红色毛癣菌对角质形成细胞TLR2及信号通路的调控机制

Dermatophytosis is one of the most common human diseases resulting in substantial therapeutic expenses and the low quality of life. Among the pathogenic dermatophyte species, Trichophyton rubrum is the most commonly isolated from humans. T. rubrum primarily affects the stratum corneum of epidermis, it’s pathogen-associated molecular patterns (PAMPs) are recognized by pattern-recognition receptors (PRRs) in keratinocytes and subsequently drive a series of immune response. Among these PRRs, TLR2 constitutively expressed on keratinocytes and play a pivotal role in the course and prognosis of this disease..In the previous studies, we have confirmed that T. rubrum can modulate the expression and translocation of TLR2, and to the end, repress the immune response of keratocytes. On this basis, we will investigate the impact of T. rubrum on TLR2 and its signal transduction pathways and the way it takes place. The research mainly focused on NF-κB and MAPK signal pathways, which conduct the signal of TLR2 in keratocytes. Furthermore, we will compare the different effect of T.rubrum conidia and hyphae on TLR2 and its signal pathways and the difference of immune response of keratinocyte. By this way, we explore the influences of morphologic shift of T.rubrum on TLR2 and its signal pathways, as well as the chronic aspect of T. rubrum infection. .T. rubrum is a typical anthropophilic dermatophyte. It’s hard to infect the immunocompetent animal to construct an animal infection model. What's more, the immune response to T.rubrum in humans and animals is quite species-specific. Therefor, besides the monolayer keratinocytes model, for the first time a new dermatophytosis model, the reconstructed human epidermis infected by T.rubrum will be created for our study to overcome the disadvantages of traditional dermatophytosis models. Our work initiated the investigation how T.rubrum manipulate TLR2 and its signal pathways to evade the immunological surveillance of the host. It’s important for both the understanding of pathogenesis of dermatophytosis and the preventions and treatments of dermatophytosis based on the immunological mechanisms.

红色毛癣菌是人类皮肤癣菌病最主要致病菌，其感染多呈慢性且极易复发，严重影响患者的身心健康并导致巨额医疗支出，免疫逃避机制是当前研究热点。TLR2具有重要的抗真菌免疫作用，构成性表达于角质形成细胞，对浅部皮肤癣菌病的发病和转归有重要影响。前期研究发现红色毛癣菌能调控TLR2的表达和分布并最终抑制角质形成细胞的免疫应答。在此基础上申请开展红色毛癣菌调控TLR2及信号通路的具体机制研究，重点针对NF-κB、MAPK信号通路，明确调控的位点和方式。通过比较红色毛癣菌孢子和菌丝调控TLR2及信号通路的差异，探讨形态转化在红色毛癣菌逃避宿主免疫杀伤中的作用。红色毛癣菌是亲人性皮肤癣菌，动物模型并非本项目的最佳选择。因此我们采用人角质形成细胞构建人工表皮感染红色毛癣菌，创新性的以此为平台开展研究，克服了传统模型的局限。本项目对于揭示红色毛癣菌免疫逃避机制，针对性开发皮肤癣菌病防治药物和方法有重要意义。

红色毛癣菌是导致人类皮肤癣菌病的重要的致病真菌。TLR2以及其信号通路，在人体针对皮肤癣菌产生免疫应答的过程中有重要的作用。本项目中我们构建了红色毛癣菌感染人工表皮的研究模型，克服了传统皮肤癣菌病感染模型的局限，很好的在体外模拟皮肤癣菌感染人体表皮的各项表现。并利用此模型进行转录组测序分析，筛选出相关分子。然后比较亲人性和亲土性皮肤癣菌调控TLR2以及其信号通路的差异，发现JNK/JUN通路的活化有明显差异，可能是不同生态学分类皮肤癣菌感染导致临床表现差异的分子机制。以上研究结果已经发表。.考虑到抗体封闭不能完全阻断人工表皮的TLR2以及信号通路。我们采用CRISPR技术构建了角质形成细胞TLR2基因敲除以及再表达细胞株，验证TLR2在识别红色毛癣菌产生免疫应答中的作用。再结合人类对红色毛癣菌普遍易感而动物（小鼠）对红色毛癣菌天然抵抗的特点，推测TLR2胞外配体识别区的物种差异是导致人类和小鼠对红色毛癣菌易感性差异的重要原因。为此我们采用无缝克隆技术，将人、小鼠角质形成细胞TLR2的胞外配体识别区相互替换，研究表明：TLR2胞外配体识别区的替换增强了人体细胞对红色毛癣菌的识别和免疫应答，同时降低了小鼠细胞对红色毛癣菌的免疫识别和应答，通过体外研究验证了我们的假设。我们拟构建TLR2人源化小鼠，在此基础上通过外用载体重新表达鼠源TLR2，比较两种小鼠对红色毛癣菌感染的免疫应答。进一步通过体内研究验证我们的假说。目前已经成功的将rAAV-GFP转染小鼠表皮，表明外用rAAV-mTLR2在人源化小鼠局部皮肤稳定表达小鼠TLR2是可行的。.本项目明确TLR2以及JNK/JUN通路在皮肤癣菌病发病机制中的作用；明确TLR2的物种差异是导致红色毛癣菌易感性差异的重要原因，并据此探讨通过改造局部表皮细胞TLR2，增强人体识别红色毛癣菌的能力并诱导保护性免疫的可行性。为进一步开展相关人体研究提供临床前研究资料，对于防治人类皮肤癣菌病有重要意义。

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