Function And Mechanism Study Of Epidermal Growth Factor Receptor In Streptococcus Suis 2 Induced Meningitis

Streptococcus suis serotype 2(SS2)is an important zoonotic bacterial pathogen which cause high morbidity and mortality in both humans and animals.Meningitis is a major clinical manifestation of SS2 infection,but the specific molecular mechanisms require further investigation.In this study,we mainly investigated the potential role of EGFR in mediating neuroinflammation and cytoskeleton destruction in SS2 strain SC19-induced meningitis.The main findings are listed below.1.SS2 strain SC19 infection caused severe neuroinflammatory response.We used h BMEC and new-born mice as our in vitro and in vivo model for SS2 stain SC19 infection.In vitro,we observed an increasing adhesion of h BMEC by SC19 strain along with infection.In vivo,SC19 infection of CD1 mice led to histopathological lesions such as meninge thickening,meningorrhagia,gliocyte proliferation,inflammatory cell accumulation,and slight neuronophagia.Moreover,the production of cytokines and chemokines in both serum and brain in vivo,as well as in h BMEC in vitro,were significantly increased.2.Intracellular tyrosine phosphorylation of h BMEC in response to SC19We next investigated the intracellular proteins tyrosine phosphorylation in h BMEC in response to SS2 infection,and observed several intracellular proteins at approximately 180,100,and 60 k Da being tyrophosphorylated.The tyrophosphorylated protein around 180 k Da was finally proved to be EGFR.3.SC19 infection mediated ligand-dependent activation and dimerization of EGFRWe next investigated the transcription and translation of Erb B family members(Erb B1/EGFR,Erb B2,Erb B3,Erb B4)in response to SC19 infection,and found that EGFR,Erb B2,and Erb B4 maintained constant along with infection,while Erb B3 showed decrased expression.Moreover,immunoprecipitation and sh RNA approaches demonstrated that SC19 infection could lead to the tyrophosphorylation of both EGFR and Erb B3,as well as the formation of their heterodimerazation.Furthermore,in contrast to the obvious EGFR activation by live SC19,we found heat-inactivated SC19 strain was not able to activate EGFR.Meanwhile,the live SC19 could stimulate the upregulation of EGFR-associated ligands AREG,EREG,and HB-EGF,while the inactived SC19 could not.Also,through blocking the release of EGFR-associated ligands via MMPs inhibitor Batimastat treatment,the EGFR activation was demonstrated to be attenuated.Therefore,these indicated that SC19 infection mediated ligand-dependent activation of EGFR.4.Inhibition of EGFR does not significantly affect bacterial adherence and colonization in vivo and in vitroTo further investigate the effects of EGFR in SS2-induced meningitis,we pretreated h BMEC with EGFR inhibitor AG1478 and detected cellular adhesion by SC19,and we found AG1478 did not significantly decrease bacterial adhesion.In vivo,we also evaluated bacterial colonization in multiple tissues like blood,brain or lung,and no significant differences were observed with and without AG1478 treatment.5.EGFR activation contributes to neuroinflammatory responseWe next analyzed the possible effects of EGFR on SC19-induced neuroinflammation.In vitro,we found AG1478 treatment could dramatically attenuate the production of cytokines and chemokines(e.g.,IL-6,IL-8,MCP-1,MIP-2,TNF-?,and GRO-?).And in vivo,although the circulatory cytokines and chemokines in serum were almost unaffected,their levels in brain tissue were significantly decreased by the treatment of EGFR inhibitor AG1478.6.SC19-induced EGFR transactivation conduces to neuroinflammation through NF-?B and MAPK-ERK1/2 signaling pathwaysBy using NF-?B and MAPK-ERK1/2 specific inhibitors,we found that SC19-induced generation of IL-6 and MCP-1 were significantly prevented.Meanwhile,the p65 and ERK1/2 exhibited an increasing phosphorylation in response to SC19,and this activation could be largely blocked by AG1478 treatment.Moreover,our immunofluorescence assay further indicated the nuclear translocation of p65 subunit,supporting the activation of NF-?B signaling.And the activation of NF-?B signaling did not interplay with MAPK-ERK1/2 signaling cascade.Together,these data suggest that SC19-induced EGFR transactivation conduces to neuroinflammation through NF-?B and MAPK-ERK1/2 signaling pathways.7.Transactivated EGFR competitively recruits ACTN4 to affect the stability of cytoskeleton in h BMECFinally,via immunoprecipitation approach,we observed the time-dependent activation of EGFR in company with the increased ACTN4 binding.Meanwhile,ACTN4 also showed an increased tyrophosphorylation along with SC19 infection.ACTN4 initially helps to maintain the cytoskeleton stability via binding to intracellular actin,however upon infection,ACTN4 was largely and competitively recruited by activated EGFR,thus resulted in the destruction of the cytosklelton.Taken altogether,we have demonstrated from above data that SS2 strain SC19 infection of h BMEC could upregulate the EGFR-associated ligands AREG,EREG and HB-EGF to mediate transactivation of EGFR as well as the EGFR/Erb B3 heterodimerization.Transactivation of EGFR results in the activation of both MAPK-ERK1/2 signaling cascade and NF-?B pathway,which contribute to the robust production of proinflammatory cytokines and chemokines in h BMEC and in brain tissue,thus mediating the development of neuroinflammation.In addition,activated EGFR competitively recruits the ACTN4 which initially bind to cellular actin to maintain the cytoskeleton,therefore resulting in the destruction of the actin cytoskeleton.