The Mucosal Adjuvant Mechanism Of Flagellin In Modulation Of Airway DC To Enhance IgA Response

Flagellin is a major structural protein of the flagella filaments.As a type of pathogen associated molecular patterns(PAMPs),flagellin can activate both TLR5 and NLRC4 signaling pathways.Many studies has proved that flagellin can performs its adjuvant activity through TLR5 and/or NLRC4 and enhance antigen-specific immune response.In recent years,a series of studies has shown that recombinant flagellin protein is an effective mucosal adjuvant when administered via the intranasal route(i.n.),which can induce higher specific secretory Ig A and enhance the final immunological protection.However,the mechanism by which flagellin drives a mucosal Ig A immune response when acting as an i.n.adjuvantremains undetermined.Using a mouse model of intranasal immunizaton,this paper conducted a systematic research on the uptake of recombianiat flagellin protein in murine epithelial cells,the interactions of flagellin and mucosal immunocompetent cells and the mechanism of flagellin induced mucosal Ig A response.First,we observed and determined the uptake and distribution of flagellin in mouse nasal mucous epithelium.Using immunohistochemical staining of the mouse nasal cavity,we found that the TLR5 protein was expressed highly on the apical surface of nasal epithelium.However,luminal flagellin can across the nasal epithelim and enter the nasal lamina propria quickly both in WT mice and TLR5 knockout mice(0.5h).Using flow cytometry,we found that the fusion protein flagellin-EGFP can be uptaked by nasal DC in nasal mucosa after intransal immunization in mice.Thus,we have proved that flagellin can be uptaked by nasal epithelium and distributed in the nasal lamina propria in a TLR5-independent way,and can be intaked by nasal DC.Second,we further observed and determined the interation of flagellin and airway DC after flagellin administered via the i.n.route.Using recombiniant flagellin protein as mucosal adjuvant and immunized with antigen OVA-Alexa Fluor647 in mice,we found that flagellin cannnot increase the ration of OVA+ DC or the amount of OVA in nasal DCs,but can increase the number of OVA+ DC in CLN of WT mice.Moreover,using an ex vivo experiment by sorting DCs and co-cultured with na?ve B Ⅸcells,we isolated and sorted CLN DCs from flagellin immunized WT mice or TLR5 KO mice separately at 18 h post i.n.immunization,and tested whether flagellin-activated airway DCs could exert this driving force on B cells and enhance antibody response.We found that DCs which isolated from WT mice immunized with flagellin had a significantly higher ability to increase Ig A+ cells and Ig A production from B cells.The results showed that the modulatory effects on airway DCs by flagellin is not through the enhancement of antigen uptake.Flagellin moudlated airway DCs in the fucntion of migration、activation and the capability of Ig A induction.The third part of this study,we tried to find out the specific mechanism of flagellin modulate airway DC and the consequential Ig A response.We found that flagellin direct stimulation of airway DCs only triggers slight up-regulation of CD80 but no CD86,and was insufficient to affect DC-mediated Ig A response.By using an in vitro system for culturing mouse primary NECs(m NECs),we demonstrated that flagellin-activated m NECs but not flagellin itself modulated airway DCs and subsequent enhancement of Ig A production.The modulation of the airway DCs and enhancement of Ig A production were dependent on TLR5 activation of m NECs rather than direct TLR5 activation of airway DCs by flagellin.We further identified GM-CSF is an essential cytokine secreted from TLR5-activated m NECs for mediating the activation of m NECs to airway DCs and the subsequent mucosal Ig A enhancement by cytokine array and anti-cytokine antibody blocking assays.Our data directly demonstrate that nasal epithelial GM-CSF contributes to TLR5-mediated modulation of airway DCs and subsequent Ig A response.Taken together,this study demonstrates that when immunized through nose,uminal flagellin and antigens can enter the nasal lamina propria in TLR5 independent way.After inter the nasal mucosa,flagellin could not increase the uptake of antigens by nasal DCs,but can modulate DCs’ function and enhance Ig A response through GM-CSF secreted by TLR5-activated m NECs.This study provides a theoretical foundation for mucosal vaccine design with flagellin,and further develops our understanding of mucosal immune response and mucosal adjuvant mechanism.