Illuminating IL-31-producing cells in allergic skin disease

项目总结/文摘

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.