Pilot Study On Proinflammatory Mechanism And Adjuvant Potential Of Treponema Pallidum Flagellins

Background and objective: Syphilis is a serious multistage sexually transmitted disease(STD)caused by Treponema pallidum subsp.pallidum that causes multiple system organ failure.Moreover,it also increases the risk of human immunodeficiency virus(HIV)transmission and acquisition.In China,syphilis has become one of the top five most reported infectious diseases and the most frequently reported STDs.Thus,how to prevent and control syphilis has become a major public health problem.The primary task of preventing and controlling syphilis is to develop an effective vaccine.However,the molecular mechanisms underlying T.pallidum pathogenesis are poorly understood,which cause the slow development of syphilis vaccine.During early infection,T.pallidum is able to elicit a vigorous inflammation and the ensuing adaptive immune response.It is probable that inflammation and the ensuing adaptive immune cooperate in damaging the tissues.However,which proteins in triggering inflammation and orchestrating adaptive immune response have not been identified.Bacterial flagellin,the main structural protein of bacterial flagella,which is not only a potent immunogen that activates the innate immune system and generates strong inflammation responses,but also acts as a vaccine carrier protein or vaccine adjuvant to enhance the immunoprotective effect of related antigens.In this study,we explored the interaction of flagellins and human monocytes to determine the proinflammatory effect and molecular mechanism of T.pallidum flagellins in the pathogenicity of T.pallidum infection.Subsequently,we screened for effective syphilis vaccine candidates and explored the potential of T.pallidum flagellin as an adjuvant for syphilis vaccine.This study will provide new insight into elucidating the pathogenesis of syphilis and the design of syphilis vaccine,and it is of great significance for the prevention and control of syphilis.Methods: 1.THP-1 cells were stimulated with Fla B1,Fla B2,or Fla B3,then the m RNA transcription levels of IL-6,IL-8,IL-10,IL-1β,and TNF-α were measured by using q RT-PCR.THP-1 cells were stimulated with Fla B1,Fla B2,or Fla B3,then the gene and protein expression levels of IL-6 and IL-8 were analyzed by q RT-PCR and ELISA,respectively.THP-1 cells were transfected with dominant negative plasmid encoding My D88(p De Ny-h My D88),TLR2(p ZERO-h TLR2),psi RNA-h TLR4,or psi RNA-h TLR5,then stimulated with Fla B1,Fla B2,or Fla B3 to measure the m RNA transcription levels of IL-6 and IL-8.2.THP-1 cells were treated with Fla B1,Fla B2 or Fla B3 for different time periods.The expression of ERK1/2,p38,JNK,and IκBα were analyzed by Western Blot.Cells were fixed and permeabilized,then stained with anti-NF-κB p65 antibody,followed by incubation with 4’-6-diamidino-2-phenylindole(DAPI)and cy3-conjugated goat-anti-rabbit antibody.Images were obtained using a confocal microscope.3.THP-1 cells were stimulated with purified Fla B1,Fla B2,or Fla B3 mutant flagellins.The gene transcription levels of IL-6 and IL-8 were analyzed by q RT-PCR.Then the expression of nonphosphorylated and phosphorylated forms of ERK1/2,p38,and IκBα were analyzed by Western Blot.Co-immunoprecipitation was performed to assess the binding of the mutated proteins to human TLR5.THP-1 cells were transfected with psi RNA-h TLR5,then stimulated with the Fla B1,Fla B2,or Fla B3 mutant flagellins to analyze the expressions of nonphosphorylated and phosphorylated forms of ERK1/2,p38,and IκBα,and the transcription levels of IL-6 and IL-8 m RNA.4.THP-1 cells were stimulated with site-directed mutant Fla B1,Fla B2 or Fla B3 flagellins for 24 h.The gene transcription levels of IL-6 and IL-8 were analyzed by q RT-PCR.Co-immunoprecipitation was performed to assess the binding of the mutated proteins to human TLR5.5.New Zealand rabbits were randomly divided into 3 groups(PBS control group,Tp0136-immunized group,and Tp0663-immunized group),with 3 animals in each group.Each animal was injected 3 times at 2 week intervals(0,2,4,and 6 weeks)with 150 μg of test antigen or PBS emulsified in a 1 : 1 mixture with complete Freund’s adjuvant system.Blood was drawn from the rabbit ear veins,the Ig G antibody levels and IFN-γ level in the blood were determined by indirect ELISA.6.21 days after the final immunization,rabbits were sedated and intradermally challenged at 8 sites along their shaved backs with 8×106 freshly isolated T.pallidum(Nichols strain).Challenge sites were examined daily and photographed every 3 days to document lesion development.At day 21 post-challenge,all of the rabbits were killed,and then q RT-PCR were used to assess the burden of T.pallidum in biopsies,blood,spleen,liver,and testicles.The skin and testicle tissues were sectioned and stained with S-P immunohistochemistry or stained with H&E.7.C57BL/6 mice were randomly divided into 4 groups(PBS,Fla B3,Tp0663,and Fla B3+Tp0663 immunized group),with eight animals in each group.Each animal was injected three times at 2 week an interval(0,2,4,and 6 weeks)with 100 μg of test antigen.The blood was drawn from the mice tail vein,and the Ig G antibody levels were determined by indirect ELISA.At week 2 after the last immunization,splenocytes were isolated from 3 mice of each group.Flow cytometry was used to assay for Tp0663 specific CD4+ T cell response.CCK8 kit was used to detect lymphocyte proliferation and ELISA was used to detect the cytokines in mouse spleen cell supernatant.8.14 days after the final immunization,mice were inoculated intradermally,intraperitoneally,intrarectally and corpus cavernosa with 1×107 organisms.At day 30 post-challenge,all of the mice were killed,and then q RT-PCR were used to assess the burden of T.pallidum in the rectum,blood,brain,liver,lymph node,spleen,and testicle.The liver,spleen and testicle tissues were sectioned and stained with S-P immunohistochemistry.At day 30 post-challenge,mouse lymph nodes were removed,extracted in saline and injected into the testicles of randomly assigned rabbits.All animals were monitored daily for orchitis and assayed at least every one week for seroconversion by using rapid plasma regain(RPR)test and T.pallidum particle agglutination(TPPA)assay.9 weeks after injection,three rabbits from each group were euthanized,darkfield microscopy was performed on testicular extracts from the rabbits to detect T.pallidum.Results: 1.Fla B1,Fla B2,or Fla B3 induced the expressions of IL-6 and IL-8(but not IL-10,IL-1β,or TNF-α)m RNA in THP-1 cells.IL-6 and IL-8 gene and protein expressions were significantly increased when treated with 1.0 μg/m L Fla B1,10.0 μg/m L Fla B2,or 5.0 μg/m L Fla B3.The highest levels of IL-6 and IL-8 m RNA and protein expressions were observed after 24 h or 48 h,respectively.After THP-1 cell were transfected with p De Ny-h My D88 or psi RNA-h TLR5,the expressions of IL-6 and IL-8 induced by Fla B1,Fla B2,or Fla B3 were significantly downregulated.In contrast,THP-1 cell transfected with p ZERO-h TLR2 or psi RNA-h TLR4 did not show any clear differences in downregulating both IL-6 and IL-8 productions.2.After THP-1 cells were treated with Fla B1,Fla B2,or Fla B3 for different time periods,Fla B1,Fla B2 or Fla B3 were found to induce ERK1/2,p38,and IκBα phosphorylation,as well as the NF-κB subunit p65 nuclear translocation.After THP-1 cells were pretreated with specific inhibitors,there were no significant changes in IL-6 and IL-8 levels when the cells were pretreated with JNK inhibitor,whereas pretreatment with ERK1/2,p38,and IκBα inhibitors result in a significant decrease of three flagellins-induced IL-6 production and Fla B2-induced IL-8 production.3.Recombinant flagellins containing the D1 domain(NC,△N0,△C0,and △N0C0)could induce a significant IL-6 and IL-8 transcriptions,as well as ERK1/2,p38 and IκBα phosphorylation.However,recombinant flagellins without the D1 domain(N,△N,△NC,△C,and C)failed to induce IL-6 and IL-8 transcriptions,as well as ERK1/2,p38 and IκBα phosphorylation.In addition,mutant flagellins containing ND1 domain(NC,△C,△C0,N,△N0,and △N0C0)successfully bind to the TLR5,while the truncated flagellins lacking of ND1 domain(△N,△NC,and C)affected the binding efficiency of flagellin molecule to TLR5.All mutant flagellins containing D1 domain(NC,△C,△C0,N,△N0,and △N0C0)led to significant downregulations of the transcriptions of IL-6 and IL-8 after the silencing of TLR5,which is consistent with the activation of ERK1/2,p38,and IκBα.4.All site-directed mutations have varying degrees of impact on the IL-6 and IL-8 transcription levels as well as the binding efficiency of those flagellin molecules to TLR5.5.Immunization with Tp0136 or Tp0663 generated a strong humoral immune response with high titers of Ig G.Compared to that of rabbits immunized with PBS alone,the production of IFN-γ was significantly increased in the rabbits immunized with Tp0136 or Tp0663.6.Tp0136-or Tp0663-immunized animals presented smaller-size swellings and lower lesion ulceration rate compared to the control group.The treponemal burden in the primary lesion sites,blood,spleen,liver,and testicles was lower in the Tp0136-or Tp0663-immunized than in the non-immunized controls.Tp0136-or Tp0663-immunized rabbits had increased levels of immune cell infiltration in the primary lesion sites relative to PBS-immunized rabbits.Furthermore,the inflammatory infiltrations in the distant testicles were notably reduced in vaccinated animals compared to PBS-immunized animals.7.The Tp0663-specific Ig G production in C57BL/6 mice immunized with Fla B3+Tp0663 was higher than that in the mice group immunized with Tp0663 or PBS.CD4+ T cells secreted only IFN-γ but not IL-4.The percentage of CD4+ T cells secreting IFN-γ,the IFN-γ levels secreted by mouse spleen lymphocytes,and the proliferation and differentiation ability of lymphocytes in C57BL/6 mice immunized with Fla B3+Tp0663 were significantly higher than those in the Tp0663 immunized group and the PBS control group.8.Treponemal burden in the blood,brain,liver,lymph node,spleen,or testicle was lower in the Fla B3+Tp0663 immunized group than in the Tp0663 and PBS immunized groups,while treponemal burden in the rectum has no difference in the Fla B3+Tp0663 immunized group than in the Tp0663 and PBS immunized groups.Testicles inoculated with lymph nodes from PBS,Tp0663,Fla B3 and Fla B3+Tp0663 immunized animals provided evidence of seroconversion by day 20,32,32,and 52,respectively.Darkfield analysis confirmed the presence of motile T.pallidum in the testicles of control recipient animals.Conclusions: 1.T.pallidum flagellins Fla B1,Fla B2 or Fla B3 induce THP-1 cells to express IL-6 and IL-8 via TLR5/My D88-dependent MAPKs and NF-κB signaling pathways.2.The D1 domain of T.pallidum flagellins Fla B1,Fla B2 or Fla B3 is essential for the activation of TLR5.R89,L93 and E113 of T.pallidum flagellins Fla B1,Fla B2 or Fla B3 are invoved in the interaction of T.pallidum flagellin with TLR5.3.New Zealand rabbits immunized with Tp0136 or Tp0663 exhibited attenuated lesion development and inhibition of treponemal dissemination to distant organs.4.T.pallidum flagellin Fla B3 could increase the immunogenicity of Tp0663 and has the potential of vaccine adjuvant,and C57BL/6 mice immunized with Fla B3+Tp0663 exhibited inhibition of treponemal dissemination to distant organs.