Gyohei Egawa

Molecular characterization of melanocyte stem cells in their niche

Emerging evidence from stem cell (SC) research has strengthened the idea that SC fate is determined by a specialized environment, known as the SC niche. However, because of the difficulty of identifying individual stem cells and their surrounding components in situ, the exact mechanisms underlying SC regulation by the niche remain elusive. To overcome this difficulty, we employed melanocyte stem cells (MSCs), which allow the identification of individual SCs in the niche, the lower permanent portion of the hair follicle (HF). Here, we present molecular makers that can distinguish MSCs from other melanocyte (MC) subsets in the HF. We also describe a simple and robust method that allows gene expression profiling in individual SCs. After isolating individual MSCs from transgenic mice in which the MCs are marked by green fluorescence protein (GFP), we performed single-cell transcript analysis to obtain the molecular signature of individual MSCs in the niche. The data suggest the existence of a mechanism that induces the downregulation of various key molecules for MC proliferation or differentiation in MSCs located in the niche. By integrating these data, we propose that the niche is an environment that insulates SCs from various activating stimuli and maintains them in a quiescent state.

Activated regulatory T cells are the major T cell type emigrating from the skin during a cutaneous immune response in mice

Tregs play an important role in protecting the skin from autoimmune attack. However, the extent of Treg trafficking between the skin and draining lymph nodes (DLNs) is unknown. We set out to investigate this using mice engineered to express the photoconvertible fluorescence protein Kaede, which changes from green to red when exposed to violet light. By exposing the skin of Kaede-transgenic mice to violet light, we were able to label T cells in the periphery under physiological conditions with Kaede-red and demonstrated that both memory phenotype CD4+Foxp3- non-Tregs and CD4+Foxp3+ Tregs migrated from the skin to DLNs in the steady state. During cutaneous immune responses, Tregs constituted the major emigrants and inhibited immune responses more robustly than did LN-resident Tregs. We consistently observed that cutaneous immune responses were prolonged by depletion of endogenous Tregs in vivo. In addition, the circulating Tregs specifically included activated CD25hi Tregs that demonstrated a strong inhibitory function. Together, our results suggest that Tregs in circulation infiltrate the periphery, traffic to DLNs, and then recirculate back to the skin, contributing to the downregulation of cutaneous immune responses.

Update of Immune Events in the Murine Contact Hypersensitivity Model: Toward the Understanding of Allergic Contact Dermatitis

Allergic contact dermatitis (ACD) is one of the most common skin diseases, consisting of sensitization and elicitation phases. With the advancement of technology and the discovery of new types of immune cells, our knowledge of the immunological mechanisms of contact hypersensitivity (CHS) as a murine model of ACD has expanded significantly in the past decade. For example, by introducing regulatory T cells, CD4(+) T-helper 17 cells, and Langerin-positive dermal dendritic cells, the initiation and termination mechanism of CHS has been revealed. In addition, the role of mast cells in CHS, long a matter of debate, has become apparent by developing conditional mast cell-deficient mice. Moreover, the role of the innate immunity system, such as that of Toll-like receptor signaling, has made a breakthrough in this field. In this review, we will integrate the recent advancement of immunological mechanisms of both the sensitization and elicitation phases of CHS into the classic view, and we will discuss updated mechanisms on its development and future directions.

Requirement of Interaction between Mast Cells and Skin Dendritic Cells to Establish Contact Hypersensitivity

The role of mast cells (MCs) in contact hypersensitivity (CHS) remains controversial. This is due in part to the use of the MC-deficient Kit W/Wv mouse model, since Kit W/Wv mice congenitally lack other types of cells as a result of a point mutation in c-kit. A recent study indicated that the intronic enhancer (IE) for Il4 gene transcription is essential for MCs but not in other cell types. The aim of this study is to re-evaluate the roles of MCs in CHS using mice in which MCs can be conditionally and specifically depleted. Transgenic Mas-TRECK mice in which MCs are depleted conditionally were newly generated using cell-type specific gene regulation by IE. Using this mouse, CHS and FITC-induced cutaneous DC migration were analyzed. Chemotaxis assay and cytoplasmic Ca2+ imaging were performed by co-culture of bone marrow-derived MCs (BMMCs) and bone marrow-derived dendritic cells (BMDCs). In Mas-TRECK mice, CHS was attenuated when MCs were depleted during the sensitization phase. In addition, both maturation and migration of skin DCs were abrogated by MC depletion. Consistently, BMMCs enhanced maturation and chemotaxis of BMDC in ICAM-1 and TNF-α dependent manners Furthermore, stimulated BMDCs increased intracellular Ca2+ of MC upon direct interaction and up-regulated membrane-bound TNF-α on BMMCs. These results suggest that MCs enhance DC functions by interacting with DCs in the skin to establish the sensitization phase of CHS.

Perivascular leukocyte clusters are essential for efficient activation of effector T cells in the skin

It remains largely unclear how antigen-presenting cells (APCs) encounter effector or memory T cells efficiently in the periphery. Here we used a mouse contact hypersensitivity (CHS) model to show that upon epicutaneous antigen challenge, dendritic cells (DCs) formed clusters with effector T cells in dermal perivascular areas to promote in situ proliferation and activation of skin T cells in a manner dependent on antigen and the integrin LFA-1. We found that DCs accumulated in perivascular areas and that DC clustering was abrogated by depletion of macrophages. Treatment with interleukin 1α (IL-1α) induced production of the chemokine CXCL2 by dermal macrophages, and DC clustering was suppressed by blockade of either the receptor for IL-1 (IL-1R) or the receptor for CXCL2 (CXCR2). Our findings suggest that the dermal leukocyte cluster is an essential structure for elicitating acquired cutaneous immunity.

Intravital analysis of vascular permeability in mice using two-photon microscopy

Blood vessel endothelium forms a semi-permeable barrier and its permeability controls the traffics of plasma contents. Here we report an intravital evaluation system for vascular permeability in mice using two-photon microscopy. We used various sizes of fluorescein-conjugated dextran as a tracer and its efflux was quantified by measuring the changes of fluorescent intensity both on the blood vessel area and the interstitial space. Using this system, we demonstrated that skin blood vessels limited the passage of dextran larger than 70 kDa under homeostatic conditions. We evaluated the kinetics of vascular permeability in histamine- or IgE-induced type I allergic models and a hapten-induced type IV allergic model. In such inflammatory conditions, the hyperpermeability was selectively induced in the postcapillary venules and dextran as large as 2000-kDa leaked from the bloods. Taken together, our study provides a convenient method to characterize the skin blood vessels as a traffic barrier in physiological conditions.

IL-17A as an Inducer for Th2 Immune Responses in Murine Atopic Dermatitis Models

Atopic dermatitis (AD) is generally regarded as a type 2 helper T (Th2)-mediated inflammatory skin disease. Although the number of IL-17A-producing cells is increased in the peripheral blood and in acute skin lesion of AD patients, the role of IL-17A in the pathogenesis of AD remains unclear. To clarify this issue, we used murine AD models in an IL-17A-deficient condition. In a repeated hapten application-induced AD model, skin inflammation, IL-4 production in the draining lymph nodes (LNs), and hapten-specific IgG1 and IgE induction were suppressed in IL-17A-deficient mice. Vγ4(+) γδ T cells in the skin-draining LNs and Vγ5(-) dermal γδ T cells in the skin were the major sources of IL-17A. Consistently, in flaky-tail (Flg(ft/ft) ma/ma) mice, spontaneous development of AD-like dermatitis and IgE induction were attenuated by IL-17A deficiency. Moreover, Th2 differentiation from naive T cells was promoted in vitro by the addition of IL-17A. Taken together, our results suggest that IL-17A mediates Th2-type immune responses and that IL-17A signal may be a therapeutic target of AD.

Basophils are required for the induction of Th2 immunity to haptens and peptide antigens

The relative contributions of basophils and dendritic cells in Th2 skewing to foreign antigen exposure remain unclear. Here we report the ability of basophils to induce Th2 polarization upon epicutaneous sensitization with different antigens using basophil conditionally depleted Bas TRECK transgenic mice. Basophils are responsible for Th2 skewing to haptens and peptide antigens, but not protein antigens in vivo. Consistent with this, basophils cannot take up or process ovalbumin protein in significant quantities, but present ovalbumin peptide to T cells for Th2 differentiation via major histocompatibility complex class II. Intriguingly, basophils promote Th2 skewing upon ovalbumin protein exposure in the presence of dendritic cells. Taken together, our results suggest that basophils alone are able to induce Th2 skewing with haptens and peptide antigens but require dendritic cells for the induction of Th2 for protein antigens upon epicutaneous immunization.

Skin as a Peripheral Lymphoid Organ: Revisiting the Concept of Skin-Associated Lymphoid Tissues

Antigen presentation to T cells is essential for the induction of adaptive immunity. This event takes place not solely in the lymph node (LN) but also in the skin. Recent in vivo trafficking studies using Kaede-transgenic mice reveal that skin-homing effector memory T cells alter their effector function and homing ability by transitioning to a central memory T cell-like phenotype through antigen recognition that occurs in the skin. In addition, these cells travel back and forth between the skin and draining LNs. These studies are evocative of the classic concept of skin-associated lymphoid tissues and underscore the critical role of skin as a peripheral lymphoid organ.

Protease Activity Enhances Production of Thymic Stromal Lymphopoietin and Basophil Accumulation in Flaky Tail Mice

Epidermal barrier abnormality due to filaggrin deficiency is an important predisposing factor in the development of atopic dermatitis (AD). In addition, the expression of thymic stromal lymphopoietin (TSLP) in keratinocytes (KCs), induced by barrier disruption, can promote type 2 helper T-cell polarization. Protease activity, including protease-activated receptor-2 (PAR-2), is also known to be involved in epidermal barrier function in AD. However, to our knowledge, the relationship between protease activity and filaggrin deficiency from the perspective of AD has not been elucidated. Flaky tail (Flg(ft)) mice, known to have a mutation in the filaggrin gene, were used to assess the role of protease in KCs in the steady state and the mite-induced AD-like skin inflammation model. In the steady state, the expression and activity levels of endogenous proteases, kallikreins 5, 7, and 14, in the skin and TSLP were higher in Flg(ft) than in control mice. In addition, activation of PAR-2 by its agonist induced the production of TSLP in KCs of Flg(ft) mice, which was abrogated by a newly developed PAR-2 antagonist. Application of the PAR-2 antagonist improved symptoms and basophil accumulation in Flg(ft) mice treated with mite extracts. These results suggest that possibly through the PAR-2 activation in KCs, filaggrin deficiency induces TSLP production and basophil accumulation, which play important roles in the establishment of AD.