Linking Nicotinic Activation with Skin Innate Immunity and Atopic Dermatitis

描述（由申请人提供）：我们对抗菌肽（AMP）的负调节如何导致皮肤病相关临床症状的理解存在重大突破。在这一领域的研究知识的持续缺乏意味着一个重要的挑战，以开发改进的治疗皮肤感染和疾病。长期目标是确定乙酰胆碱烟碱受体（nAChR）激活如何抑制AMP表达和活性，并将AMP调节中的这些变化与AMP失调相关的人类皮肤病的因果治疗联系起来。本研究的目的是确定参与角质形成细胞AMP抑制的nAChR亚型，评估nAChR激活改变的AMP谱，并将此机制与特应性皮炎（AD）的发病机制联系起来。我们的假设是角质形成细胞中过量的nAChR活化损害了皮肤AMP的正常加工和功能，这有助于AD的发病机制。我们的假设是根据初步数据制定的，建立nAChR激活显着降低AMP活性和皮肤感染的抵抗力。AD患者的皮肤屏障受损，其中包括AMP表达减少，这被认为是引发炎症和感染的原因。应激可能通过增加表皮ACh和nAChR活化而加重AD的临床表现，从而损害正常的AMP调节和微生物易感性。我们进行这些研究的理由是，大多数研究都集中在AMP的正调控，以增加其表达和活性，但确定那些抑制或减少AMP表达的分子。 AMP活性对于开发更好的临床疗法以改善与AD和其他炎性皮肤病相关的症状至关重要。在令人信服的初步数据的驱动下，我们的假设将通过解决三个具体目标进行评估：1）确定哪些nAChR亚型参与AMP抑制，并确定体外原代正常人表皮角质形成细胞（NHEK）中的AMP谱; 2）确定nAChR激活在AMP对体内感染的反应中的作用; 3）将nAChR信号传导机制与AD的发病机制联系起来。对于目标1，将用nAChR激动剂和拮抗剂刺激NHEK，以评估已知的AMP并使用已建立的生物化学技术鉴定新的抗微生物分子。目的2将使用nAChR激活状态改变的小鼠，使用已建立的蛋白质组学方法分析AMP对感染的反应。目的3将证明，nAChR激活参与抑制AMP反应感染的皮肤AD患者使用分子方法。我们的方法是创新的，因为它利用新的蛋白质组学和分子技术来进一步确定nAChR激活如何影响表皮AMP对感染的反应。拟议的研究是重要的，因为它们预计将确定负调节皮肤中AMP活性的主要nAChR信号传导机制，并确定疾病进展的替代途径。

DESCRIPTION (provided by applicant): An important breach exists in our understanding of how the negative regulation of antimicrobial peptides (AMPs) leads to the clinical symptoms associated with skin disease. The persistent lack of knowledge in this area of research signifies an important challenge to develop improved therapies for cutaneous infection and disease. The long-term goal is to identify how acetylcholine nicotinic receptor (nAChR) activation suppresses AMP expression and activity, and relate these changes in AMP regulation to the causal treatment of human skin diseases associated with AMP dysregulation. The objective of the proposed research is to identify the nAChR subtype(s) involved in AMP suppression in keratinocytes, evaluate the AMP profile altered by nAChR activation, and link this mechanism to the pathogenesis of Atopic Dermatitis (AD). Our hypothesis is that excess nAChR activation in keratinocytes impairs the normal processing and function of cutaneous AMPs, which contributes to the pathogenesis of AD. Our hypothesis was formulated based on preliminary data establishing that nAChR activation significantly reduced AMP activity and the resistance to cutaneous infection. Patients with AD develop a compromised skin barrier, which includes decreased AMP expression thought to precipitate inflammation and infection. Stress likely exacerbates the clinical manifestations of AD by increasing epidermal ACh and, consequently, nAChR activation to compromise normal AMP regulation and microbial susceptibility. Our rationale for these studies is that most research has focused on the positive regulation of AMPs to increase their expression and activity, yet identifying those molecules that inhibit or diminish AMP activity is critical to develop better clinical remedies to ameliorate the symptoms associated with AD and other inflammatory skin diseases. Driven by compelling preliminary data, our hypothesis will be evaluated by addressing three Specific Aims: 1) Identify which nAChR subtypes are involved in AMP suppression and identify the AMP profile in primary normal human epidermal keratinocytes (NHEKs) in vitro; 2) Determine the role of nAChR activation in the AMP response to infection in vivo; 3) Link mechanisms of nAChR signaling to the pathogenesis of AD. For Aim 1, NHEKs will be stimulated with nAChR agonists and antagonists to assess known AMPs and identify new antimicrobial molecules using established biochemical techniques. Aim 2 will use mice with altered states of nAChR activation to analyze the AMP response to infection using established proteomic approaches. Aim 3 will demonstrate that nAChR activation participates in the suppression of the AMP response to infection in skin from AD patients using molecular approaches. Our approach is innovative because it utilizes novel proteomic and molecular techniques to further define how nAChR activation influences the epidermal AMP response to infection. The proposed studies are significant because they are anticipated to identify the major nAChR signaling mechanism that negatively regulates AMP activity in skin, and identify alternative pathways for disease progression.