The Mechanism Of TLR4-Dependent Humoral Immune Response Induced By Rabies Vaccines

Rabies is a fatal zoonosis caused by rabies virus（RABV）damaging the central nervous system（CNS）.So far,prophylaxis is the most effective measure to prevent and control this disease,and rabies vaccine-induced humoral response is a critical indicator of the protection ability.TLR4 molecule expresses on various immune cells which play an important role in the recognition of molecule patterns,pathways activation,cytokines release,humoral response,and so on.TLR4 not only recognizes bacterial lipopolysaccharide（LPS）and structural proteins of various viruses but also responds to a variety of endogenous warning molecules（DAMPs）.Conventional dendritic cells（c DCs）belong to antigen-presenting cells（APCs）and play a critical role in the initiation of the humoral response.However,it has not been reported how TLR4 pathway of c DCs subsets including c DC1（CD8α⁺CD11b^-）and c DC2（CD8α^-CD11b⁺）participates in immune response induced by rabies vaccines.In this study,we compared TLR4^-/-mice with wild type（WT）mice,investigated functions of TLR4 molecule in rabies vaccines induced-humoral response including virus neutralizing antibody（VNA）,total Ig G and protection ability,maturation of c DCs,recruitment of c DC1/c DC2 and differentiation of helper follicular T cells（Tfh）and differentiation of type I/II helper T cells（Th1/Th2）,proliferation of germinal center B cell（GC B）and production of plasma cells（PCs）,these main results are as follows:1.TLR4 promotes humoral response induced by live rabies vaccineLive rabies vaccines induce T cell-dependent immune response in mice,APCs bridge innate and adaptive immunity.Here,although TLR4 molecule did not affect the development and maturation of APCs（including BMDCs,B cells and c DCs）after stimulation with live rabies vaccine,it significantly affected VNA level and immune protection.Live rabies vaccine significantly increased c DC2 in secondary lymphoid organs（SLOs）of mice,resulting in a significant decrease of c DC1 proportion,indicating that c DC2 was the main APC for processing RABV antigen and presenting antigen-peptide complex（p MHC-II）to na?ve T cells（Tn）.RABV infection-induced TLR4-dependent migration of p MHC-II bearing c DC2 into SLOs for activation of Tn cells,leading to the differentiation of Tfh and Th1 cells.c DC2-dependent Tfh differentiation promoted GCs formation and GC B proliferation,induced more antibody-secreting PCs,VNA and total Ig G.Therefore,TLR4-dependent c DC2 migration/recruitment promoted humoral immune response after stimulation with live rabies vaccine and improved the ability of mice against virulent RABV infection.However,after infection with RABV,it remains to be further studied that the molecular mechanism of the migration of c DC2 from peripheral tissue to SLOs with the help of endogenous TLR4 ligands（such as HSP70,HS and HMGB1）.Besides,we also found that TLR4 molecule promoted Th1 immune response（high-affinity Ig G2a）,but inhibited Th2 immune response（low-affinity Ig G1）,and balanced Th1/Th2homeostasis.2.TLR4 agonist enhances humoral response induced by inactivated rabies vaccineThe immune response induced by inactivated rabies vaccine was significantly weaker than that induced by live rabies vaccines because the inactivated rabies vaccine did not induce maturation of c DCs,recruitment of c DC2,differentiation of Tfh and Th1/Th2.MPLA,as a specific TLR4 agonist,promoted maturation of c DCs,recruitment of c DC2,differentiation of Tfh,proliferation GC B and PCs through TLR4 pathway,increased VNA level and production of RABV-specific Ig G,Ig G2a and Ig G2b,and induced a strong Th1immune response.MPLA enabled the inactivated vaccine to mimic immune induced by live rabies vaccine and improved immune efficiency and protection of inactivated vaccine for mice.