Kallikrein-PAR interactions in skin inflammation

激肽释放酶-PAR 在皮肤炎症中的相互作用

基本信息

批准号：
10449979
负责人：
Nicole Leanne Ward
金额：
$ 68.42万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10449979
关键词：
Ablation Acanthosis Address Adverse reactions Affect Atopic Dermatitis Attenuated Autoimmunity Backcrossings Biological Bone Marrow CRISPR/Cas technology Cell Separation Cells Coculture Techniques Coupled Cre lox recombination system Cutaneous Data Set Disease remission Experimental Genetics F2R gene FDA approved Genetic Genetically Engineered Mouse Hematopoietic Histologic Human Hyperactivity IL17 gene In Vitro Inflammation Inflammatory Kininogenase Knock-out Knockout Mice Measures Mediating Messenger RNA Modeling Molecular Molecular Genetics Mus Organ Culture Techniques Organ Transplantation PAR-1 Receptor Pathogenesis Pathogenicity Pathway interactions Patients Peptide Hydrolases Phenotype Population Prevalence Proteinase-Activated Receptors Proteins Psoriasiform Dermatitis Psoriasis Publishing Research Resistance Rho-associated kinase Role Serine Protease Severities Severity of illness Signal Pathway Signal Transduction Skin Small Interfering RNA T cell infiltration T-Lymphocyte Testing Therapeutic Time Transcript Translating Work Xenograft Model Xenograft procedure chronic inflammatory disease cytokine immune cell infiltrate improved in vivo inflammatory milieu inhibitor innovation interleukin-23 mouse model new therapeutic target novel overexpression prevent receptor reduce symptoms small molecule therapeutic development therapeutically effective transcriptome sequencing whole genome

项目摘要

Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease hypothesized to promote inflammation and autoimmunity via cleavage of protease-activated receptors (PAR)1 and PAR2. KLK6 is expressed in skin, however its biological activities in vivo remain largely unknown. Recent work published by our lab identified increases in KLK6 mRNA and protein in psoriasis patient lesional skin and primary KCs that decrease rapidly with treatment and correspond with disease severity. Despite KLK6 being a highly regulated cutaneous transcript/protein, the pathogenic significance of KLK6 in psoriasis remains unknown. To address this question, we genetically engineered mice to overexpress KLK6 in KCs (modeling lesional psoriasis skin). KLK6+ mice spontaneously develop a psoriasiform skin phenotype at the histological, cellular and molecular levels and RNAseq analyses of KLK6+ mouse skin revealed high correspondence with human psoriasis and identified significant increases in T cell derived-cytokines Il22 and Il17a/f. To identify the mechanisms mediating KLK6-elicited inflammation, KLK6+ mice were backcrossed with either PAR1- or PAR2-deficient (KO) mice. KLK6+PAR2KO mice develop similar levels of skin inflammation as KLK6+ mice. In contrast, KLK6+PAR1KO mice have attenuated skin inflammation demonstrating a critical pathogenic role for PAR1, and not PAR2 in KLK6-induced skin inflammation. PAR1 is found on KCs and T cells and signals through Rac1 and Rho associated kinase (ROCK)2. Using this innovative new mouse model, combined with genetic knockout approaches, cre-lox technologies and small molecule inhibition targeting strategies coupled with in vitro co-culture, CRISPR-Cas9 and cell signaling approaches, we will test the hypothesis that KLK6 cleaves PAR1 on KCs and T cells and activates Rac1 and ROCK2, initiating a self-sustaining proinflammatory loop between KCs and T cells that results in a psoriasis-like proinflammatory environment. Ultimately, we will show using human psoriasis skin organ cultures and xenograft models that interfering with new targets in this pathway will improve human psoriasis. The work proposed herein will identify the cellular mechanism(s) underlying KLK6-PAR1-mediated inflammation. Successful completion of our aims will identify KLK6 as a critical protease for psoriasis pathogenesis and KLK6-PAR1 signaling as a new target for therapy. This pathway is profoundly different than currently targeted cytokine pathways being investigated for the treatment of chronic inflammatory disease and offers a new direction in psoriasis research and autoimmunity research as a whole.

激肽释放酶相关肽酶 6 (KLK6) 是一种分泌性丝氨酸蛋白酶，推测可促进炎症通过蛋白酶激活受体 (PAR)1 和 PAR2 的裂解产生自身免疫。 KLK6 在皮肤中表达，然而其在体内的生物活性仍然很大程度上未知。我们实验室最近发表的工作确定银屑病患者皮损皮肤和原发性 KC 中 KLK6 mRNA 和蛋白增加，但迅速减少治疗并与疾病严重程度相对应。尽管 KLK6 是一种高度调控的皮肤转录物/蛋白质，KLK6 在银屑病中的致病意义仍不清楚。为了解决这个问题，我们对小鼠进行了基因改造，使其在 KC 中过度表达 KLK6（模拟病变牛皮癣皮肤）。 KLK6+小鼠在组织学、细胞学方面自发地形成牛皮癣样皮肤表型 KLK6+小鼠皮肤的分子水平和RNAseq分析显示与人类高度一致银屑病并发现 T 细胞衍生的细胞因子 Il22 和 Il17a/f 显着增加。为了识别介导 KLK6 引发炎症的机制，KLK6+ 小鼠与 PAR1- 或 PAR1- 回交 PAR2 缺陷 (KO) 小鼠。 KLK6+PAR2KO 小鼠出现与 KLK6+ 小鼠相似水平的皮肤炎症。在相比之下，KLK6+PAR1KO 小鼠减轻了皮肤炎症，这表明其具有关键的致病作用。 KLK6 诱导的皮肤炎症中是 PAR1，而不是 PAR2。 PAR1 存在于 KC 和 T 细胞和信号上通过 Rac1 和 Rho 相关激酶 (ROCK)2。使用这种创新的新型鼠标模型，结合基因敲除方法、cre-lox 技术和小分子抑制靶向策略相结合通过体外共培养、CRISPR-Cas9 和细胞信号传导方法，我们将检验 KLK6 的假设裂解 KC 和 T 细胞上的 PAR1 并激活 Rac1 和 ROCK2，启动自我维持的促炎症反应 KC 和 T 细胞之间的循环导致银屑病样促炎环境。最终，我们将表明使用人类牛皮癣皮肤器官培养物和异种移植模型干扰了这一领域的新目标途径将改善人类牛皮癣。本文提出的工作将确定 KLK6-PAR1 介导的细胞机制炎。成功完成我们的目标将使 KLK6 成为银屑病的关键蛋白酶发病机制和 KLK6-PAR1 信号传导作为新的治疗靶点。这条道路与目前正在研究用于治疗慢性炎症疾病的靶向细胞因子途径为牛皮癣研究和自身免疫研究提供了一个新的整体方向。