Depletion of Treg by the Diphtheria Toxin System.

Specific cell ablation by the diphtheria toxin (DT) system is widely used to analyze the in vivo function of target cells in mice. In this chapter, we describe the methods of depleting regulatory T cells (Tregs) systemically or selectively in the skin. Since it has been difficult to conclude the importance of tissue-residing Tregs with systemic Treg ablation, we sought to selectively deplete cutaneous Tregs to investigate their function in the skin without the depletion of Tregs in non-target organs. Here, we describe protocols for the depletion of Tregs by the DT system, and subsequent analysis of Tregs in the skin and skin-draining lymph node (dLN) by flow cytometry. This procedure of selective depletion of cutaneous Tregs can be applicable to other tissues and cells, to allow investigation of the role of tissue-resident cells in mice.

Infiltration and local differentiation of bone marrow-derived integrinß7-positive mast cell progenitors in atopic dermatitis-like skin.

BACKGROUND: Mast cells (MCs) are tissue-resident cells with various immunologic functions. MCs are increased in atopic dermatitis (AD) skin and can contribute to the inflammation. Although skin MCs are inducible from bone marrow (BM) cells in vitro, they are maintained locally by self-proliferation in the steady state in vivo. However, how skin MCs are increased in AD skin, including the infiltration of BM-derived MC progenitors (MCps) and their differentiation, remains unclear. OBJECTIVE: We sought to identify and characterize BM-derived MCps in AD skin. METHODS: BM-derived MCps in AD skin were analyzed by flow cytometry using BM-chimeric mice and parabiosis in an MC903-induced AD model. BM-derived MCps in AD-like skin were compared with resident MCs for gene expression by RNA- sequencing analysis. RESULTS: We observed local proliferation of resident MCs and an increase in BM-derived MCs in AD-like skin. BM-derived MCs in the skin were derived from circulating MCps and were distinguishable from resident MCs by integrinß7. RNA- sequence analysis showed that integrinß7+ MCs (BM-derived MCps) in the skin shared the characteristics of both mucosal-type MCs and connective tissue-type MCs, and increased the expression of genes related to MCp migration. BM-derived MCps proliferated in situ, gradually lost the integrinß7 expression, and acquired connective tissue-type MC phenotypes during the remission phase of inflammation. CONCLUSIONS: BM-derived integrinß7+ MCps migrate to AD-like skin and contribute to the maintenance of skin MCs.

CCL2‒CCR2 Signaling in the Skin Drives Surfactant-Induced Irritant Contact Dermatitis through IL-1ß‒Mediated Neutrophil Accumulation.

Surfactant-induced cumulative irritant contact dermatitis (ICD) is a common and clinically important skin disorder. CCL2 is known to mediate inflammation after tissue damage in various organs. Thus, we investigated whether and how CCL2 contributes to the development of murine cumulative ICD induced by a common surfactant, SDS. Wild-type mice treated topically with SDS for 6 consecutive days developed skin inflammation that recapitulated the features of human cumulative ICD, including barrier disruption, epidermal thickening, and neutrophil accumulation. CCL2 was upregulated in SDS-treated skin, and local CCL2 blockade attenuated SDS-induced ICD. SDS-induced ICD and neutrophil accumulation were also attenuated in mice deficient in CCR2, the receptor for CCL2. Neutrophil depletion alleviated SDS-induced ICD, suggesting that impaired neutrophil accumulation was responsible for the amelioration of ICD in CCR2-deficient mice. In RNA-sequencing analyses of SDS-treated skin, the expression levels of Il1b in Ccr2-deficient mice were highly downregulated compared with those in wild-type mice. Furthermore, the intradermal administration of IL-1ß in the SDS-treated skin of CCR2-deficient mice restored the local accumulation of neutrophils and the development of ICD. Collectively, our results suggest that CCL2‒CCR2 signaling in the skin critically promotes the development of SDS-induced ICD by inducing IL-1ß expression for neutrophil accumulation.

Autoimmune Pemphigus: Latest Advances and Emerging Therapies.

Pemphigus represents a group of rare and severe autoimmune intra-epidermal blistering diseases affecting the skin and mucous membranes. These painful and debilitating diseases are driven by the production of autoantibodies that are mainly directed against the desmosomal adhesion proteins, desmoglein 3 (Dsg3) and desmoglein 1 (Dsg1). The search to define underlying triggers for anti-Dsg-antibody production has revealed genetic, environmental, and possible vaccine-driven factors, but our knowledge of the processes underlying disease initiation and pathology remains incomplete. Recent studies point to an important role of T cells in supporting auto-antibody production; yet the involvement of the myeloid compartment remains unexplored. Clinical management of pemphigus is beginning to move away from broad-spectrum immunosuppression and towards B-cell-targeted therapies, which reduce many patients' symptoms but can have significant side effects. Here, we review the latest developments in our understanding of the predisposing factors/conditions of pemphigus, the underlying pathogenic mechanisms, and new and emerging therapies to treat these devastating diseases.

Single-Cell Profiling Reveals Divergent, Globally Patterned Immune Responses in Murine Skin Inflammation.

Inflammatory response heterogeneity has impeded high-resolution dissection of diverse immune cell populations during activation. We characterize mouse cutaneous immune cells by single-cell RNA sequencing, after inducing inflammation using imiquimod and oxazolone dermatitis models. We identify 13 CD45+ subpopulations, which broadly represent most functionally characterized immune cell types. Oxazolone pervasively upregulates Jak2/Stat3 expression across T cells and antigen-presenting cells (APCs). Oxazolone also induces Il4/Il13 expression in newly infiltrating basophils, and Il4ra and Ccl24, most prominently in APCs. In contrast, imiquimod broadly upregulates Il17/Il22 and Ccl4/Ccl5. A comparative analysis of single-cell inflammatory transcriptional responses reveals that APC response to oxazolone is tightly restricted by cell identity, whereas imiquimod enforces shared programs on multiple APC populations in parallel. These global molecular patterns not only contrast immune responses on a systems level but also suggest that the mechanisms of new sources of inflammation can eventually be deduced by comparison to known signatures.

Percutaneous sensitization is limited by in situ inhibition of cutaneous dendritic cell migration through skin-resident regulatory T cells.

BACKGROUND: Percutaneous sensitization is associated with various allergic diseases, including asthma and food allergies. However, the immunologic mechanisms underlying how the skin regulates percutaneous sensitization are still unclear. OBJECTIVE: We aimed to investigate whether and how CD4+Foxp3+ regulatory T (Treg) cells residing in the skin regulate percutaneous sensitization in the skin. METHODS: Selective reduction of numbers of cutaneous Treg cells was achieved by means of intradermal injection of diphtheria toxin into the ear skin of Foxp3DTR mice, in which Treg cells specifically express the diphtheria toxin receptor fused with green fluorescent protein. RESULTS: Thirty percent to 40% of cutaneous Treg cells were capable of IL-10 production in both mice and human subjects. Selective reduction of cutaneous Treg cells at the sensitization site promoted migration of antigen-bearing dendritic cells (DCs) to the draining lymph nodes (dLNs). Accordingly, sensitization through the skin with reduced numbers of Treg cells led to enhanced antigen-specific immune responses in the dLNs, including both effector T-cell differentiation and T cell-dependent B-cell responses, such as the development of germinal center B cells expressing IgG1 and IgE. Furthermore, antigen-bearing cutaneous DC migration was enhanced in mice with IL-10 deficiency restricted to the cutaneous Treg cell compartment, suggesting an important role of cutaneous IL-10+ Treg cells in limiting percutaneous sensitization. Treg cells with a skin-homing phenotype in skin dLNs expressed high levels of IL-10, suggesting that they contribute to renewal and maintenance of the cutaneous IL-10+ Treg cell population. CONCLUSION: Skin-resident Treg cells limit percutaneous sensitization by suppressing antigen-bearing DC migration through in situ IL-10 production.

Resolvin E1 inhibits dendritic cell migration in the skin and attenuates contact hypersensitivity responses.

Resolvin E1 (RvE1) is a lipid mediator derived from ω3 polyunsaturated fatty acids that exerts potent antiinflammatory roles in several murine models. The antiinflammatory mechanism of RvE1 in acquired immune responses has been attributed to attenuation of cytokine production by dendritic cells (DCs). In this study, we newly investigated the effect of RvE1 on DC motility using two-photon microscopy in a contact hypersensitivity (CHS) model and found that RvE1 impaired DC motility in the skin. In addition, RvE1 attenuated T cell priming in the draining lymph nodes and effector T cell activation in the skin, which led to the reduced skin inflammation in CHS. In contrast, leukotriene B4 (LTB4) induced actin filament reorganization in DCs and increased DC motility by activating Cdc42 and Rac1 via BLT1, which was abrogated by RvE1. Collectively, our results suggest that RvE1 attenuates cutaneous acquired immune responses by inhibiting cutaneous DC motility, possibly through LTB4-BLT1 signaling blockade.

Generation of Helios reporter mice and an evaluation of the suppressive capacity of Helios(+) regulatory T cells in vitro.

Helios is a member of the Ikaros transcription factor family and has been reported to be a marker of thymus-derived regulatory T cells (Treg). Helios is an intracellular protein, however, and hence cannot be used as a marker to separate living Tregs. To solve this problem, we generated Helios reporter mice in which Helios+ cells selectively express Venus, a variant of green fluorescent protein. Most of the Tregs in the thymus expressed Helios, whereas its expression was varied in peripheral lymphoid organs. The Helios+ Treg-population was superior in ability to suppress both antigen-specific and TCR-stimulated T cell responses. We also showed that Helios+ Tregs inhibited the cytokine production by T cells more efficiently than Helios- Tregs. We conclude that Helios reporter mouse strain is a useful tool to study function of Helios and that Helios+ Tregs represent the highly suppressive population.

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.

Classification of inflammatory skin diseases: a proposal based on the disorders of the three-layered defense systems, barrier, innate immunity and acquired immunity.

The host defense system of the skin is composed of (1) a barrier, (2) innate immunity, and (3) acquired immunity. Inflammatory skin diseases can be classified into one of the disorders of these layers of the defense system, unless there is an ordinary response to specific infectious agents or internal/external injury. Any inflammatory skin disease partly simulates the response to real infections or dangers. Disorders of acquired immunity can be classified into (1) immunodeficiency, (2) immunohyperactivity (allergy), and (3) qualitative disorder (autoimmunity). Disorders of innate immunity can be classified into (1) innate immunodeficiency, (2) innate immunohyperactivity (general or local autoinflammation), and (3) qualitative disorder (general or local innate autoimmunity). The barrier of the skin is composed of (1) the physical barrier and (2) the chemical barrier. Several diseases, such as atopic dermatitis, are attributed to the disorder of these components of the barrier. Here, we propose an algorithm to classify the pathology of inflammatory skin diseases by means of what disorder in the specific layer of the host defense system is truly responsible.

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.