Linking Nicotinic Activation with Skin Innate Immunity and Atopic Dermatitis

描述(由申请人提供)：我们对抗菌肽(AMPs)的负面调节如何导致与皮肤病相关的临床症状的理解存在一个重要的漏洞。在这一研究领域长期缺乏知识表明，开发改进的皮肤感染和疾病治疗方法是一个重要的挑战。长期目标是确定乙酰胆碱烟碱受体(NAChR)激活如何抑制AMP的表达和活性，并将这些AMP调节的变化与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角质形成细胞中参与AMP抑制的nAChR亚型(S)，评价nAChR激活改变的AMP谱，并将这一机制与特应性皮炎(AD)的发病机制联系起来。我们的假设是角质形成细胞中nAChR的过度激活损害了皮肤AMPs的正常加工和功能，这可能是AD的发病机制之一。我们的假设是基于初步数据提出的，即nAChR激活显著降低AMP活性和对皮肤感染的抵抗力。AD患者会出现一种受损的皮肤屏障，其中包括AMP表达减少，被认为会导致炎症和感染。应激可能通过增加表皮ACh，从而激活nAChR来损害正常的AMP调节和微生物敏感性，从而加重AD的临床表现。我们进行这些研究的理由是，大多数研究都集中在AMP的正向调节以增加它们的表达和活性，但识别出那些抑制或减弱它们的分子 AMP活性对于开发更好的临床药物以改善与AD和其他炎症性皮肤病相关的症状至关重要。在令人信服的初步数据的推动下，我们的假设将通过以下三个具体目标进行评估：1)确定哪些nAChR亚型参与AMP抑制，并在体外确定原代正常人类表皮角质形成细胞(NHEK)中的AMP谱；2)确定nAChR激活在体内AMP感染反应中的作用；3)nAChR信号与AD发病机制的联系。对于目标1，NHEK将被nAChR激动剂和拮抗剂刺激，以评估已知的AMP并使用现有的生化技术识别新的抗微生物分子。目的2将利用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用分子方法证明nAChR的激活参与抑制AD患者皮肤AMP的感染反应。我们的方法是创新的，因为它利用新的蛋白质组和分子技术来进一步定义nAChR激活如何影响表皮AMP对感染的反应。这项拟议的研究意义重大，因为他们有望确定负向调节皮肤AMP活性的主要nAChR信号机制，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.