The Preliminary Of The Molecular Mechanisms Of Levamisole Regulate Dendritic Cells Induce Th1Immune Response In Vitro

Levamisole (LMS) as an adjuvant, can induce the antigen-presenting cells produce the inflammatory cytokines and upregulated the expression of co-stimulatory molecules CD80and CD86, but the cellular basis and the molecular mechanisms of how levamisole strengthening the Thl cellular immune responses was not determined. So whether levamisole as adjuvant regulating dendritic cell maturation by TLRs-mediated signaling pathways, resulting in the induction of Thl-type cytokine microenvironment, thereby enhancing the Thl immune response, has yet to see the research report that about this aspect. Therefore, the project intends to explore the mechanisms that levamisole enhance cellular immune response, especially Thl-type cellular immune response.The cell membrane molecules TLRs of DC as target, researching the effect of levamisole as a adjuvant on DC, and on the basis of this to explore both receptor and signal transduction mechanisms, aims to providing new information for the mechanisms of the adjuvant action and the development and application of new vaccine.First, we cultured murine bone marrow derived dendritic cells (DC) by GM-CSF and IL-4, treatment of DC with levamisole.Then cell morphology was observed under optical microscopy, flow cytometry analysis to measure the cell membrane molecules, finally detecting cytokines secreted in the supernatant by ELISA; Second, to test whether the levamisole-treated murine DC is sufficient to promote activation and stimulating of spleen naive T cells by MTT. Then detecting the secretion of IFN-y and IL-4in the DC and T cells co-culture supernatant by ELISA,in order to determine the direction of T cell differentiation; Finally, LMS treated DC, detecting the cell-surface molecules TLR2,4,6,9and the myeloid differentiation primary response88(MyD88)、nuclear factor-kappa B (NF-kB) by Real-time PCR and Western boltting.The results are as follows:1、we successfully cultured dendritic cells in vitro.Treatment of DC with levamisole increased the presentation of CD11C、CD80、CD86and MHC-II molecules on the cell membrane, as well as the production of interleukin IL-12p70. This determined that levamisole can induce DC maturation in vitro.2、 Levamisole-treated human DC also enhanced T cell activation towards type1T helper immune response by inducing interferon-ysecretion but not the IL-4.3、We determined LMS acts on TLR2that onimmature DC, which activates signal transduction pathways.Anti-TLR-2mAb blocked levamisole-induced IL-12p70production.4、After levamisole activation TLR2,TLR2stimulate MyD88and NF-kB upregulation. The study will provide theoretical guidance for the further reveal the mechanism of levamisole stimulate maturation of immature dendritic cells.