Kallikrein-PAR interactions in skin inflammation

激肽释放酶-PAR 在皮肤炎症中的相互作用

• 批准号: 10615327
• 负责人: Nicole Leanne Ward
• 金额: $ 35.79万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 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/10615327
• 关键词: Kallikrein; PAR; interactions; skin; inflammation

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（KLK 6）是一种分泌型丝氨酸蛋白酶，被推测促进炎症 以及通过蛋白酶激活受体（PAR）1和PAR 2的裂解的自身免疫。KLK 6在皮肤中表达， 然而，其体内生物学活性仍然很大程度上未知。我们实验室最近发表的研究表明， 银屑病患者皮损皮肤和原发性KC中KLK 6 mRNA和蛋白的增加， 与治疗和疾病的严重程度相对应。尽管KLK 6是一种高度调节的皮肤 尽管KLK 6在银屑病中的转录/蛋白质的表达水平很低，但KLK 6在银屑病中的致病意义仍然未知。 为了解决这个问题，我们对小鼠进行了基因工程改造，使其在KC中过表达KLK 6（模拟病变）。 牛皮癣皮肤）。KLK 6+小鼠自发地在组织学、细胞学和免疫组织化学上形成银屑病样皮肤表型。 KLK 6+小鼠皮肤的分子水平和RNAseq分析显示与人类皮肤的高度对应性。 银屑病，并确定了T细胞衍生的细胞因子IL 22和IL 17 a/f的显著增加。识别 由于介导KLK 6引起的炎症的机制，将KLK 6+小鼠与PAR 1-或 PAR 2缺陷（KO）小鼠。KLK 6 + PAR 2KO小鼠与KLK 6+小鼠发生相似水平的皮肤炎症。在 相比之下，KLK 6 + PAR 1 KO小鼠的皮肤炎症减弱，证明了KLK 6 + PAR 1 KO的关键致病作用。 KLK 6诱导的皮肤炎症中是PAR 1，而不是PAR 2。PAR 1存在于KC和T细胞上， 通过Rac 1和Rho相关激酶（ROCK）2。使用这种创新的新小鼠模型，结合 基因敲除方法、cre-lox技术和小分子抑制靶向策略耦合 通过体外共培养，CRISPR-Cas9和细胞信号传导方法，我们将测试KLK 6 切割KC和T细胞上的PAR 1，并激活Rac 1和ROCK 2，启动自我维持的促炎反应。 KCs和T细胞之间的环，导致银屑病样促炎环境。最终，我们将 显示使用人类银屑病皮肤器官培养物和异种移植物模型， pathway途径将改善人类牛皮癣。 本文提出的工作将确定KLK 6-PAR 1介导的细胞内机制。 炎症成功完成我们的目标将确定KLK 6作为银屑病的关键蛋白酶 发病机制和KLK 6-PAR 1信号传导作为治疗的新靶点。这条道路与 目前正在研究用于治疗慢性炎性疾病的靶向细胞因子途径， 为银屑病研究和自身免疫研究提供了一个新的方向。