The Mechanism Of Heparin-binding Hemagglutinin Induces IL-17 Exert Immune Protective Effect Against Tuberculosis

Tuberculosis(TB)is an infectious disease caused by Mycobacterium tuberculosis(MTB).The End TB Strategy remains a priority workfor the World Health Organization(WHO).Bacillus Calmette-Guérin(BCG)is the only vaccine approved for the prevertion of TB,which can prevent TB in children but is ineffective against pulmonary TB in adults.At present,the lack of new vaccines that can effectively prevent TB is one of the main obstacles to prevention and control the disease.Therefore,it is particularly important to study of MTB-specific antigens that can deepen the understanding of TB infection and immune mechanisms,and also help in vaccines development,disease diagnosis,immunotherapy,etc.Heparin-binding hemagglutinin(HBHA)is an antigen on the surface of MTB,mediates the adhesion of MTB to non-phagocytic cells,promotes the replication and dissemination of MTB in lung epithelial cells.Therefore,it participates in the occurrence and development of TB.Previous studies by our group had shown that HBHA can interact with lung epithelial cells and macrophages,and they both play an important protective role in the early stage of MTB infection.Using HBHA as a candidate vaccine,it was found that HBHAcan induce Thl and Th17 cell-type immune responses.Recent studies have found that Th17 cells are critical for vaccine-induced protection,especially in early stageof infection.In addition,IL-17 secreted by Th17 cells can promote the recruitment of T and B cells by chemokines to participate in the formation of Tertiary Lymphoid Structures(TLS).Inducible bronchus-associated lymphoid tissue(iBALT)is one of the types of TLS in the lungs and has been shown to be associated with the control of pulmonary MTB infection.In summary,we hypothesized that HBHA may promote the formation of iBALT by inducing IL-17,thereby exerting an early immune protective effect against MTB infection.This study will elucidate the mechanism of immune effect induced by HBHA through the vitro experiments and animal immune models,and demonstrate the mechanism at tissue,cellular and molecular levels.Part 1 The effect of HBHA on stimulation of IL-17 productionObjective:To verify whetherHBHA can promote the enhancement of IL-17 expression.Methods:Female C57BL/6 mice aged 6-8 weeks were randomLy divided into 5 groups with 6 mice in each group.The control group,the early secreting antigen target-6(ESAT-6)intranasal group,and the HBHA intranasal group,respectively received 30μg(20μL)of PBS,ESAT-6,and HBHA,once every 7 days for a total of 4 times.For mice receiving prime-boost immunization,first subcutaneously inoculate 5×105 CFU of BCG,28 days later,intranasally immunize with 30μg(20μL)of PBS(BCG/PBS control group)or HBHA(BCG/HBHA boost group),once every 7 days for a total of 4 times.On the 14th day after the last immunization,the mice were sacrificed to immunological analysis,including ELISA was used to detect the concentration of IL-17A,IL-6,TNF-α,IFN-γ and TGF-β in the plasma;real-time quantitative PCR(qPCR)was used to detect the relative quantity(RQ)mRNA of IL-17A,IL-6,IL-23,IL-22,IL-1α,and IL-1β in the lung;flow cytometry was used to detect the frequency of Thl and Th17 cells in the peripheral blood mononuclear cells(PBMCs)and the spleen.In order to preliminarily verified the ability of HBHA to induce IL-17A in human vitro experiment,6 healthy controls(HC)and 6 patients with active tuberculosis(ATB)were collected.The peripheral blood was collected and co-cultured with PBS or HBHA,and then the levels of IL-17A were detected.Results:Compared with other groups,in the HBHA intranasal group or the BCG/HBHA boost group,the level of plasma IL-17A in mice was significantly increased,and the transcription levels of IL-17A,IL-22,and IL-23 in lung were significantly up-regulated.Except for the PBS control group,plasma IL-6 levels were increased in all groups;plasma TGF-β levels were significantly decreased in the BCG/HBHA boost group.In addition,the frequency of Th17 cell subsets increased in the BCG/HBHA boost group.The vitro experiments showed that HBHA could promote the secretion of IL-17 in peripheral blood of HC patients,and the inductive ability was stronger in the peripheral blood of ATB patients.Conclusion:HBHA can induce the production of IL-17in both animal immune model and human vitro experiment.Part 2 Mucosal booster immunization with HBHA induces iBALT formationObjective:To study the effect of mucosal immunity with HBHA to promote the formation ofiBALT,and explore the mechanism of its formation.Methods:Thirty female C57BL/6 mice were randomLydivided into the control group,the ESAT-6 intranasal group,the HBHA intranasal group,the BCG/PBS control group,and the BCG/HBHA boost group,each group with 6 mice.On the 14th day after the last immunization,the lung tissue sections were stained with HE and immunofluorescence to detectthe formation of iBALT.qPCR was performed to detect the relative quantity of mRNA in the lung,including chemokines,chemokine receptors and intercellular adhesion molecules.Finally,the transcriptional level of IL-10,which inhibits iBALT formation,was detected in the lung,as well as the frequency of Treg cells in spleen.Results:By HE staining and immunofluorescence staining,it was found that iBALT was formed in the lung of the BCG/HBHA boost group,and well-structured iBALT could not be formed in other groups.The transcription levels of chemokines CXCL13 and CCL19 related to the formation of iBALT in the HBHA intranasal group and the BCG/HBHA boost group were significantly higher than those in other groups;the transcription levels of lymphocyte function-associated antigen-1(LFA-1)and intercellular adhesion molecule-1(MCP-1)in the BCG/HBHA boost group were higher than those in other groups.Only loose lymphocyte aggregation was observed in the HBHA intranasal group,which may be related to the up-regulation of IL-10 transcriptional level and the increased frequency of Treg cell subsets in this group.Conclusion:HBHA intranasal booster immunization of BCG-vaccinated mice can effectively induce the formation of iBALT dependent on CXCL13,CCL19,LFA-1 and MCP-1.Part 3 Early protective effect of mucosal booster HBHA on BCG imitative infectionObjective:To investigate the protective effect of booster immunization with HBHA on BCG imitative infection.Methods:C57BL/6 mice received prime-boost immunization were divided into BCG/PBS control group and BCG/HBHA boost group,30 mice in each group.On the 14th day after the last immunization,the mice received 1×106 CFU BCG intranasal imitative infection.On the 4th,8th,12th,24th,and 36th days after infection,the weight of mice,the transcriptional level of IL-17Ain lung,and the concentration of IL17A and IFN-γ in plasma,and the frequency of Thl and Th17 cellsubsets in spleen were detected at each time point.On the 8th,12th,24th and 36th days after infection,the number of bacteria in the lungs was detected by plate count method and expressed as the logarithm of colony forming units(CFUs).On the 36th day after infection,the transcription levels of molecules related to tuberculous granuloma formationwere detected by qPCR;HE staining was used to observe the formation of granulomas in lung tissue.Results:On the 4th day after the imitativeinfection,the infection was confirmed by modified acid-fast staining,and the body weight of the mice was significantly lower than that before infection in the both of BCG/PBS control group and BCG/HB HA boost group.The time of weight recovery and increase of mice in BCG/HBHA boost group occurred earlier than that in BCG/PBS control group.On days 12th and 24th after imitative infection,the bacterial load in the lungs of mice in the BCG/HBHA boost group was lower than that in the BCG/PBS control group.On the 36th day after infection,the transcription levels ofintercellular adhesion molecule-1(ICAM-1)and LFA-1 in lung were increased,and more tuberculous granulomas were observed in the lung tissue of the mice in the BCG/HBHA boost group.Conclusion:HBHA intranasal immunization of BCGvaccinated mice may be an effective immunization method,which can reduce the lung bacterial load and promote the formation of granulomas in the early stage of imitative infection.Conclusion:In animal immune models and human vitro experiment,HBHA had the ability to promot the production of IL-17.HBHA intranasal boosted immunization of BCG-vaccinated mice induced IL-17 production,promoted iBALT formation,and reduced lung bacterial load at an early stage when challenged with imitative infection.To sum up,the mucosal boosting immunity of HBHA has an immune protective effect against MTB infection.