Illuminating IL-31-producing cells in allergic skin disease

项目概要/摘要 靶向细胞因子白细胞介素 31 (IL-31) 受体的生物制剂在晚期疾病中显示出有希望的结果 多种炎症性皮肤病的临床试验。我的新实验室的皮肤免疫学研究项目是 专注于连接 IL-31 产生细胞和 IL-31 反应细胞的途径的表征 慢性组织炎症。在这个项目中，我们将使用我们开发的R03资助机制和工具 在 NIAMS K08 奖学金资助的研究期间，我致力于探索有关细胞的基本开放问题 产生 IL-31 的皮肤细胞群的身份。这里概述的实验将阐明特定的 IL-31 来源 人群及其调节的皮肤细胞和分子网络。 该提案的第一个目标是对所有基因进行公正的高分辨率表型分析 体内产生皮肤IL-31的细胞。为此，我们将利用新型 IL31 转基因报告基因 我们通过敲入/敲除基因靶向策略开发了小鼠品系。 IL31报告基因转基因 能够对单个活 IL-31 产生细胞进行可视化。然而，由于表达 IL-31 的细胞稀疏 在没有组织炎症的情况下，IL31报告基因转基因动物将受到局部挑战 在我们的实验中，明确表征的过敏原屋尘螨会引发皮肤炎症。读数将 包括高参数流式细胞术和 scRNA 测序。 CD4 T 细胞预计表达 报告基因转基因，但造血和非造血谱系的其他细胞也已被 建议作为 IL-31 来源。如果皮肤中存在 IL31 报告基因，这些细胞和其他先前的细胞会表达 IL31 报告基因。 我们公正的绘图工作将捕获未检测到的 IL-31 来源，例如先天淋巴细胞。 我们的第二个目标是解决 IL-31 产生细胞对其他皮肤细胞的下游影响 人口。为此，我们将利用嵌入 IL31 报告转基因的 Cre 重组酶来 选择性改变表达 IL31 的细胞。具体来说，小鼠对 IL31 报告基因和 Cre- 可诱导的“命运映射器”转基因 Ai14 将以转基因方式标记所有曾经表达 IL31 的细胞。小鼠双 IL31报告基因转基因和Cre诱导“删除”转基因Rosa26-DTA将细胞自主 删除所有曾经表达 IL31 的细胞。通过交叉 scRNA 测序数据集，从“IL31 命运”中解析出 映射器”和“IL31删除器”小鼠皮肤，我们将能够将表达IL31的细胞与其他细胞区分开来。 代表直接或间接反应者的辍学人群。计划的数据集生物信息分析 来自应答细胞群的细胞可以识别由表达 IL31 的细胞施加的基因表达程序 体内应答细胞网络。 如果成功，本提案中概述的实验将为我的新研究产生重要的新数据。 实验室并确定未来对 IL-31 介导的途径进行更具选择性的治疗靶向的角度。

PROJECT SUMMARY/ABSTRACT Biologics targeting the receptor for cytokine interleukin-31 (IL-31) show promising results in advanced clinical trials for diverse inflammatory skin diseases. My new laboratory's skin immunology research program is focused on characterization of pathways that connect IL-31 producing cells and IL-31 responsive cells in chronic tissue inflammation. In this project, we will use the R03 funding mechanism and tools we developed during my NIAMS K08 fellowship-funded research to pursue fundamental open questions about the cellular identity of IL-31-producing skin cell populations. Experiments outlined here will illuminate specific IL-31 source populations and the cutaneous cellular and molecular networks they modulate. The first objective of this proposal is to perform unbiased high-resolution phenotypic analysis of all cutaneous IL-31-producing cells in vivo. To do so, we will take advantage of a novel IL31 reporter transgenic mouse strain we developed by a knock-in/knock-out gene targeting strategy. The IL31 reporter transgene enables visualization of individual live IL-31-producing cells. However, since cells that express IL-31 are sparse in the absence of tissue inflammation, IL31 reporter transgenic animals will be challenged topically with the well-characterized allergen house dust mite to provoke skin inflammation in our experiments. Readouts will include high-parameter flow cytometry plus scRNA-sequencing. CD4 T cells are expected to express the reporter transgene, but additional cells of both hematopoietic and non-hematopoietic lineages have also been proposed as IL-31 sources. If present in skin, IL31 reporter expression by these cells and other previously- undetected IL-31 sources such as innate lymphoid cells will be captured by our unbiased mapping efforts. Our second objective is to resolve downstream effects of IL-31-producing cells on other skin cell populations. To do so, we will take advantage of Cre recombinase embedded in the IL31 reporter transgene to selectively alter cells that express IL31. Specifically, mice double transgenic for the IL31 reporter and the Cre- inducible 'fate-mapper' transgene Ai14 will transgenically flag all cells that ever expressed IL31. Mice double transgenic for the IL31 reporter and Cre-inducible 'deleter' transgene Rosa26-DTA will cell-autonomously delete all cells that ever expressed IL31. By intersecting scRNA-sequencing datasets resolved from 'IL31 fate- mapper' and 'IL31 deleter' mouse skin, we will be able to distinguish IL31-expressing cells from additional dropout populations that represent direct or indirect responders. Planned bioinformatic analyses of datasets from responder cell populations can identify gene expression programs imposed by IL31-expressing cells on responder cell networks in vivo. When successful, experiments outlined in this proposal will generate significant new data for my new laboratory and identify angles for more selective therapeutic targeting of IL-31-mediated pathways in the future.