FimH Regulate Dendritic Cells Activity Via TLR4 And Acts As A Vaccine Adjuvant For Dental Caries

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) whose primary function is to capture, process, and present antigens(Ags) to unprimed T cells and link innate and adaptive immunity. The activation and maturation of DCs play a key role on the enhancement of antigen-induced mucosal immune responses. Therefore, the specific immune response of anti-caries vaccines might be improved to some extent by targeting DCs'activation and maturation. Using Toll-like receptors (TLRs), DCs rapidly recognize and respond to pathogen-derived Toll-like receptor ligands (TLR-Ls) by undergoing maturation and enhancing secretion of proinflammatory and immunoregulatory cytokines. Due to these DC-stimulating functions, TLR-Ls are used as potent immune adjuvants to modulate the quality of the immune responses as well as enhance the magnitude and overall effectiveness of vaccine-induced immune responses.Bacteria encode numerous pathogen-associated molecular patterns (PAMPs), including LPS, flagellin, peptidoglycan and bacterial DNA, which can somehow activate the innate immune system via TLRs. FimH, the adhesion portion of type 1 fimbriae, is involved in bacterial attachment to epithelial cells through interactions with mannose. It has been proved that it can be used as a candidate vaccine or adjuvant for the prevention of urinary tract infections caused by Uropathogenic Escherichia coli (UPEC). Moreover, studies have recently suggested FimH, as a novel TLR4 ligand, can directly activates macrophages and natural killer (NK) cells in vitro.Based on the background, we firstly investigated whether or not the two recombinant FimH, FimH-K12 from E.coli K-12 and FimH-S.T. from Salmonella typhimurium, can affect the maturation and functional properties of DC2.4. And then we explained the underlying molecular mechanism involved. Ultimately, the anti-caries vaccines developed by our scientific crews were utilized along with FimH-K12 or FimH-S.T. aiming at assessing and evaluating the effectiveness and type of the immune response induced by them.Three parts are included in the study: Part One In vitro DC2.4 cell line activation by recombinant FimH The recombinant plasmids pET28a-FimH-K12 and pET28a-FimH-S.T. were expressed in DH5a competent E.coli. FimH-K12 and FimH-S.T. protein were purified by Metal-chelate affinity chromatograph. SDS-PAGE electrophoresis analysis was used for Verification. Proteins were added to DC2.4 at the concentration:FimH-K12, l00ng/ml; FimH-S.T., l00ng/ml; LPS as a positive control, lOng/ml. The relative mRNA expression of IL-6, IL-10, IL-12p35 and IL-12p40 were measured by realtime RT-PCR after 6 hours. The expression of CD40, CD80, CD83, CD86 and MHC-II on the surfaces of DC2.4 were measured by flow cytometry and production of IL-6, IL-10 and IL-12 in the supernatants were tested by ELISA all after 24 hours. Results: The mRNA expression and production of cytokines were increased in the groups FimH-K12, FimH-S.T. and LPS as well as the upregulation of CD40, CD80, CD83, CD86 and MHC-II on the surfaces compared with untreated DC2.4. It suggested that both FimH-K12 and FimH-S.T. enhance the maturation and immunostimulatory activity of DC2.4.Part Two Recombinant FimHs activate DC2.4 cell line via TLR4Flow cytometry was usedto test the TLR4 expression after stimulation by FimH-K12, FimH-S.T. and LPS for 24 hours. In order to confirm that FimH-induced responses are TLR4-dependent, DC2.4 were preincubated with the anti-TLR4 mAb for 1 hour before the treatment with FimH-K12, FimH-S.T. and LPS. And the cell-free supernatants were assayed for IL-6, IL-10 and IL-12 by ELISA. Results: TLR4 expression was upregulated by FimH-K12 and FimH-S.T. which was the same as LPS. Moreover, the TLR4 neutralization significantly inhibit the FimH-induced cytokines production, and the same results were also found about LPS. These results suggested the FimH activates DC2.4 via TLR4 in the same way in which LPS might contribute to.To gain additional insight into the molecular mechanism underlying the effects of FimH on DC2.4 activation and maturation, we sought to study the effect of FimH-K12, FimH-S.T. treatment on the MAPK and NF-kB pathways in DC2.4. DC2.4 were respectively stimulated with FimH-K12, FimH-S.T. and LPS, the levels of MAPK phosphorylation and activation of NF-kB pathways were assessed by western blot. Results:Both FimH-K12 and FimH-S.T. can upregulate the phosphorylation of the MAPKs p38, ERK and JNK in very early stimulation stage. Also FimH-K12 and FimH-S.T. treatment resulted in an increase of NF-kB activity. Therefore, these results suggested that FimH induces DC activation, probably through activation of the MAPK and NF-kB pathways.Part Three Recombinant FimHs enhance the mucosal immune response of anti-caries vaccine in vivoIn this study, groups of BALB/c mice (6-8 weeks of age) were immunized with a 'DNA (the DNA vaccine pCIA-P)primer-protein (rPAc) boosting'strategy. Each mouse was immunized at OW and 2W via intranasal (i.n.). In the negative control group, mice were immunized with PBS for two times. In the groups with FimH, FimH-K12, FimH-S.T. were added for 10ug/mouse/time as adjuvant respectively. Levels of anti-PAc antibodies in serum and saliva were determined by ELISA. And the spleen cell suspensions were prepared at 8 weeks (6 weeks after final immunization) and the production of IFN-y and IL-4 were tested to measure the Th1-and Th2-type immune responses. Results:FimH-K12, FimH-S.T. promoted the production of PAc-specific IgG in serum and IgA in saliva of anti-caries vaccines by intranasal immunization. The immunization with'DNA pCIA-P primer-rPAc boosting'combined no adjuvant induced a Th2-type response mainly. Nevertheless, anti-caries vaccine with FimH-K12 or FimH-S.T. as a adjuvant was indicated a mixed Thl and Th2-type response in this study.