Aire-dependent regulation of spontaneous autoimmune-mediated alopecia

摘要 这项建议将使申请者能够获得有指导的研究培训，并发展成为一个富有成效的、 独立内科医生-皮肤科领域的科学家。 斑秃(AA)是一种自身免疫性皮肤病，以斑片状无疤痕脱发为特征。 解药。再生障碍性贫血是由自身反应性T细胞的不适当激活引起的，这种T细胞以角质形成细胞为靶点并损伤角质形成细胞 生长期毛囊(HFs)由于HF免疫豁免权(IP)的崩溃。为什么生长期HFs经历IP崩溃 还没有被很好地理解。最近，Janus激酶抑制剂(Jaki)通过以下方式显示出对再障患者的临床疗效 然而，Jaki抑制HF的免疫细胞活性，并不是对所有的AA患者都有效。更好的机械师 了解JAK-STAT调控可以导致更有效的AA治疗。我们的初步数据显示 我们有一种新的类似AA的小鼠模型，在遗传缺失的小鼠中表现出自发性脱发 自身免疫调节剂(Aire-/-)。AIRE是一种在髓质胸腺上皮细胞中表达的转录调节因子 这就消除了自体反应性T细胞。我们和其他人最近发现，Aire也在表皮中表达 毛囊角质形成细胞。越来越多的临床证据也支持Aire在再障中的重要性，作为患者 随着功能丧失，AIRE的突变会增加患再生障碍性贫血的风险。我们观察成年女性 C57BL/6 Aire-/-(生殖系)小鼠自发发生AA样病变(n=35/73)。脱发患者的皮肤活检 AIRE-/-小鼠在宏观、组织病理学和分子水平上类似于人类AA损伤。此外，我们 观察到JAK-STAT信号在Aire/-皮肤损伤和AIRE缺陷培养的角质形成细胞中上调 同时下调STAT1抑制剂PIAS1的表达。这些发现表明：1)艾尔是一种 2)AIRE是角质形成细胞JAK-STAT信号的关键抑制因子。 这项建议将解决目前我们对AA中的HF IP和JAK-STAT信号理解的空白。在目标1中， 我们假设胸腺或角质形成细胞Aire的丢失是导致HF IP崩溃的原因之一。为了测试这一点，我们 将从脱发Aire-/-小鼠体内采集CD8+NKG2D+T细胞，皮下注射给Aire+/+小鼠，并 监测他们是否出现再生障碍性贫血。溶细胞T细胞活性将通过角质形成细胞共培养实验进行评估。这个 这些研究的结果将揭示胸腺Aire丢失是否足以引发HF IP崩溃。同时，我们 将测试皮肤特异性的Aire缺失是否足以通过使用他莫昔芬治疗来触发HF IP崩溃 Airefl/flK5-CreERT2小鼠。在目标2中，我们假设AIRE参与了干扰素γ-JAK-STAT的调节 培养角质形成细胞中的信号转导。为了测试这一点，我们将确定AIRE如何影响PIAS1、STAT1和 PIAS1-STAT1复合体，评估JAK-STAT信号需要哪些AIRE域，并确定 人角质形成细胞在干扰素γ刺激下表达AA细胞因子和趋化因子。这些研究的结果 研究将揭示再生障碍性贫血中JAK-STAT信号的新调控机制。

ABSTRACT This proposal will enable the applicant to acquire mentored research training and develop into a productive, independent physician-scientist in the field of dermatology. Alopecia areata (AA) is an autoimmune skin disease, characterized by patchy non-scarring hair loss, with no cure. AA is caused by inappropriate activation of autoreactive T cells that target and damage keratinocytes of anagen hair follicles (HFs) due to the collapse of HF immune privilege (IP). Why anagen HFs undergo IP collapse is not well-understood. Recently, Janus kinase inhibitors (JAKi) have shown clinical efficacy in AA patients by suppressing immune-cell activity at the HF, however, JAKi do not work for all AA patients. A better mechanistic understanding of JAK-STAT regulation can lead to more effective AA treatments. Our preliminary data indicates that we have a novel AA-like mouse model that exhibits spontaneous alopecia in mice genetically null for autoimmune regulator (Aire-/-). Aire is a transcriptional regulator expressed in medullary thymic epithelial cells that eliminates autoreactive T cells. We and others have recently shown that Aire is also expressed in epidermal and follicular keratinocytes. Increasing clinical evidence also supports the importance of Aire in AA, as patients with loss-of-function mutations in AIRE have an increased risk of developing AA. We observe adult female C57BL/6 Aire-/- (germline) mice spontaneously develop AA-like lesions (n=35/73). Skin biopsies from alopecic Aire-/- mice resemble human AA lesions on macroscopic, histopathologic, and molecular levels. Additionally, we observed upregulation in JAK-STAT signaling in Aire-/- skin lesions and AIRE-deficient cultured keratinocytes alongside downregulated expression of PIAS1, a STAT1 inhibitor. These findings suggest that 1) Aire is a critical regulator of HF IP in AA and 2) AIRE is a key suppressor of JAK-STAT signaling in keratinocytes. This proposal will address current gaps in our understanding of HF IP and JAK-STAT signaling in AA. In Aim 1, we hypothesize that the loss of either thymic or keratinocyte Aire contribute to HF IP collapse. To test this, we will harvest CD8+NKG2D+ T cells from alopecic Aire-/- mice, subcutaneously inject them into Aire+/+ mice and monitor them for AA onset. Cytolytic T cell activity will be assessed via keratinocyte co-culture experiments. The results of these studies will reveal whether thymic Aire loss is sufficient to trigger HF IP collapse. In parallel, we will test whether skin-specific deletion of Aire is sufficient to trigger HF IP collapse by utilizing tamoxifen-treated Airefl/flK5-CreERT2 mice. In Aim 2, we hypothesize that AIRE contributes to the regulation of IFNγ-JAK-STAT signaling in cultured keratinocytes. To test this, we will determine how AIRE influences the PIAS1, STAT1, and the PIAS1-STAT1 complex, assess which AIRE domains are required for JAK-STAT signaling, and identify the AA cytokines and chemokines expressed by AIRE-/- keratinocytes upon IFNγ stimulation. The results of these studies will uncover novel regulatory mechanisms of JAK-STAT signaling in AA.