Michiko K. Oyoshi

Animal models of atopic dermatitis

Atopic dermatitis (AD) is characterized by allergic skin inflammation. A hallmark of AD is dry itchy skin due, at least in part, to defects in skin genes that are important for maintaining barrier function. The pathogenesis of AD remains incompletely understood. Since the description of the Nc/Nga mouse as a spontaneously occurring model of AD, a number of other mouse models of AD have been developed. They can be categorized into three groups: (1) models induced by epicutaneous application of sensitizers; (2) transgenic mice that either overexpress or lack selective molecules; (3) mice that spontaneously develop AD-like skin lesions. These models have resulted in a better understanding of the pathogenesis of AD. This review discusses these models and emphasizes the role of mechanical skin injury and skin barrier dysfunction in eliciting allergic skin inflammation.

Filaggrin-deficient mice exhibit TH17-dominated skin inflammation and permissiveness to epicutaneous sensitization with protein antigen

BACKGROUND Filaggrin is important for skin barrier function and is mutated in 15% to 20% of patients with atopic dermatitis. OBJECTIVE To examine whether filaggrin deficiency predisposes to skin inflammation and epicutaneous sensitization with protein antigen. METHODS Skin histology in filaggrin-deficient flaky tail (ft)/ft mice and wild-type controls was assessed by Hematoxylin and Eosin (H&E) staining and immunohistochemistry. Cytokine mRNA expression was examined by quantitative RT-PCR. Serum antibody levels and splenocyte secretion of cytokines were measured by ELISA. RESULTS The ft/ft mice developed eczematous skin lesions after age 28 weeks and a progressive increase in serum IgE and IgG(1) levels. Normal-appearing skin from 8-week-old ft/ft mice had epidermal thickening and increased dermal infiltration with CD4(+) cells and expression of mRNA for IL-17, IL-6, and IL-23, but not IL-4, IL-13, or IFN-gamma. Lesional skin of 32-week-old ft/ft mice exhibited qualitatively similar, but more pronounced, changes, and elevated IL-4 mRNA levels. Epicutaneous application of ovalbumin to shaved skin of 8-week-old ft/ft mice, but not WT mice, resulted in increased epidermal thickening, dermal infiltration by CD4(+) cells but not eosinophils, and expression of IL-17, IL-6, IL-23, IL-4, and IFN-gamma, but not IL-5 or IL-13, mRNA. Splenocytes from epicutaneously sensitized ft/ft mice, but not controls, secreted cytokines in response to ovalbumin stimulation, and their sera, but not those of controls, contained ovalbumin-specific IgE and IgG(1) antibodies. CONCLUSION Filaggrin-deficient mice exhibit T(H)17-dominated skin inflammation and eczematous changes with age, and are permissive to epicutaneous sensitization with protein antigen.

TSLP acts on infiltrating effector T cells to drive allergic skin inflammation

Thymic stromal lymphopoietin (TSLP) is a cytokine expressed by epithelial cells, including keratinocytes, and is important in allergic inflammation. Allergic skin inflammation elicited by epicutaneous immunization of mice with ovalbumin (OVA), a potential model of atopic dermatitis, was severely impaired in TSLPR−/− mice, as evidenced by decreased infiltration of eosinophils and decreased local expression of T helper 2 (Th2) cytokines. However, secretion of Th2 cytokines by splenocytes from epicutaneous sensitized TSLPR−/− mice in response to OVA was normal. Skin dendritic cells from TSLPR−/− mice were normal in their ability to migrate to draining lymph nodes, express activation markers, and induce proliferation and Th2 cytokine production by naïve T cells. CD4+ T cells from TSLPR−/− mice expressed the skin homing receptor E-selectin ligand normally, and homed to the skin normally, but failed to transfer allergic skin inflammation to WT recipients. TSLP enhanced Th2 cytokine secretion in vitro by targeting TSLPR on antigen specific T cells. Intradermal injection of anti-TSLP blocked the development of allergic skin inflammation after cutaneous antigen challenge of OVA immunized WT mice. These findings suggest that TSLP is essential for antigen driven Th2 cytokine secretion by skin infiltrating effector T cells and could be a therapeutic target in allergic skin inflammation.

Epicutaneous antigen exposure induces a Th17 response that drives airway inflammation after inhalation challenge

IL-17 has been implicated in a number of inflammatory diseases, but the conditions of antigen exposure that drive the generation of Th17 responses have not been well defined. Epicutaneous (EC) immunization of mice with ovalbumin (OVA), which causes allergic skin inflammation with many characteristics of the skin lesions of atopic dermatitis, was found to also drive IL-17 expression in the skin. EC, but not i.p., immunization of mice with OVA drove the generation of IL-17-producing T cells in draining lymph nodes and spleen and increased serum IL-17 levels. OVA inhalation by EC-sensitized mice induced IL-17 and CXCL2 expression and neutrophil influx in the lung along with bronchial hyperreactivity, which were reversed by IL-17 blockade. Dendritic cells trafficking from skin to lymph nodes expressed more IL-23 and induced more IL-17 secretion by naïve T cells than splenic dendritic cells. This was inhibited by neutralizing IL-23 in vitro and by intradermal injection of anti-TGFβ neutralizing antibody in vivo. Our findings suggest that initial cutaneous exposure to antigens in patients with atopic dermatitis may selectively induce the production of IL-17, which, in turn, drives inflammation of their airways.

Cellular and molecular mechanisms in atopic dermatitis.

Atopic dermatitis (AD) is a pruritic inflammatory skin disease associated with a personal or family history of allergy. The prevalence of AD is on the rise and estimated at approximately 17% in the USA. The fundamental lesion in AD is a defective skin barrier that results in dry itchy skin, and is aggravated by mechanical injury inflicted by scratching. This allows entry of antigens via the skin and creates a milieu that shapes the immune response to these antigens. This review discusses recent advances in our understanding of the abnormal skin barrier in AD, namely abnormalities in epidermal structural proteins, such as filaggrin, mutated in approximately 15% of patients with AD, epidermal lipids, and epidermal proteases and protease inhibitors. The review also dissects, based on information from mouse models of AD, the contributions of the innate and adaptive immune system to the pathogenesis of AD, including the effect of mechanical skin injury on the polarization of skin dendritic cells, mediated by keratinocyte-derived cytokines such as thymic stromal lymphopoietin (TSLP), IL-6, and IL-1, that results in a Th2-dominated immune response with a Th17 component in acute AD skin lesions and the progressive conversion to a Th1-dominated response in chronic AD skin lesions. Finally, we discuss the mechanisms of susceptibility of AD skin lesions to microbial infections and the role of microbial products in exacerbating skin inflammation in AD. Based on this information, we discuss current and future therapy of this common disease.

Mechanical injury polarizes skin dendritic cells to elicit a TH2 response by inducing cutaneous thymic stromal lymphopoietin expression

BACKGROUND Atopic dermatitis is characterized by scratching and by T(H)2-dominated immune response to cutaneously introduced antigens. Antigen application to skin mechanically injured by tape stripping results in T(H)2-dominated skin inflammation. OBJECTIVE To examine the effect of tape stripping on the capacity of skin dendritic cells (DCs) to polarize T cells toward a T(H)2 phenotype. METHODS CD11c(+) DCs were isolated from skin of BALB/c or C57BL/6 mice. Fluorescein isothiocyanate (FITC)(+) and FITC(-) DCs were isolated from draining lymph nodes (DLNs) 24 hours after painting the skin with FITC. DCs were assessed for their ability to induce cytokine secretion by ovalbumin-stimulated naive CD4(+) T cells from T cell receptor-ovalbumin transgenic DO11.10 mice. Cytokine mRNA levels were examined by quantitative PCR. RESULTS Dendritic cells isolated from the skin of wild-type, but not thymic stromal lymphopoietin (TSLP) receptor(-/-) or IL-10(-/-), mice 6 hours after tape stripping elicited significantly more IL-4 and IL-13 and significantly less IFN-γ production by CD4(+) cells than DCs isolated from unmanipulated skin, and expressed significantly more mRNA for the T(H)2 skewing molecules IL-10, Jagged1, and Jagged2, but significantly less mRNA for the T(H)1 skewing cytokine IL-12. CD11c(+)FITC(+) cells isolated from DLNs of shaved and tape stripped skin of wild-type, but not TSLP receptor(-/-) or IL-10(-/-), mice polarized T cells significantly more toward T(H)2 and expressed significantly more IL-10, Jagged1, and Jagged2 mRNA than CD11c(+)FITC(+) cells isolated from DLNs of shaved skin. Tape stripping significantly increased TSLP levels in the skin, and TSLP was shown to play an essential role in the T(H)2 polarization of skin DCs by tape stripping. CONCLUSIONS Tape stripping upregulates TSLP levels in the skin, which polarizes skin DCs to elicit a T(H)2 response via the induction of IL-10.

Common cytological and cytogenetic features of Epstein-Barr virus (EBV)-positive natural killer (NK) cells and cell lines derived from patients with nasal T/NK-cell lymphomas, chronic active EBV infection and hydroa vacciniforme-like eruptions

Summary. In this study, we describe the cytological and cytogenetic features of six Epstein‐Barr virus (EBV)‐infected natural killer (NK) cell clones. Three cell clones, SNK‐1, ‐3 and ‐6, were derived from patients with nasal T/NK‐cell lymphomas; two cell clones, SNK‐5 and ‐10, were isolated from patients with chronic active EBV infection (CAEBV); and the other cell clone, SNK‐11, was from a patient with hydroa vacciniforme (HV)‐like eruptions. An analysis of the number of EBV‐terminal repeats showed that the SNK cell clones had monoclonal EBV genomes identical to the original EBV‐infected cells of the respective patients, and SNK cells had the type II latency of EBV infection, suggesting that not only the cell clones isolated from nasal T/NK‐cell lymphomas but also those isolated from CAEBV and HV‐like eruptions had been transformed by EBV to a certain degree. Cytogenetic analysis detected deletions in chromosome 6q in five out of the six SNK cell clones, while 6q was not deleted in four control cell lines of T‐cell lineage. This suggested that a 6q deletion is a characteristic feature of EBV‐positive NK cells, which proliferated in the diseased individuals. The results showed that EBV‐positive NK cells in malignant and non‐malignant lymphoproliferative diseases shared common cytological and cytogenetic features.

Exaggerated IL-17 response to epicutaneous sensitization mediates airway inflammation in the absence of IL-4 and IL-13

BACKGROUND Atopic dermatitis (AD) is characterized by local and systemic T(H)2 responses to cutaneously introduced allergens and is a risk factor for asthma. Blockade of T(H)2 cytokines has been suggested as therapy for AD. OBJECTIVES We sought to examine the effect of the absence of IL-4 and IL-13 on the T(H)17 response to epicutaneous sensitization in a murine model of allergic skin inflammation with features of AD. METHODS Wild-type, IL4 knockout (KO), IL13 KO and IL4/13 double KO (DKO) mice were subjected to epicutaneous sensitization with ovalbumin (OVA) or saline and airway challenged with OVA. Systemic immune responses to OVA, skin and airway inflammation, and airway hyperresponsiveness were examined. RESULTS OVA-sensitized DKO mice exhibited impaired T(H)2-driven responses with undetectable OVA-specific IgE levels and severely diminished eosinophil infiltration at sensitized skin sites but intact dermal infiltration with CD4(+) cells. DKO mice mounted exaggerated IL-17A but normal IFN-gamma and IL-5 systemic responses. Airway challenge of these mice with OVA caused marked upregulation of IL-17 mRNA expression in the lungs, increased neutrophilia in bronchoalveolar lavage fluid, airway inflammation characterized by mononuclear cell infiltration with no detectable eosinophils, and bronchial hyperresponsiveness to methacholine that were reversed by IL-17 blockade. IL-4, but not IL-13, was identified as the major T(H)2 cytokine that downregulates the IL-17 response in epicutaneously sensitized mice. CONCLUSION Epicutaneous sensitization in the absence of IL-4/IL-13 induces an exaggerated T(H)17 response systemically and in lungs after antigen challenge that results in airway inflammation and airway hyperresponsiveness.

Leukotriene B4-Driven Neutrophil Recruitment to the Skin Is Essential for Allergic Skin Inflammation

Scratching triggers skin flares in atopic dermatitis. We demonstrate that scratching of human skin and tape stripping of mouse skin cause neutrophil influx. In mice, this influx was largely dependent on the generation of leukotriene B4 (LTB4) by neutrophils and their expression of the LTB4 receptor BLT1. Allergic skin inflammation in response to epicutaneous (EC) application of ovalbumin to tape-stripped skin was severely impaired in Ltb4r1(-/-) mice and required expression of BLT1 on both T cells and non-T cells. Cotransfer of wild-type (WT) neutrophils, but not neutrophils deficient in BLT1 or the LTB4-synthesizing enzyme LTA4H, restored the ability of WT CD4(+) effector T cells to transfer allergic skin inflammation to Ltb4r1(-/-) recipients. Pharmacologic blockade of LTB4 synthesis inhibited allergic skin inflammation elicited by cutaneous antigen challenge in previously EC-sensitized mice. Our results demonstrate that a neutrophil-T cell axis reliant on LTB4-BLT1 interaction is required for allergic skin inflammation.

IL-21R is essential for epicutaneous sensitization and allergic skin inflammation in humans and mice

Atopic dermatitis (AD) is a common allergic inflammatory skin disease caused by a combination of intense pruritus, scratching, and epicutaneous (e.c.) sensitization with allergens. To explore the roles of IL-21 and IL-21 receptor (IL-21R) in AD, we examined skin lesions from patients with AD and used a mouse model of allergic skin inflammation. IL-21 and IL-21R expression was upregulated in acute skin lesions of AD patients and in mouse skin subjected to tape stripping, a surrogate for scratching. The importance of this finding was highlighted by the fact that both Il21r-/- mice and WT mice treated with soluble IL-21R-IgG2aFc fusion protein failed to develop skin inflammation after e.c. sensitization of tape-stripped skin. Adoptively transferred OVA-specific WT CD4+ T cells accumulated poorly in draining LNs (DLNs) of e.c. sensitized Il21r-/- mice. This was likely caused by both DC-intrinsic and nonintrinsic effects, because trafficking of skin DCs to DLNs was defective in Il21r-/- mice and, to a lesser extent, in WT mice reconstituted with Il21r-/- BM. More insight into this defect was provided by the observation that skin DCs from tape-stripped WT mice, but not Il21r-/- mice, upregulated CCR7 and migrated toward CCR7 ligands. Treatment of epidermal and dermal cells with IL-21 activated MMP2, which has been implicated in trafficking of skin DCs. These results suggest an important role for IL-21R in the mobilization of skin DCs to DLNs and the subsequent allergic response to e.c. introduced antigen.