Protective Regulatory T Cell Immune Response Induced By Intranasal Immunization With Live Attenuated Pneumococcal Vaccine SPY1 Via TGF-?1-Smad2/3 Pathway

Objective:To investigate the generation of SPY1-specific Treg cells and the underlying mechanisms responsible for the protection induced by SPY1-specific Treg cells.Background:Vaccine effectiveness is mainly dependent on the mechanism of immunizing protection elicited,unraveling the protective mechanism remains a critical part of new pneumococcal vaccine development.Streptococcus pneumoniae(S.pneumoniae)strain SPY1 is a live attenuated pneumococcal vaccine we previously reported,and the vaccine is characterized by highly reduced virulence,reliable genetic stability,high safety and excellent protection against pneumococcal infection in mice model.In previous study,we have detected the protective role of regulatory T(Treg)cell immune response elicited by SPY1,however,the underlying mechanisms remains unknown.TGF-?is essential for the differentiation of Treg cells.Previously we have proved that the SPY1-elicited protection against invasive S.pneumoniae infection could be attenuated by a synthetic short peptide P17 through inhibition of TGF-?1activity,suggesting the importance of SPY1-induced TGF-?1 to the protective Treg cell immune response in acquired immunity from a certain perspective.Consequently,we present questions that by which signal did TGF-?1 mediating the generation of SPY1-specific Treg cells,and which mechanisms are responsible for the protection induced by SPY1-specific Treg cells?Therefore,in this study,we will further explore the generation and effection of SPY1-induced Treg cells,providing theoretical basis for clarifying the protective effect of SPY1 vaccine.Methods:1.Female C57BL/6J mice were intranasally received vaccine(group CT+SPY1)or adjuvant alone(group CT)for four times at 1-week intervals.During the whole vaccination progress,half of mice from group CT+SPY1were randomly chosen to be intraperitoneally injected with 100?g of peptide P17 daily(group CT+SPY1+P17).The other half of mice in group CT+SPY1 received sterile PBS as control.Similarly,mice from CT group were also injected with P17 or PBS daily.All the mice were mucosally challenged with either strain 19F(1×10~8 CFU)or strain D39 at one week after the final immunization,with P17-treated mice receiving 500?g of P17both 2 h and 4 h before challenge.2.Mice weights were monitored during the whole immune process.After challenged by pneumococcal strain D39,mice weights and survival time were observed to detect the effect of P17 on SPY1 protection.3.Cytokines secreted by mice splenocytes stimulating with 70%ethanol-killed SPY1 were measured with ELISA(IL-6,TNF-?,IL-12p70,IFN-?,IL-4,IL-5,IL-17A,IL-10).Pulmonary injuries were analyzed by H&E staining post 19F challenge and cytokine levels in lung homogenate were detected by ELISA.4.The levels of active TGF-?1 in lung homogenate,spleen homogenate and splenocyte supernatant were detected by ELISA on day 7post the final SPY1 vaccination,respectively.After strain 19F challenge,levels of tgf1-?1 mRNA and active TGF-?1 in lung were detected by PCR and IHC,respectively.The related molecule expressions of Smad-dependent and Smad-independent pathways in TGF-?signalling were measured by PCR,WB and IHC were used to further verification.5.The role of PD-1 and CTLA-4 were analyzed by flow cytometry post 19F challenged.Results:1.In murine model of pathogenic S.pneumoniae strain D39 infection,the survival time of P17-treated SPY1-immunized mice were significantly shorter than that of untreated immunized mice,further proofed the immune protection of SPY1.2.P17 treatment further increased the immune cytokines(IL-12p70,IFN-?,IL-4,IL-5,and IL-17A)and infection-associated inflammatory cytokine TNF-?in SPY1-immunized mice.The upregulation of both immunosuppressive cytokine IL-10 and anti-inflammatory cytokine IL-6were reversed by P17 to some extent.H&E staining showed that pulmonary injuries and inflammatory response in P17-treated SPY1-immunized mice were more intensive than that in untreated immunized mice.The changes of cytokines detected in lung homogenates were consistent with those in splenocytes supernatant.3.By using PCR and IHC,results showed that SPY1 immunization significantly up-regulated the production of Foxp3 in lungs.The increased levels of active TGF-?1 in lungs,spleens and splenocytes of SPY1immunized mice were detected by ELISA.Simultaneously,the upregulation of TGF-?1 by SPY1 immunization reversed by P17 treatment were further proofed by PCR and IHC post 19F infection.4.In SPY1 immunized mice,the mRNA levels of smad2,smad3,and smad4 were increased,the mRNA level of smad7 was decreased.The increased productions of Smad2/3 and phosphor-Smad2/3 and decreased Smad7 in SPY1-vaccinated mice were further proved by WB.All the changes could be inverted by P17 treatment,demonstrating SPY1immunization active the TGF-?1-Smad2/3 signaling pathway.5.Flow cytometry results showed that the expression of PD-1 and CTLA-4 on SPY1-induced Treg cells was elevated post 19F challenged.Conclusions:Inhibition of TGF-?1 dramatically attenuates the SPY1-induced acquired protection against pulmonary injury caused by pneumococcal colonization.Besides,SPY1-elicited TGF-?1 is essential for the balances among systemic protective immune responses triggered by SPY1vaccination.The activated TGF-?1-Smad2/3 signaling is responsible for the generation of SPY1-specific Treg cells,which plays protective role in SPY1 protection through the elevated expressions of PD-1 and CTLA-4.