Study On Pseudomonas Aeruginosa And Pyocyanin Induced Immunity Reaction Of Human Monocytic U937 Cells And Lung Infection In Rats

ObjectivePseudomonas aeruginosa(PA)is a Gramnegative bacterium that colonises the lower rspiratory tract in patients with bronchiectasis,cystic fibrosis and chronic obstructive pulmonary disease.It is an important pathogen causing a wide range of acute and chronic infections.It is believed to stimulate a persistent host immune response leading to progressive airway destruction in the pathogenesis of bronchiectasis.The bacteria are widely distributed and have characteristic of multidrug resistant.Once infected,clinic therapies are of difficulty.Moreover,Most P.aeruginosa strains secrete pyocyanin(N-methyl-l-Hydroxy-phenazine),the pigment that gives blue-green color to the bacterial colonies.High concentrations of pyocyanin are detected in pulmonary secretions of patients with bronchiectasis and CF whose lungs are colonised by this organism,where it exerts a proinflammatory effect and impairs ciliary function.Pyocyanin plays important role in acute invasive infection.So it is of significance to study pathogenesis of host infected by P.aeruginosa and Pyocyanin.Alveolar macrophages are significant defense cells and inflammation regulation cells which switch on multiplicity mediators of inflammation and cytokine and then cause acute lung injury.Thus,intervention in NF-κB has become therapic focus.However,human medullary system cell line U937 cells are of characteristic with monoblast and pedomonocyte.U937cells activated with phorbol 12-myristate 13-acetate(PMA)were induced and differentiated to form macrophages.Thus, differentiated U937cells are ideal model as studying human macrophages.Pseudomonas infections are characterized by a marked influx and excessive inflammatory response. The CXC chemokines,of which interleukin(IL)-8 is the prototype,act predominantly on neutrophils.IL-6 and IL-1βare important pro-inflammatory cytokines.It is knowned that muli-cytokine secreted by PA induced pro-inflammatory cytokines production in epithelial cells of respiratory tract and monocyte.However,in vitro studies concentrated on airway epithelial cells and neutrophilic leukocyte about P.aeruginosa and pyocyanin.There are fewer studies on monocytes induced by pyocyanin.It is not clear about the production of IL-8 and IL-6 induced by P.aeruginosa live strains and pyocyanin as well as the signaling pathway in human monocytic leukemia cell lines which were differentiated.The molecular mechanisms by which these factors exert their effects are poorly understood.IL-1βis a procytokine that plays important role in host defense pathogenic bacteria infection.In vitro studies showed that the reduction or disappear of endogenous IL-1 reactive can inhibit inflammatory reaction and counteract P.aeruginsa pneumonsia.The result suggested that IL-1βpossibly participate in pathogenesy of P.aeruginsa pneumonsia.However,it is poorly understood the role of IL-1βon immune reaction against P.aeruginosa infection in U937 cells.Therefore,in this study,we examined the ability of P.aeruginosa and pyocyanin to induce inflammation reaction and the signaling pathway of IL-8 and IL-6 release in PA infected U937 cells and IL-8 release in PCN infected U937 cells,and explored the production of the cytokines in host cells induced by P.aeruginosa and pyocyanin as well as the regulatory effect of cytokines IL-1βon host defense against P.aeruginosa infection.Moreover,we also investigated the expression of NF-κB and cytokines in PA-induced pneumoneia of rats,and the effect of pyrrolidine dithiocarbamate(PDTC) on its expression.MethodsThe experiments were divided into two parts,including U937 cells model in vitro and rats model in vivo.1.In vitro studies(1)Pyocyanin was prepared by photolysis of phenazine methosulphate and purified pyocyanin was judged by HPLC and UV absorbance.(2)Differently differentiated U937 cells were infected by P.aeruginosa at an multiplicity of infections(MOI)of 10:1,20:1 and 50:1.The expression of IL-8 mRNA and IL-6mRNA was quantified by semi-quantitative RT-PCR analysis afte two hours of P.aeruginosa infection,usingβ-actin mRNA as an internal standard.Total cellular RNA was isolated from cells by using TRI reagent.ELISA was performed to test the expression of IL-8 and IL-6 in infected U937 cells after 8,16 and 24 hours of P.aeruginosa infection.(3)Westem blotting method was used to test the expression of NF-κB protein in PMA-differentiated U937cells infected by P.aeruginosa.In addition, PMA-differentiated U937 cells were cultured together with PKC inhibitor,calphotin C or NF-κB inhibitor,pyrrolidine dithiocarbamate(PDTC),respectively before P. aeruginosa infection 60min,then the protein expression of NF-κB were assayed by Westem blotting method,and the protein secretion of IL-8 and IL-6 was measured by ELISA method.(4)Immunocytochemistry method was employed to examine the expression of NF-κB p65 protein after P.aeruginosa infection.(5)RT-PCR and ELISA methods were performed to test the expression of IL-8 in differentiated U937 cells infected by various concentrations of Pyocyanin.After incubations for 1 up to 2 h,cells were harvested for total RNA extraction and expression of IL-SmRNA was assayed by RT-PCR methods.For IL-8 release,the cell-free supernatants were harvested after treatments for 8,16 and 24h,and assayed for IL-8 by ELISA method.(6)Western blotting method was employed to examine the phosphorylation of MAPKs including ERK1/2,P38 and protein expression of NF-κBp65.In addition, PMA-differentiated U937 cells were cultured together with MAPK inhibitor,including ERK1/2(PD98059)and P38(SB203580)or NF-κB inhibitor(PDTC)respectively before Pyocyanin infection,then the expression of IL-8 and NF-κB were assayed by RT-PCR,ELISA and Westem blotting in order to demonstrated whether or not MAPKs and NF-κB signaling pathyways participate IL-8 expression induced by pyocyanin in PMA-differentiated U937 cells.(7)Morever,as positive control,TNF-αand IL-βsingle or combination with pyocyanin or P.aeruginosa induced IL-8 expression in U937 cells by RT-PCR and ELISA methods in order to observe the role of IL-1βor TNF-αin P.aeruginos a or pyocyanin infection. 2.In vivo studiesSeventy-two male SD rats in good conditions were divided into three groups at random:control group,PA group and PDTC group.There were 24 rats in each group. PA induced pneumonia models were established in SD rats.The rats in control group and PA group were intraperitoneally given saline(1ml)at 60 min before PA exposure,while the rats in PDTC group were received the same volume of PDTC(200mg.kg^-1).After 60min,the rats in PA group and PDTC group were intratracheally instilled with PA 0.2ml(6×10⁸CFU/ml),while the rats in control group were received the same volume of saline.At 3,6,16,24 h after PA exposure,the rats were examined.Then they were sacrificed and the lung were excised for routine histological analysis.Immunohistochemical staining with an antibody against activated NF-κB and Western blot were performed to detect the expression of NF-κB.The change of IL-8 protein secretion by ELISA.Results1.In vitro studies(1)P.aeruginosa was able to induce IL-8 and IL-6 mRNA expression and protein secretion in PMA-differentiated U937 cells and had marked time-and concentration-dependent relations.(2)IL-1βcan increase IL-8 production in U937 cells by P.aeruginosa strain.(3)P.aeruginosa could also significantly promote the phosphorylation of I-κBαand activation of NF-κB after 60min of P.aeruginos infection.PDTC and calphotin C could significantly decrease the activation of NF-κB.(4)PDTC and calphotin C reduce the secretion of IL-8 and IL-6 in concentration-dependent manner(P＜0.01).(5)The results of immunocytochemistry methods showed that NF-kB exists in the cytoplasm stained with well-distributed light dye in an inactive form in normal condition.Increased NF-kB activity with nuclear localization was observed after stimulated by P.aeruginosa.In addition,after PMA-differentiated U937 cells were cultured together with PKC inhibitor,Cal C,or NF-κB inhibitor,PDTC respectively for 60min before P.aeruginosa infection in vitro,NF-κB nuclear translocation was found. (6)Pyocyanin was able to induce IL-8 mRNA expression and IL-8 protein secretion in PMA-differentiated U937 cells and had marked time and concentration-dependent relations.(7)The phosphorylations of p38 was induced after 10min infection.Marximal phosphorylation occurred at 30 min,and then started to declined with time-dependent manner.Pretreament of U937 cells with specific inhibitors of P38(SB203580)could significantly diminished the pyocyanin-induced phosphorylations of p38.In contrast,the levels of total p38 protein was not changed in whole experimental period.(8)The phosphorylations of ERK was induced after 10min infection.Marximal phosphorylation occurred at 30 min,and then started to declined with time -dependent manner.Pretreament of U937 cells with specific inhibitors of ERK(PD98059)could significantly diminished the pyocyanin-induced phosphorylations of ERK.In contrast,the levels of total ERK protein was not changed in whole experimental period.(9)There was obvious expresson of NF-κB in PMA-differentiated U937 cells after 30min ofpyocyanin infection,and peak at 60 min.(10)Pretreament of U937 cells with specific inhibitors of ERK1/2(PD98059),the kinase that activates ERK1/2,P38(SB203580)and of NF-κB(PDTC)could significantly diminished the pyocyanin-induced IL-8 production(P＜0.01).(11)This study also found that pyocyanin,TNF-αor IL-1βrespectively induced IL-8 production,howerever,the combination with pyocyanin and TNFαor IL-1βdid not have synergistic effect.2.In vivo studies(1)Histology findings demonstrated that lung exposed to PA showed significant changes in Lung structure,edema and pronounced inflammatory cells infiltration.Both symptoms and damages of lung were lesser in the rats of PDTC group than that of the PA group.(2)The results of immunocytochemistry methods showed that expression of NF-kB in 3 to 24h after PA infection and marked expression at 3h.The result of western blotting corresponded with that of immunocytochemistry methods.Pretreament of U937 cells with specific inhibitors of NF-κB(PDTC)could significantly diminished the expression of NF-κB(P＜0.01).(3)Compared with PA group,Expression of IL-8 in PDTC group were significantly down regulation after PA challenge 3-24h(P＜0.01),separately. Expression of IL-8 was observed at 3-24h,and peak expression of IL-8 at 16h after PA exposure.Conclusions1.The directly induction of P.aeruginosa could promote the activation of NF-κB by PKC signal pathway,then cause the expression of IL-8 and IL-6.IL-1βprobably participate in pathogenesy of P.aeruginosa.2.Pyocyanin can induce IL-8 production in time -and dose -dependen manner. MAPK and NF-κB signaling pathway may participates in IL-8 expression induced by pyocyanin in PMA-differentiated U937 cells.3.The expression of NF-κB and pro-inflammatory mediator induced by NF-κB play an important role in the pathogenesis of P.aeruginosa pneumonia.The inhibitor of NF-κB,PDTC,can relieve the lung damages produced by P.aeruginosa.