ABSTRACT
Obesity is associated with comorbidities including type 2 diabetes, chronic nonhealing wounds and psoriasis. Normally skin homeostasis and repair is regulated through the production of cytokines and growth factors derived from skin-resident cells including epidermal γδ T cells. However epidermal γδ T cells exhibit reduced proliferation and defective growth factor and cytokine production during obesity and type 2 diabetes. One of the genes modulated in epidermal γδ T cells during obesity and type 2 diabetes is CCR6, which is the receptor for CCL20. CCL20 is elevated in the skin during obesity and type 2 diabetes. Here we identify a subset of murine epidermal γδ T cells that expresses CCR6 in response to activation in vitro and post-wounding or psoriasis induction with imiquimod in vivo. We show that CCL20 stimulates epidermal γδ T cells to produce IL-17 suggesting CCR6 regulates the IL-17 axis as in dermal γδ T cells. Further, epidermal γδ T cells upregulate CCR6 and produce IL-17 during murine models of wound repair and psoriasis. Obesity increases CCR6 and IL-17 expression by epidermal γδ T cells during wound repair but has less of an effect during psoriasis. These findings have novel implications for the regulation of a specific population of IL-17-producing epidermal γδ T cells during skin damage and inflammation.
ABSTRACT
Skin-resident and infiltrating γδ T lymphocytes are components of the cutaneous immune system that provide the first line of defense against pathogens and the environment. Research that employs the isolation and culture of T cells from murine and human skin can help delineate the molecular and cellular mechanisms utilized by T lymphocytes in skin-specific immunity. However, obtaining high numbers of T cells from epithelial tissue without resorting to long-term culture or transformation can be difficult. Here, specific approaches are described for the isolation and culture of γδ T lymphocytes from murine skin and human skin explant cultures. In addition, a protocol to assess the morphology and activation of epidermal γδ T cells in situ using immunofluorescent microscopy is detailed. These techniques can be used to analyze resident and infiltrating γδ T lymphocytes in the skin via flow cytometry, RNA-seq, or proteomics to further study inflammatory diseases, cancer, or autoimmunity. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Isolation, culture, and analysis of γδ T cells from murine epidermis Basic Protocol 2: Examination of γδ T cells in epidermal sheets to assess activation and morphology Basic Protocol 3: Preparation of human skin explant cultures for analysis of skin T cells Support Protocol 1: Counting live cells with hemocytometer Support Protocol 2: Preparing a Matrigel.
