Illuminating IL-31-producing cells in allergic skin disease

项目总结/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乳腺癌中显示出有希望的结果。 针对多种炎症性皮肤病的临床试验。我新实验室的皮肤免疫学研究项目是 集中于表征IL-31产生细胞和IL-31应答细胞之间的通路， 慢性组织炎症在本项目中，我们将使用我们开发的R 03资助机制和工具 在我的NIAMS K 08奖学金资助的研究中，我追求关于细胞的基本开放问题， 产生IL-31的皮肤细胞群的身份。本文概述的实验将阐明特定的IL-31来源 群体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的基因型进行无偏的高分辨率表型分析。 体内皮肤IL-31产生细胞。为此，我们将利用一种新的IL 31报告基因转基因技术， 我们通过敲入/敲除基因靶向策略开发了小鼠品系。IL 31报告基因转基因 能够可视化单个活的IL-31产生细胞。然而，由于表达IL-31的细胞稀少， 在不存在组织炎症的情况下，将IL 31报告基因转基因动物用 在我们的实验中，我们使用了特征明确的过敏原屋尘螨来引发皮肤炎症。读数将 包括高参数流式细胞术加scRNA测序。预期CD 4 T细胞表达 报告基因转基因，但造血和非造血谱系的额外细胞也已被 作为IL-31的来源。如果存在于皮肤中，则这些细胞和其他先前存在于皮肤中的细胞的IL 31报告基因表达可被抑制。 未检测到的IL-31来源如先天淋巴样细胞将通过我们的无偏定位努力而被捕获。 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， 人口数量。为此，我们将利用嵌入IL 31报告基因转基因中的Cre重组酶， 选择性地改变表达IL 31的细胞。具体地，小鼠对于IL 31报告基因和Cre-10基因双转基因。 诱导型“命运映射者”转基因Ai 14将转基因标记所有曾经表达IL 31的细胞。小鼠加倍 IL 31报告基因和Cre诱导的“删除”转基因Rosa 26-DTA的转基因将细胞自主地 删除所有曾经表达IL 31的细胞。通过交叉scRNA测序数据集，从IL 31命运- mapper“和”IL 31 deleter“小鼠皮肤，我们将能够区分IL 31表达细胞和其他细胞， 代表直接或间接应答者的辍学人群。计划的数据集生物信息学分析 可以鉴定由表达IL 31的细胞施加于细胞的基因表达程序。 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。 实验室和确定角度为更有选择性的治疗靶向IL-31介导的途径在未来。

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.