COX-2-sliencing macrophages,the apoptosis level of host cells was confirmed by testing the activity of caspase-3.5)Texas Red-labeled BCG were used to challenge COX-2-silencing or-overexpressing macrophages,and the colocalization of BCG with the lysosome marked by green fluorescein-coupled antibody against lysosome marker CD63 was viewed with confocal microscopy.6)After COX-2-knockdown macrophages were infected with H37Rv or BCG,the autophagic flux was detected by testing protein level of LC3 with western blot and the amount of LC3 puncta with confocal microscopy.Moreover,a selective(V)-ATPase inhibitor,Bafilomycin A1(Baf A1)was served to block LC3-?degration,the association between COX-2 and LC3-? was detected too.4.COX-2 promoted bactericidal activity of macrophages via enhancing autophagy.1)In COX-2-knockdown cells,the LC3-autophagosomes containing Texas Red-labeled BCG in macrophages was examined with confocal microscopy.2)3-methyladenine(3-MA),the specific inhibitor of autophagy,was served to decrease the induction of autophagy in RAW264.7-COX-2 cells,and then LC3 protein level and the intracellular bacterial burden were detected with western blot and CFU assay.5.COX-2 promoted autophagy via its prostaglandin products in mycobacterium-infected macrophages.1)H37Rv-infected COX-2-silenced macrophages were treated with PG(including PGD2,PGE2,PGF2?,PGI2 and PGJ2)respectively,and then LC3 protein level and the intracellular bacterial burden were detected by western blot and CFU assay.2)H37Rv-infected COX-2-silenced macrophages were treated with all of PG mixture.LC3 protein level and the intracellular bacterial burden were detected with western blot and CFU assay.3)LC3 puncta were tested with confocal microscopy in PGs-treated COX-2-knockdown macrophages.6.COX-2 enhanced autophagy through inhibiting the AKT/mTOR signaling pathway1)The phosphorylation level of AKT was examined in H37Rv-infected RW264.7-COX-2 cells by western blot.2)After PGs mixture-treated cells were infected with H37Rv,the phosphorylation level of AKT was tested with western blot.3)In COX-2-silenced or overexpressed macrophages followed by H37Rv infection,the phosphorylation levels of mTOR and eukaryotic initiation factor 4E binding protein 1(4E-BP1),the downstream substrate of mTOR signaling,were examined with western blot.4)The phosphorylation levels of mTOR and 4E-BP-1 were examined with western blot in PGs-treated COX-2-knockdown macrophages infecting with H37Rv.7.Statistical analysisSPSS statistical software version with 17.0(SPSS,Chicago,IL)was used to analyze statistically.Statistical significance was determined by one-way ANOVA,and multiple comparisons were performed using the least significant difference(LSD)post-hoc test when the variance between samples was equal,or Dunnett's T3 test when the variances were not equal.Independent experiments were performed three times,and all the data were showed as the means±standard error of the mean(SEM).The P value<0.05 was considered statistically significant.Results1.In mycobacterial infection macrophages,the levels of COX-2 were increased in a time-dependent.Results showed that both the gene and protein levels of COX-2 were increased in a time-dependent,but not dose-dependent pattern.Interestingly,protein level of COX-2 increased dramatically at 6 h in BCG-infected cells,but H37Rv infection started to induce COX-2 protein level at 16 h.2.Silence of COX-2 significantly impaired the bactericidal activity of mBMDMsSilencing COX-2 impaired bactericidal activity of mBMDMs against intracellular BCG at 48 and 72 hour-post-infection(h.p.i.).3.Levels of cytokines in BCG-infected macrophages were influenced by inhibiting COX-2 expressionIn BCG-infected macrophages,silencing COX-2 decreased expression of IFN-?and increased protein level of IL-1?,but had no effect on the level of IL-6 and TNF-?.4.Silencing COX-2 changed the secretion of ROS and NOThe results showed that,in BCG infection macrophages,inhibition of COX-2 decreased the level of NO,but had no effects on the production of ROS.5.The percentage of BCG infection-induced apoptosis had no change in COX-2-silence macrophagesResults of the activity of caspase 3 demonstrated that,inhibition of COX-2 expression was failure to change the percentage of BCG-induced apoptosis.6.COX-2 promoted the maturation of bacteria-containing phagosomesSilencing COX-2 expression inhibited maturation of bacteria-containing phagosomes.In contrast,overexpression of COX-2 promoted the maturation of mycobacterial phagosomes.7.Autophagy was enhanced by COX-2 in mycobacterium-infected macrophagesBCG or H37Rv-induced protein levels of LC3-? and the amount of LC3 puncta was decreased in COX-2 siRNA-targeted macrophages.8.Elimination of intracellular mycobacterium by COX-2 was via autophagyResults of CFU indicated that,COX-2 overexpression-induced accelerating mycobacterial elimination was dramatically abrogated by 3-MA,the specific inhibitor of autophagy.9.COX-2 enhanced autophagy process in mycobacterium-infected macrophages through its prostaglandin products.In H37Rv-infected COX-2 siRNA-targeted macrophages,PGD2,PGE,PGF2a,PGI2 and PGJ2 were used for treating cells respectively or combinedly.Results of western blot showed that,COX-2-inhibited cells,when treated with single PG(including PGD2,PGE,PGF2?,PGI2 and PGJ2),showed suppressed LC3-? level.However,such inhibition of LC3-? caused by COX-2 silencing was reversed after all of those PGs were added as a mixture,even in NC-targeted cells in mycobacterium-infected macrophages.Furthermore,suppression of mycobacterial elimination caused by COX-2 silencing was abolished by PG mixture treatment too.10.COX-2 enhanced autophagy through inhibiting the AKT/mTOR signaling pathwayActivity of AKT/mTOR signal pathway was inhibited in RAW264.7-COX-2 cells and PGs-treated macrophages.ConclusionMycobacterium-induced COX-2 promoted the process of autophagy by inhibiting the activity of the PI3K/AKT/mTOR pathway to suppress mycobacterial survival.Thus,we unraveled a novel and important role of COX-2 in autophagy regulation and mycobacterial elimination,and these results provided a novel view to intervention against M.tuberculosis infection.