Enhance Immunity Mechanism By Mannosylated Brucella Polypeptide Targeting Dendrit Cell

The genus Brucella are the etiological agents of brucellosis, a extended bacteriological disease worldwide that has a negative economic impact on animal production and human public health. Because of the serious economic and medical consequences of brucellosis, efforts have been made to prevent the infection through the use of vaccines. Brucella can survive and replicate in macrophages. Dendritic cells(DCs) are the antigen presenting cells that initiate and regulate immunity. By studying these cells, we will be able to move beyond standard approaches and design vaccines that directly harness the elaborate properties of DCs to control immunity. The mannose receptor(MR) is an important component of the immune system. The receptor plays an important role in linking both innate and adaptive immunity.BMDCs were incubated with fluorescent isothiocyanate(FITC)-labeled mannosylated Omp31,the affinity of MR and its lignads were measured by flow cytometry(FCM) and fluorescene microscope. The binding of MR and its ligands can be inhibited by the existence of D-mannose, mannan and Con A, especially by mannan. The measurement of the affinity of MR and its ligands by flow cytometry(FCM) and fluorescene microscope is a simple, quick and accurate. The affinity of mannan and MR is higher than other carbohydrate, it may be used to regulate the immunological function by targeting at MR.Omp3148-74 from Brucella cellular membrane was synthesized. Mannan derived from S. cerevisiae, its oxidized or reduced form was prepared as previously described. Mannan was oxidized into a poly aldehyde compound by sodium periodate at 4℃. The OM fraction was collected through a Sephadex G-25 M column. Addition of sodium borohydride into OM solution reduces aldehydes to amines and alcohols, resulting in formation of RM. We make sure of the glycation of ε-amino groups of lysines as the glycosylation site of Omp3137. The Omp31 were glycosylated with α-D-mannopyranosyl-phenylisothiocyanate, OM and RM, and the production was identified with the resorcinol-sulfuric acid reagents analysis. The result indicated that the Omp31 were glycosylated efficiently. We demonstrate that Th1-type(generating high CTL, low antibody) or Th2-type(the reciprocal) responses can be induced by conjugation of the Omp31 to the carbohydrate polymer mannan: Th1 responses are selected by using oxidizing conditions, Th2 responses are selected by using reducing conditions for the conjugation. Using Brucella Omp31 as a model antigen in mice, immunization with oxidized mannan-Omp31 fusion protein(MO-Omp31) led to a Th1 response with high interferon-γ(IFN-γ) secretion, no interleukin 4(IL-4), and a predominant Ig G2 a antibody response, red M-Omp31 selects for a Th2-type response with IL-4 production and a high predominant Ig G1 antibody response but no IFN-γ.Stimulants(PBS, OM, RM and LPS) were added into DC cultures at specified doses and cells were harvested at specific time-points. To evaluate systemic DC activation, each stimulant was injected intravenously at bases of tails, at specified doses into four mice. After 24 h, spleens were collected for the systemic DC analysis. To set up allogeneic MLR, stimulated non-adherent BMDCs were washed, and added as stimulatory cells into 96-well U-bottom plates containing allogeneic BALB/c na?ve CD4+ T cells in quadruplets. To evaluate phenotypic maturation of BMDCs and splenic DCs, cells were stained with anti-mouse CD11c+ conjugated to APC in combination with in-house rat anti-mouse CD40, CD80, CD86 or MHC-class II conjugated to fluoroscein isothiocyanate(FITC). Evaluation of cytokine m RNA expression via RT–PCR for evaluation of mouse DC Th1/Th2 cytokines(IL-12p35, IL-10 and IL-4). Supernatants from DC cultured with different mannan derivatives or media alone were collected at 24 h and tested for IL-12 p70, IL-10, IL-4 and IFN-γ, Cytokine levels were determined by sandwich ELISA. The adjuvant effect of mannan derivatives including OM and RM, in comparison to LPS, DCs were investigated. OM and RM were capable of stimulating mouse bone marrow-derived DC in vitro, up-regulate DCs surface markers, secrete differential T helper 1(Th1)/Th2 cytokines. OM instruct DCs to stimulate Th1 responses via IL-12p70 production, RM barely induce IL-12p70, but for IL-10 and IL-4, and activating allogenic T cell responses. Injection of mice with OM and RM induced a mature phenotype of splenic DCs. The data presented here, together with evidence reported previously on OM and RM in induction of immune responses in vivo, suggest that OM and RM exert a dual capacity to target antigen to APCs as well as mature DCs.TLR4 gene expression was reduced by RNA interference(RNAi) technology, and the impacts of RNAi on these indicators were observed to explore the regulatory mechanism. Stimulants were added into Si BMDC cultures. To evaluate phenotypic maturation of BMDCs, cells were stained with anti-mouse CD11c+ conjugated to APC in combination with in-house rat anti-mouse CD40, CD80, CD86 or MHC-class II conjugated to fluoroscein isothiocyanate(FITC). BMDCs were stimulated with the indicated Mannan derivatives for up to 30 min. Nuclear or cytoplasmic proteins were resolved by SDS PAGE and transferred to onto a nitrocellulose membrane. The membrane was blocked, washed and incubated with primary antibody against NF-κB p65, phosphorylated p38, JNK, ERK or ERK 1/2. Specific TLR4 si RNA can down-regulate the expression of TLR4 and provide an effective means to further study TLR4 function in BMDCs. The expressions of CD80 and CD40 reduced in si BMDCs with silencing TLR4 treated by mannan derivatives, Subsequent experiments demonstrated that activation of DCs was Toll-like receptor-4-dependent. Here, we demonstrate that OM and RM instruct mice BMDCs to induce distinct Th cell responses by differentially modulating mitogen-activated protein kinase signaling. OM activated DCs to depend on the phosphorylation of p38 and c-Jun N-terminal kinase 1/2. In contrast, the RM stimulate sustained duration and magnitude of extracellular signal-regulated kinase 1/2 phosphorylation. NF-κB is central to DC maturation,As expected Western blot result of OM and RM showed nuclear translocation of p65 subunit into the nucleus.