黑素细胞在巨噬细胞抗白念珠菌免疫应答中的作用

Melanocytes（MCs）- melanin-producing dendritic cells in the skin- were thought to do nothing more than providing pigmentation and ultraviolet light protection to skin.Whether MCs play a role in the skin innate immune resposes against infection has long been largely neglected in the research field of skin immunity. There has been no literature about the immunological role of MCs in the skin immunity against pathogen. Recently, we have found that MCs significantly enhance macrophages inflammatory responses to Candida albicans which is the most common opportunistic fungus in human. It is well known that macrophages are organizers and coordinators of innate skin immune repsonses against pathogens.During the early inflammatory phase, macrophages exert pro-inflammatory functions like antigen-presenting, phagocytosis and production of inflammatory cytokines and growth factors that facilitate the initiation of immune responses against pathogens. Our findings were highly suggestive that MCs may have immunological potential and are actively involved in the macrohage-mediated immunity against infection. Based on our previous findings that the presence of MCs could enhance the inflammatory cytokines release of macrophages when stimulated with Candida albicans, this project is designed to further reveal the unappreciated roles of melanocytes in macrophage-meidated host defense against Candida albicans. Experiments in vitro will be conducted to explore whether melanocytes can enhance macrophages' recognition, phagocytosis, killing of Candida albicans and the expression of co-stimulatory molecules on macrophages. The expected results of the project will hoplefully reshapge our current view of melanocytes by adding an immunological dimension to the melanocytes, and may further renew our knowledge of the skin immune system.

黑素细胞是主要分布于人体表皮的一类能够合成黑色素的细胞。长期以来黑素细胞被排除在皮肤免疫系统研究之外，认为皮肤黑素细胞通过合成黑色素仅发挥赋予皮肤颜色、保护组织细胞免受紫外线损伤的功能。黑素细胞是否参与感染免疫应答目前国内外无相关文献报道。最近我们发现黑素细胞能够明显提高巨噬细胞遇到白念珠菌菌体时分泌TNF-α等炎症因子的能力。众所周知巨噬细胞被活化后能吞噬杀伤病原体，分泌炎症因子，加工递呈抗原，招募活化其他免疫细胞，在皮肤天然免疫反应中发挥重要作用。我们的研究结果提示在感染或外伤早期黑素细胞可能通过巨噬细胞间接发挥抗感染作用。本课题以此发现为切入点，通过体外实验进一步研究黑素细胞是否能够增强巨噬细胞对白念珠菌的趋化识别、吞噬杀伤能力，对巨噬细胞的活化以及表面共刺激分子表达的影响等内容。通过本研究将初步揭示黑素细胞潜在的免疫学功能，尤其是在天然免疫应答中的作用。

黑素细胞是一种主要分布于人体表皮的能够合成黑色素的细胞。黑素细胞在皮肤内通过合成并输出黑色素至周围的角质形成细胞，赋予皮肤以不同颜色，同时发挥保护细胞和组织免受紫外线损伤的作用，但黑素细胞的免疫学功能长期被忽视。本研究通过细胞体外培养、RT-PCR、 MTT实验，免疫荧光、流式细胞术等分子生物学及免疫学实验方法，从多个不同的角度探讨黑素细胞对巨噬细胞抗白念珠菌免疫应答的影响以及其可能的分子机制。. 在体外成功将小鼠骨髓细胞诱导为巨噬细胞的基础上，我们研究发现巨噬细胞对黑素细胞条件培养基并无明显趋化作用，对白念珠菌抗原有中度趋化作用，而在黑素细胞条件培养基存在的情况下，趋化至白念珠菌抗原的巨噬细胞数量明显增加（p＜0.05），提示黑素细胞能够明显增强巨噬细胞向白念珠菌孢子趋化的能力。黑素细胞条件培养基预处理12小时后的巨噬细胞吞噬真菌能力较对照组明显提高，具有统计学意义。进一步研究结果表明黑素细胞条件培养基能够促进巨噬细胞杀伤白念珠菌，并且这种杀伤增强作用随着黑素细胞条件培养基浓度增加而增加。黑素细胞条件培养基能够上调巨噬细胞Dectin-1 mRNA表达水平，可能是黑素细胞能够促进巨噬细胞吞噬真菌抗原的分子机制之一。黑素细胞能够通过其分泌的可溶性蛋白成分增强热灭活念珠菌刺激的巨噬细胞表达TNF-a，其作用机制可能是通过上调巨噬细胞表面IL-23R，增强巨噬细胞对IL-23的反应性，从而提高TNF-a表达水平。 进一步用蛋白印迹的方法证实，黑素细胞条件培养基同时能够不同程度提高活化的巨噬细胞表达IL-2Rβ, IL-6, IL-10, IL-12, CD27 Ligand /TNFSF7, Fas/TNFRSF6等与抗真菌免疫相关分子蛋白表达水平。上述结果表明黑素细胞能够增强巨噬细胞对白念珠菌的免疫应答，提示黑素细胞具有免疫学潜能，在巨噬细胞介导的抗真菌免疫中发挥重要作用，对进一步了解黑素细胞及皮肤免疫系统具有一定理论和实际意义。