Analysis for a role of dectin-2 in ultraviolet-light-induced immune tolerance

众所周知，紫外线B光（UV）损害接触超敏反应（CHS）的诱导阶段，随后诱导耐受性。尽管这一现象几十年来一直被认为是一个重要的生物学意义，但这一事件背后的免疫学机制尚不完全清楚，Dectin-2是c型凝集素的新成员，它在表皮朗格汉斯细胞（LC）上选择性表达。在这里，我们研究了dectin-2在uvb诱导的CHS免疫耐受中的作用。为此，我们首先生成了可溶性的decin -2（sDec2）。利用重组技术生成sDec2。简单地说，将dectin-2 cDNA的胞外结构域亚克隆到表达载体上，然后将其转染到大肠杆菌中表达。从大肠杆菌裂解物中回收可溶性重组蛋白，对其进行纯化，并通过体外实验确定其对同源蛋白dectin-2的抑制活性。为了解决最初的问题，sDec2被注射到小鼠体内，然后再诱导或激发CHS。然而，无论注射与否，CHS都没有受到影响，这表明dectin-2介导的途径没有参与这一事件。相反，如果将dectin-2注射到已经连续4天暴露在1000 j /m^2紫外线下的小鼠身上。然后对这些小鼠进行半抗原致敏和激射，导致CHS的完全结果，而当只致敏和激射而不注射sdec2时，紫外线显著抑制了CHS。这表明dectin-2参与了紫外线介导的CHS损伤。为了检验其是否参与了紫外线诱导的耐受性，我们对受到紫外线损伤的CHS小鼠进行了14天的不处理，然后进行了再致敏和再刺激，结果与未暴露的小鼠相比，CHS明显受到抑制，表明诱导了耐受性。然而，在第一次致敏前注射sdec2恢复了CHS，并没有导致耐受性。基于耐受性是通过T抑制细胞（T suppressor cells, Ts）的产生介导的这一事实，上述结果表明，decin -2介导半抗原特异性T的诱导。这进一步证实了与sdec2结合的T细胞，但未结合的T细胞在转移给初始受体时不会损害对CHS的诱导。为了表征sdec2结合T细胞的表型特征，我们进行了FACS分析，发现紫外线诱导的T细胞在其表面表达CD4和CD25，类似于目前突出的调节性T细胞（Ir）。Tr是重新命名的t，它具有预防自身免疫性疾病发作的能力，包括小鼠模型的1型糖尿病或多发性硬化症。总之，我们的研究揭示了紫外线诱导的耐受性是通过Tr， Less的产生介导的

It is known that ultraviolet B light(UV) impairs the induction phase of contact hypersensitivity responses(CHS) and subsequently induces tolerance. Although this phenomenon has been appreciated for decades to be a major biological importance, immunological mechanisms underlying this event are not completely clear yet Dectin-2 is a new member of c-type lectin, which is selectively expressed on epidermal langerhans cells(LC). Here we examined roles of dectin-2 in UVB-induced immune tolerance of CHS. To do so, we first generated a soluble form of dectin-2(sDec2). sDec2 was generated by use of recombinant technology. Briefly, an extracellular domain of dectin-2 cDNA was subcloned to an expression vector, which was then transfected to and expressed in E.cli. Soluble recombinant protein was recovered from lysates of coli, purified and its inhibitory activity to cognate dectin-2 was ascertained in in vitro assays. To address the initial questions, sDec2 was injected to mice before either the … More induction or the elicitation of CHS. However, CHS was not affected regardless of the injection, indicating that dectin-2-mediated pathways are not involved in this event By contrast, if dectin-2 was injected to mice which was already UV exposed at 1,000J/m^2 for 4 consecutive days. Those mice were then hapten sensitized and challenged, leading to full outcome of CHS, while UV significantly suppressed CHS when those were only sensitized and challenged but not sDec2-injected. This indicates that dectin-2 is involved in the UV-mediated impairment of CHS. To check for its involvement in UV-induced tolerance, mice whose CHS were impaired by UV were left untreated for 14 days and then resensitized and rechallenged, resulting that CHS was significantly suppressed as compared to unexposed animals, indicating that tolerance was induced. However, sDec2-injection before the first sensitization restored CHS and did not lead to tolerance. Based on the fact that tolerance is mediated via generation of T suppressor cells(Ts), the above mentioned results indicate that dectin-2 mediates the induction of hapten specific Ts. This was further confirmed as sDec2-bound, but not unbound T cells impaired the induction of CHS upon transfer to naive recipients. To phenotypically characterize sDec2-bound Ts, FACS analysis was conducted, demonstrating that UV-induced Ts express CD4 and CD25 on their surface resembling currently hightlighted regulatory T cells(Ir). Tr are the re-nomenclatured Ts, which possess the ability to prevent the onset of autoimmune disorders, including type I diabetes mellitus or multiple sclerosis of murine models. Together, our study disclosed that UV-induced tolerance is mediated via generation of Tr, Less