Mechanisms Of Flagellar Hook Protein FlgE-induced Inflammatory Responses

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Immunostimulatory activity of FlgE in mice acute lung injuryObjective: Interactions between microbial components and host immune compartments contribute much to host-microbe interactions.In previous studies we found that pseudomonas aeruginosa flagellar hook protein E(FlgE)manifested strong immunomodulatory activities [1],such as stimulating HCEC cell line and mice lung slices to secret cytokines(i.e.IL-1b and IL-6).However the inflammation effect of host challenged with FlgE in vivo remains unclear.In this part,we employ a model of mice acute lung injury(ALI)to investigate the role of FlgE in acute inflammation.Methods: First,the recombinant FlgE protein(rm FlgE)was expressed and purified.Then(250 mg/100 mL)FlgE-induced mice acute lung injury was establised intranasally as previous.All the experiment mice were sacrificed at 24 h after infected.The Pulmonary lesions in mice after intranasal instillation were obsreved by HE staining.Flow cytometry was used to detect the percentage of immune cell subsets in lung tissue and spleen.q PCR was used to detect the expression of cytokines and chemokines in lung.ELISA was used to detect the level of both MPO in serum and cytokines in BALF(bronchoalveolar lavage fluid).The expression of ROS within neutrophils and the percentage of apoptosis neutrophils were analyzed by flow cytometry.Fluorescence microscope was used to observe the release of the NETs in lung tissue and fluorescence ELISA was used to detect the quantification of DNA content in BALF.Furthermore,the neutrophils isolated form CVA-/-mice were stimulated with FlgE and the expression of cytokines/chemokines were detedcted by q PCR.Results: The mouse model of ALI was successfully established.Compared with control group,the mice intranasal administration with rm FlgE revealed a severe pneumonia.The majority of infiltrates in BALF were neutrophils,and depletion ofneutrophils with anti-Ly6 G antibody could significantly inhibited FlgE-induced ALI development.The mRNA expression of CXCL1,CXCL2 in mice lung tissues as well as its protein levels in BALF were increased after stimulated with FlgE.The proportion of ROS +neutrophils was increased after stimulated by FlgE,while apoptosis was not obviously obaseved.Moreover,the NETs-like structure was observed under fluorescence microscope,which could not be inhibited by ROS inhibitor(DPI).The amount of NET-DNA in BALF was significantly increased in FlgE treated mice.We also found that the mRNA expression of Il1 b,Il6,Cxcl1 and Cxcl2 in neutrophil were impaired when CAV1 deficiency.Conclusion: In FlgE induced ALI in mice,neutrophils were the dominant effector cells that were recruited to inflammatory site with the NETosis formation.CAV1 that expressed on neutrophils might worked as a potential counterparter of FlgE.Part ? The role of IL-17 A in the acute lung injury induced by FlgEObjective: IL-17 A is a key cytokine involved in the host inflammatory response and is secreted by a variety of immune cells.In our study,we observed the expression of IL-17 A was increased when mice challenged with FlgE.However,the role of IL-17 A acting in vivo is not clear.Therefore,we will study this issue in this part.Methods: To establish the FlgE-induced ALI model in vivo,and the response changes between Il17a-/-mice and WT mice were described in three aspects: the degree of histopathological changes,the level of immune cell infiltration and cytokines or proteins expression.In vitro,lung slices from WT mice and Il17a-/-mice were treated with FlgE for different times,total proteins were obtained and subjected to STAT3 phosphorylation detection and the culture-supernantants were applied to detect neutrohils migration using transwell assay or applied to NETs formation detection.Neutrophils isolated from WT mice and stimulated with FlgE or preincubated with STAT3 inhibitor,the mRNA expression of Il17 a,Il17ra,as well as Il17 rc were detected by q PCR or flow cytometry.Results: Compared with those observed in WT mice,the FlgE-induced neutrophils recruitment and chemokines production were significantly impaired in Il17a-/-mice.FlgE-induced lung damage was also attenuated in neutrophil-depleted mice.Accompanied with Il6 mRNA expression,the STAT3 phosphorylation of proteins from lung tissue slices was up-regulated upon FlgE treatment in vitro,but the phosphorylation level of STAT3 was quickly attenuated into normal while IL-17 A deficiency.FlgE or the culture supernatant of FlgE-treated lung tissue slices from WT mice,but not from Il17a-/-mice,strongly enhanced neutrophils migration,which was inhibited by STAT3 inhibitor III administration.On the other hand,we demonstrated that the levels of IL-17 A,IL-17 receptor C(IL-17RC)expression and STAT3 phosphorylation were upregulated in FlgE-treated neutrophils.Blocked STAT3 activation in neutrophils from WT mice decreased IL-17 A expression.We further observed that the FlgE induced neutrophil extracellular traps(NETs)formation and reactive oxygen species(ROS)generation were impaired while IL-17 A deficiency.Conclusion: In FlgE-induced ALI model,the cytokine IL-17 induced the expression of chemokines such as CXCL1,and CXCL2 in lung epithelial cells,which then in turn triggers neutrophils migration and indirectly enhanced NETs formation,and this response was regulated by STAT3 signaling pathway.Moreover,FlgE was able to stimulate neutrophils to express IL17A/IL-17 RC and phosphorylation of STAT3.Part ? ?subunit of F1 FO ATPase expressed on endothelial cell membrane mediates FlgE-induced inflammatory responseObjective: The immunomodulatory activities of aeruginosa flagellar hook protein FlgE,which might mediated by Be site or Fe site,has been proved in our study,however the binding receptor of FlgE was remain unclear.Our previous work found several membrane proteins such as ATP synthase,Rab family members,CAV1,or prohibitin might served as the potential membrane counterparts of FlgE to elicit biological reactions including inflammation or immune responses.In this study,we aimed to demonstrate whether the beta-subunit of F1F0 ATPase expressed on endothelial membrane could mediate FlgE induced inflammation.Methods: Generation and purification of polyclonal rabbit antibody against ATP5 B peptide(Pc ATP5BAb),flow cytometry analysis the expression of ecto-beta subunit of F1F0 ATPase on HUVEC and SVEC cells.Total membrane proteins were prepared from HCEC and SVEC cells,and pull-down assay was used to identify the affinity between FlgE,FlgEM and the beta-subunit of ATPase expressed on endothelial cell membrane.HCEC and SVEC of logarithmic phase was stimulated with FlgE,FlgEM,Be or Fe,in someexperiments,Pc ATP5 BAb was used to block the binding between experiment regents and ATP5 B.The proliferation of HCEC and SVEC was assayed by MTT.The permeality of endothelial cells was determined on the mean fluorescence intensity(MEI)of FITC-Dextran by transwell.The ATP production synthesized by cell surface ATPase of ECs was evaluated using luminescence assay.The migration of endothelial cells was measured by scratch-wound experiment.The apoptosis of endothelial was detected by staining with Annexin V and 7AAD.Results: Flow cyto results shown that ecto-ATP5 B was distrubuted on HUVEC and SVEC cell plasma membrane.Pull-down assay indicated that FlgE,instead of FlgEM,was able to bind with the beta-subunit of ATPase(ATP5B)which distributed on the surface of endothelial cell membrane.The ATP synthesized activity performed by ecto-ATPase was inhibited by Pc ATP5 BAb,Be peptide or Fe peptide via using luminescence assay.The Pc ATP5 BAb sucsseccfully blocked the affinity between rm FlgE,Be peptide,Fe peptide and ATP5 B,subsequently inhibited the response of endothelial cells which triggered by FlgE stimulation.Conclusion: Our results demonstrated that the ATP5 B expressed on endothelial cell membrane was one of the critical counterpartner of the Be site and Fe site of recombinant FlgE protein,which alter the inflammatory response induced by FlgE upon blocked by ATP5 B antibody.