Study On The Immune Escape Of Pseudomonas Aeruginosa Biofilm To Polymorphonuclears And Mononuclear Macrophages And Its Mechanisms

PART ? Immune escape of Pseudomonas aeruginosa biofilm from PMNs and its mechanismObjectives:Because of biofilm?BF?formation on the endotracheal tube?ETT?in newborns,the ventilator-associated pneumonia?VAP?is still a serious problem.In the previous study,we found that one of the most common bacterias in the BF on the ETT was Pseudomonas aeruginosa?P.aeruginosa?.In addition,P.aeruginosa is easy to form BF in the ETT,and PMNs are closely related with the formation,development and pathogenicity of BF in host.However,the effect and mechanism of PMNs on BF caused by mucoid P.aeruginosa is unclear.The present study was to investigate the effects of PMNs and their major products of oxidative burst,H₂O₂,on mucoid P.aeruginosa BF and its mechanism.Methods:There were four groups:FRD1 group?control group?,FRD1+PMNs group,FRD1+1 mM H₂O₂ group and FRD1+2 mM H₂O₂group.Effect of PMNs or H₂O₂ on the adhesion of mucoid P.aeruginosa was observed by plate counting method and crystal violet assay.The formation and the mature of mucoid P.aeruginosa BF were observed by plate counting method and confocal laser scanning microscopy?CLSM?.The influence of PMNs or H₂O₂ on the content of alginate was measured with Sulphuric acid-Oxybenzene Method,and the expressions of important genes such as algD?algR?algU were detected by RT-PCR.Results:The adhesion and the formation of mucoid P.aeruginosa in the groups treated with activated PMNs or H₂O₂ were significantly greater than that in the control groups?P<0.05?.Moreover,the live P.aeruginosa and the biofilm thickness in the groups treated with activated PMNs or H₂O₂ were significantly increased,compared with control groups?P<0.05?.The content of alginate in the groups treated with activated PMNs or H₂O₂were more than that in the control groups?P<0.05?,and PMNs or H₂O₂could upregulate the expression of genes?algD?algR?algU??P<0.05?.Conclusions:PMNs are well known to be the first line of host defense against bacterial infection,activated PMNs or the main product?H₂O₂?could significantly promote mucoid bacterial adhesion and the formation of BF instead of removing them.In addition,activated PMNs or H₂O₂ could increase the production of alginate and the expression of genes related with alginate production.Suppressing the excessive oxidative respiratory burst of PMNs may be a promising approach to accelerate to eliminate biofilm infections by mucoid P.aeruginosa.PART ? Immune escape of wide type Pseudomonas aeruginosa biofilm from mononuclear macrophage and its mechanismObjective: We demonstrated in the part? that activated PMNs or the main product?H2O2?could significantly promote mucoid bacterial adhesion and the formation of BF instead of removing them.Innate immune is the first line of host defense against bacterial infection,and two of the main cells in innate immune are PMNs and mononuclear macrophages.It is reported that planktonic P.aeruginosa could activate the inflammasome of mononuclear macrophages,which can impair P.aeruginosa clearance in acute pneumonia.Therefore,we wonder whether the BF of P.aeruginosa can cause the activation of inflammasome ? and what is the mechanism? In this part,we would prove this problem.Methods: We divided the mononuclear macrophages THP-1 into three groups: THP-1 group?control group?,THP-1+ PAO1 group and THP-1 + PAO1 BF group.Gene transcription of IL-1? and IL-18 were detected by RT-PCR.The transcription of key proteins NLRC4,NLRP3,Caspase-1 and Caspase-4 in THP-1 were measured by RT-PCR.The expression of IL-1? and IL-18 was detected by ELISA,and the expression of Caspase-1 ? Caspase-4 ? Caspase-1 p20 and Caspase-4 p20 was measured by western blot.Results: Compared with THP-1 + PAO1 group,the IL-1? m RNA transcription of THP-1 in the THP-1 + PAO1 BF group was significantly increased?P<0.05?,but the IL-18 m RNA transcription were not significantly changed.Wide-type P.aeruginosa BF promote the transcription and production of IL-1? and IL-18?P<0.05?.Compared with THP-1 group,PAO1 BF could enhance the gene expression of NLRP3,Caspase-1 and Caspase-4?P<0.05?,however,the expression of NLRC4 was decreased in THP-1 + PAO1 BF group.The content of Caspase-1?Caspase-4?Caspase-1 p20 and Caspase-4 p20 were increased in THP-1 +PAO1 BF group,compared with control groups?P<0.05?.Conclusions: Wide-type P.aeruginosa BF causes mononuclear macrophage THP-1 to produce IL-1? and IL-18 and the expression of NLRP3?Caspase-4 were upregulated when THP-1was treated with widetype P.aeruginosa BF,however,the expression of NLRC4 was decreased,which indicated that wide-type P.aeruginosa BF may activate the NLRP3 and Caspase-4 inflammasomes,not the traditional NLRC4 inflammasome.PART ? Immune escape of mucoid Pseudomonas aeruginosa biofilm from mononuclear macrophage and its mechanismObjectives: We demonstrated in the part ? that wide-type P.aeruginosa BF could activate the NLRP3 inflammasome of mononuclear macrophages.Mucoid morphology is more common in the strains isolated from patients with chronic P.aeruginosa BF infection,and the main extracellular matrix of mucoid P.aeruginosa BF is alginate.Therefore,whether mucoid P.aeruginosa BF or alginate could cause inflammasome of mononuclear macrophage? and what is the mechanism?Methods: The mononuclear macrophage groups were as follows:THP-1 group?control group?,THP-1 + FRD1 BF group,THP-1+ alginate group.Gene transcription of IL-1? and IL-18 were detected by RT-PCR.The transcription of key proteins NLRC4,NLRP3,Caspase-1 and Caspase-4 in THP-1 were measured by RT-PCR.The expression of IL-1? and IL-18 was detected by ELISA,and the expression of Caspase-1?Caspase-4?Caspase-1 p20 and Caspase-4 p20 was measured by western blot.Results: Mucoid P.aeruginosa BF or alginate could promote the transcription and production of IL-1? and IL-18?P<0.05?.Compared with the wild-type P.aeruginosa BF,the mucoid P.aeruginosa BF promoted the more production of IL-1? and IL-18?P<0.05?.Compared with THP-1group,THP-1 + FRD1 BF could enhance the gene expression of IL-1? and IL-18?P<0.05?,and the expression of NLRP3,Caspase-1 and Caspase-4were also increased?P<0.05?.A similar trend was observed in the THP-1+alginate group.Conclusions: Mucoid P.aeruginosa BF causes mononuclear macrophage THP-1 to produce IL-1? and IL-18.The expression of NLRP3 ? Caspase-4 were upregulated when THP-1was treated with mucoid P.aeruginosa BF,which indicated that mucoid P.aeruginosa BF may activate NLRP3 inflammasome through Caspase-4 pathway,according to the main extracellular matrix of mucoid P.aeruginosa BF,alginate.