念珠菌形态转换中DC-SIGN介导的免疫识别通过Raf-1激酶调控CD4+Th0细胞定向分化的机制

The transition of Candida albicans (C. albicans) from yeast phase into mycelial phase will induce the up-regulation of IL-10 expression on human dentritic cells (DCs) in a manner dependent on TLR2/6 signalling pathway, and trigger the bias of protective immunity against C. albicans. Notiveably, upon recognition of α-mannose moiety on the cell wall of C. albicans by DC-SIGN, a C-type lectin receptor, the associated Raf-1 signalling pathway is activated and consequently induce the up-regulation of IL-10 mediated by TLRs. Accordingly, we infer that DC-SIGN may be the target of C. albicans during the transition into the mycelial phase, which will induce the up-regulation of anti-inflammatory cytokines including IL-10 mediated by TLR2 and TLR4, and facilitates the development of initial CD4+ helper T lymphocytes (Th0) into the Th2 cells..In this project, the blockage of TLRs associated signalling pathway, RNAi and Chromatin Immunoprecipitation (CHIP) techniques will be introduced to analyze whether the expression of inflammatory cytokines mediated by TLR2 and TLR4 is down-regulated in a DC-SIGN/Raf-1 dependent manner, and therefore, whether the negetively biased development of co-cultured CD4+ helper T lymphocytes is triggered. Furthermore, the animal experiments are designed to assess the effects of DC-SIGN/Raf-1 blockage on the protective immunity mediated by murine DCs, and accordingly, we hope to provide the assistance for the hyphothesis that DC-SIGN might be the effective target for the immunological therapy of candidiasis.

白色念珠菌由酵母相向菌丝相转换会引起机体抗原递呈细胞中TLR2/6介导的IL-10表达上调，并诱导机体保护性免疫应答偏倚；C型凝集素样受体DC-SIGN可识别念珠菌细胞壁中α-甘露糖并通过Raf-1途径诱导多种TLR介导IL-10表达上调。我们推测：念珠菌向菌丝相转换中，以DC－SIGN为免疫识别靶点并通过Raf-1途径诱导TLR2、4介导IL－10等抗炎因子表达上调，导致CD4+Th0细胞向Th2细胞分化。.本课题拟采用TLR信号阻断、RNAi和染色质免疫共沉淀等技术分析：念珠菌向菌丝相转换过程中是否通过DC-SIGN/Raf-1途径负向调控TLR2、4介导的炎性因子并诱导共培养的CD4+Th0细胞分化的偏倚；并通过动物实验进一步验证DC-SIGN/Raf-1的阻断对小鼠树突状细胞介导的保护性免疫应答的影响，从而为DC-SIGN能否作为免疫治疗念珠菌病的有效靶位提供实验室依据。

【研究背景】. 在机体抗念珠菌免疫应答中，Th1亚群分化和IFN-γ的释放可增强嗜中性粒细胞或巨噬细胞氧依赖性杀灭病原真菌的能力，进而促进真菌病原体的清除；而Th2亚群分化和IL-4的释放则会增加感染播散的风险。研究证实，部分病原真菌可通过胞壁甘露糖成分特异性结合宿主树突状细胞（Dc）表面的DC-SIGN受体，并利用其下游信号通路诱导宿主Th2型细胞免疫应答的偏倚。近期研究表明，大剂量糖皮质激素的临床应用会显著影响宿主树突状细胞的抗原递呈功能和嗜中性粒细胞的杀菌效能，进而增加念珠菌血症的发生概率。. 然而，1、在念珠菌形成侵袭或播散性感染过程中，DC-SIGN受体是否参与介导Th亚群的分化？2、糖皮质激素的应用对Dc细胞表面DC-SIGN的表达及下游信号通路的影响如何？3、糖皮质激素是通过改变抗原递呈细胞的炎性因子谱系抑或直接调控宿主CD4+Th亚群的分化，进而影响到念珠菌感染的转归？迄今尚需进一步探索。.【主要研究内容】. 在本研究中，我们通过体外细胞学实验分析在白念珠孢子作用下，小鼠骨髓源性树突状细胞（BMDC）培养上清中相关炎性因子的分泌和表达；作用前后细胞表面DC-SIGN的表达水平；建立白念珠菌致BALB/c小鼠腹腔感染模型，进而分析腹腔注射大剂量（20 mg/kg）糖皮质激素：1、对脾脏Dc细胞表面DC-SIGN表达的影响；2、对脾脏感染灶内抗原递呈细胞及Th细胞相关炎性因子分泌谱系的影响；3、对腹腔感染灶内病原体寄生形态、感染模式及转归的影响。.【重要实验结果】. 1、在白念珠菌孢子作用前后，未检测到DC-SIGN在体外培养BMDC及小鼠脾脏感染灶Dc细胞中的表达；. 2、在糖皮质激素作用下，小鼠脾细胞IFN-γ的生成和分泌受到显著性抑制；. 3、糖皮质激素直接而非通过炎性因子网络抑制IFN-γ的表达和分泌；. 4、糖皮质激素作用后，念珠菌孢子在小鼠腹腔接种灶内向菌丝相转化，并形成持续性感染，而在对照组BALB/c小鼠腹腔接种灶内，念珠菌孢子则被完全清除。.【科学意义】. 本研究从小鼠实验层面初步揭示，在宿主抗念珠菌侵袭性感染中，DC-SIGN受体并未参与调控机体Th细胞亚群分化；大剂量糖皮质激素可直接抑制宿主T淋巴细胞的分化和IFN-γ的生成，进而诱导感染的侵袭性发展或播散。