Mast cell-derived IL-13 downregulates IL-12 production by skin dendritic cells to inhibit the TH1 cell response to cutaneous antigen exposure.

BACKGROUND: Atopic dermatitis (AD) is characterized by a skin barrier defect aggravated by mechanical injury inflicted by scratching, a TH2 cell-dominated immune response, and susceptibility to viral skin infections that are normally restrained by a TH1 cell response. The signals leading to a TH2 cell-dominated immune response in AD are not completely understood. OBJECTIVE: Our aim was to determine the role of IL-13 in initiation of the TH cell response to cutaneously encountered antigens. METHODS: Wild-type, Il13-/-, Il1rl1-/-, and Il4ra-/- mice, as well as mice with selective deficiency of IL-13 in mast cells (MCs) were studied; in addition, dendritic cells (DCs) purified from the draining lymph nodes of tape-stripped and ovalbumin (OVA)-sensitized skin were examined for their ability to polarize naive OVA-TCR transgenic CD4+ T cells. Cytokine expression was examined by reverse-transcriptase quantitative PCR, intracellular flow cytometry, and ELISA. Contact hypersensitivity to dinitrofluorobenzene was examined. RESULTS: Tape stripping caused IL-33-driven upregulation of Il13 expression by skin MCs. MC-derived IL-13 acted on DCs from draining lymph nodes of OVA-sensitized skin to selectively suppress their ability to polarize naive OVA-TCR transgenic CD4+ T cells into IFN-γ-secreting cells. MC-derived IL-13 inhibited the TH1 cell response in contact hypersensitivity to dinitrofluorobenzene. IL-13 suppressed IL-12 production by mouse skin-derived DCs in vitro and in vivo. Scratching upregulated IL13 expression in human skin, and IL-13 suppressed the capacity of LPS-stimulated human skin DCs to express IL-12 and promote IFN-γ secretion by CD4+ T cells. CONCLUSION: Release of IL-13 by cutaneous MCs in response to mechanical skin injury inhibits the TH1 cell response to cutaneous antigen exposure in AD.

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-22 promotes allergic airway inflammation in epicutaneously sensitized mice.

BACKGROUND: Serum IL-22 levels are increased in patients with atopic dermatitis, which commonly precedes asthma in the atopic march. Epicutaneous sensitization in mice results in TH2-dominated skin inflammation that mimics atopic dermatitis and sensitizes the airways for antigen challenge-induced allergic inflammation characterized by the presence of both eosinophils and neutrophils. Epicutaneous sensitization results in increased serum levels of IL-22. OBJECTIVE: We sought to determine the role of IL-22 in antigen-driven airway allergic inflammation after inhalation challenge in epicutaneously sensitized mice. METHODS: Wild-type (WT) and Il22-/- mice were sensitized epicutaneously or immunized intraperitoneally with ovalbumin (OVA) and challenged intranasally with antigen. OVA T-cell receptor-specific T cells were TH22 polarized in vitro. Airway inflammation, mRNA levels in the lungs, and airway hyperresponsiveness (AHR) were examined. RESULTS: Epicutaneous sensitization preferentially elicited an IL-22 response compared with intraperitoneal immunization. Intranasal challenge of mice epicutaneously sensitized with OVA elicited in the lungs Il22 mRNA expression, IL-22 production, and accumulation of CD3+CD4+IL-22+ T cells that coexpressed IL-17A and TNF-α. Epicutaneously sensitized Il22-/- mice exhibited diminished eosinophil and neutrophil airway infiltration and decreased AHR after intranasal OVA challenge. Production of IL-13, IL-17A, and TNF-α was normal, but IFN-γ production was increased in lung cells from airway-challenged and epicutaneously sensitized Il22-/- mice. Intranasal instillation of IFN-γ-neutralizing antibody partially reversed the defect in eosinophil recruitment. WT recipients of TH22-polarized WT, but not IL-22-deficient, T-cell receptor OVA-specific T cells, which secrete both IL-17A and TNF-α, had neutrophil-dominated airway inflammation and AHR on intranasal OVA challenge. Intranasal instillation of IL-22 with TNF-α, but not IL-17A, elicited neutrophil-dominated airway inflammation and AHR in WT mice, suggesting that loss of IL-22 synergy with TNF-α contributed to defective recruitment of neutrophils into the airways of Il22-/- mice. TNF-α, but not IL-22, blockade at the time of antigen inhalation challenge inhibited airway inflammation in epicutaneously sensitized mice. CONCLUSION: Epicutaneous sensitization promotes generation of antigen-specific IL-22-producing T cells that promote airway inflammation and AHR after antigen challenge, suggesting that IL-22 plays an important role in the atopic march.

IL-22 derived from γδ T cells restricts Staphylococcus aureus infection of mechanically injured skin.

BACKGROUND: Staphylococcus aureus is an opportunistic pathogen that colonizes the skin of patients with atopic dermatitis (AD) and aggravates their disease. Neutrophils and the cytokines IL-17A and IL-17F, which drive the expression of the neutrophil-attracting chemokines, are important for the clearance of S aureus infection. The cytokine IL-22 is often coproduced by IL-17-secreting cells. The levels of IL-22 are elevated in AD skin lesions. OBJECTIVE: We sought to determine the role of IL-22 in the clearance of S aureus infection of mouse skin subjected to tape stripping, a surrogate for scratching, a cardinal feature of AD. METHODS: S aureus was applied to the tape-stripped skin of wild-type and Il22-/- mice. Bacterial burden was evaluated by enumerating colony-forming units. Quantitative PCR and ELISA were performed to quantify Il22 mRNA and IL-22 protein in mouse and human skin. Flow cytometry was used to enumerate neutrophils in the skin. RESULTS: Scratching the skin of healthy adults and tape stripping of mouse skin induced local expression of Il22 mRNA and IL-22 protein. Induction of Il22 expression by tape stripping was dependent on IL-23 and γδ T cells. Clearance of S aureus from tape-stripped skin was significantly impaired in Il22-/- mice. Neutrophil infiltration and upregulation of expression of genes encoding the antimicrobial peptides antigen-6/urokinase-type plasminogen activator receptor related protein-1 and ß-DEFENSIN 14 and the chemokine (C-X-C motif) ligand following tape stripping were significantly impaired in Il22-/- mice. CONCLUSIONS: These findings show that IL-22 is important for limiting the growth of S aureus on mechanically injured skin and caution that IL-23 and IL-22 blockade in patients with AD may enhance susceptibility to staphylococcal skin infection.

IL-33 promotes food anaphylaxis in epicutaneously sensitized mice by targeting mast cells.

BACKGROUND: Cutaneous exposure to food allergens predisposes to food allergy, which is commonly associated with atopic dermatitis (AD). Levels of the epithelial cytokine IL-33 are increased in skin lesions and serum of patients with AD. Mast cells (MCs) play a critical role in food-induced anaphylaxis and express the IL-33 receptor ST2. The role of IL-33 in patients with MC-dependent food anaphylaxis is unknown. OBJECTIVE: We sought to determine the role and mechanism of action of IL-33 in patients with food-induced anaphylaxis in a model of IgE-dependent food anaphylaxis elicited by oral challenge of epicutaneously sensitized mice. METHODS: Wild-type, ST2-deficient, and MC-deficient KitW-sh/W-sh mice were epicutaneously sensitized with ovalbumin (OVA) and then challenged orally with OVA. Body temperature was measured by means of telemetry, Il33 mRNA by means of quantitative PCR, and IL-33, OVA-specific IgE, and mouse mast cell protease 1 by means of ELISA. Bone marrow-derived mast cell (BMMC) degranulation was assessed by using flow cytometry. RESULTS: Il33 mRNA expression was upregulated in tape-stripped mouse skin and scratched human skin. Tape stripping caused local and systemic IL-33 release in mice. ST2 deficiency, as well as ST2 blockade before oral challenge, significantly reduced the severity of oral anaphylaxis without affecting the systemic TH2 response to the allergen. Oral anaphylaxis was abrogated in KitW-sh/W-sh mice and restored by means of reconstitution with wild-type but not ST2-deficient BMMCs. IL-33 significantly enhanced IgE-mediated degranulation of BMMCs in vitro. CONCLUSION: IL-33 is released after mechanical skin injury, enhances IgE-mediated MC degranulation, and promotes oral anaphylaxis after epicutaneous sensitization by targeting MCs. IL-33 neutralization might be useful in treating food-induced anaphylaxis in patients with AD.

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.

Innate antifungal immunity of human eosinophils mediated by a beta 2 integrin, CD11b.

Eosinophils produce and release various proinflammatory mediators and also show immunomodulatory and tissue remodeling functions; thus, eosinophils may be involved in the pathophysiology of asthma and other eosinophilic disorders as well as host defense. Several major questions still remain. For example, how do human eosinophils become activated in diseased tissues or at the site of an immune response? What types of host immunity might potentially involve eosinophils? Herein, we found that human eosinophils react vigorously to a common environmental fungus, Alternaria alternata, which is implicated in the development and/or exacerbation of human asthma. Eosinophils release their cytotoxic granule proteins, such as eosinophil-derived neurotoxin and major basic protein, into the extracellular milieu and onto the surface of fungal organisms and kill the fungus in a contact-dependent manner. Eosinophils use their versatile beta(2) integrin molecule, CD11b, to adhere to a major cell wall component, beta-glucan, but eosinophils do not express other common fungal receptors, such as dectin-1 and lactosylceramide. The I-domain of CD11b is distinctively involved in the eosinophils' interaction with beta-glucan. Eosinophils do not react with another fungal cell wall component, chitin. Because human eosinophils respond to and kill certain fungal organisms, our findings identify a previously unrecognized innate immune function for eosinophils. This immune response by eosinophils may benefit the host, but, in turn, it may also play a role in the development and/or exacerbation of eosinophil-related allergic human diseases, such as asthma.

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.

A novel IL-1 family cytokine, IL-33, potently activates human eosinophils.

BACKGROUND: Eosinophils are likely key cells involved in the pathogenesis of asthma and allergic diseases; however, the mechanisms that regulate eosinophil dynamics and functions in mucosal tissues are incompletely understood. IL-33, which is produced by mucosal cells, is a new member of the IL-1 cytokine family. Mice injected with IL-33 display profound mucosal eosinophilia with associated pathologic changes. Although mast cells and T(H)2 cells express the IL-33 receptor, ST2, the roles of IL-33 and ST2 in eosinophil biology are unknown. OBJECTIVES: We investigated the effects of IL-33 on human eosinophils in vitro. METHODS: Eosinophils and neutrophils were isolated from blood of normal individuals and mildly atopic patients. Real-time RT-PCR and flow cytometry were used to detect ST2. Granulocyte responses to IL-33 were monitored by superoxide anion production and by degranulation; IL-5, IL-1beta, and TNF-alpha served as controls. Eosinophil survival and cytokine production were assessed by flow cytometry and ELISA, respectively. RESULTS: ST2 mRNA and protein were detected on eosinophils. IL-33 induced eosinophil superoxide anion production and degranulation as potently as IL-5. IL-33 also increased eosinophil survival and induced production of IL-8. Anti-ST2 inhibited eosinophil responses to IL-33. Neutrophils did not express ST2, nor did they respond to IL-33. CONCLUSION: IL-33 and its receptor, ST2, may play important roles in eosinophil-mediated inflammation; they may provide new therapeutic targets for controlling mucosal eosinophilic inflammation.

From Bench to Clinic: the Potential of Therapeutic Targeting of the IL-22 Signaling Pathway in Atopic Dermatitis.

Atopic dermatitis (AD) is the most common pruritic inflammatory skin disease characterized by thickening of epidermis and dermis as well as by the infiltration of multiple pathogenic polarized T lymphocytes, including Th2, Th17, and Th22 cells. Significant progress has been made to develop targeted therapeutics for treating AD, e.g., Food and Drug Administration-approved dupilumab, an antibody for dual targeting of IL-4 and IL-13 signaling pathways. Additionally, a growing body of published evidence and a promising result from the early stage of the clinical trial with ILV-094, an anti-IL-22 antibody, strongly support the notion that IL-22 is a potential therapeutic target for treating AD. Moreover, we also experimentally proved that IL-22 contributes to the pathophysiology of AD by employing a murine model of AD induced by epicutaneous sensitization. Here, we review recent preclinical and clinical findings that have advanced our understanding of the roles of IL-22 and Th22 cells in skin inflammation. We conclude that blockade of IL-22 signaling may be a promising therapeutic approach for the treatment of AD.

Discovery of a Novel Highly Selective Histamine H4 Receptor Antagonist for the Treatment of Atopic Dermatitis.

The histamine H4 receptor (H4R), a member of the G-protein coupled receptor family, has been considered as a potential therapeutic target for treating atopic dermatitis (AD). A large number of H4R antagonists have been disclosed, but no efficient agents controlling both pruritus and inflammation in AD have been developed yet. Here, we have discovered a novel class of orally available H4R antagonists showing strong anti-itching and anti-inflammation activity as well as excellent selectivity against off-targets. A pharmacophore-based virtual screening system constructed in-house successfully identified initial hit compound 9, and the subsequent homology model-guided optimization efficiently led us to discover pyrido[2,3- e]tetrazolo[1,5- a]pyrazine analogue 48 as a novel chemotype of a potent and highly selective H4R antagonist. Importantly, orally administered compound 48 exhibits remarkable efficacy on antipruritus and anti-inflammation with a favorable pharmacokinetic (PK) profile in several mouse models of AD. Thus, these data strongly suggest that our compound 48 is a promising clinical candidate for treatment of AD.

IL-23 induced in keratinocytes by endogenous TLR4 ligands polarizes dendritic cells to drive IL-22 responses to skin immunization.

Atopic dermatitis (AD) is a Th2-dominated inflammatory skin disease characterized by epidermal thickening. Serum levels of IL-22, a cytokine known to induce keratinocyte proliferation, are elevated in AD, and Th22 cells infiltrate AD skin lesions. We show that application of antigen to mouse skin subjected to tape stripping, a surrogate for scratching, induces an IL-22 response that drives epidermal hyperplasia and keratinocyte proliferation in a mouse model of skin inflammation that shares many features of AD. DC-derived IL-23 is known to act on CD4(+) T cells to induce IL-22 production. However, the mechanisms that drive IL-23 production by skin DCs in response to cutaneous sensitization are not well understood. We demonstrate that IL-23 released by keratinocytes in response to endogenous TLR4 ligands causes skin DCs, which selectively express IL-23R, to up-regulate their endogenous IL-23 production and drive an IL-22 response in naive CD4(+) T cells that mediates epidermal thickening. We also show that IL-23 is released in human skin after scratching and polarizes human skin DCs to drive an IL-22 response, supporting the utility of IL-23 and IL-22 blockade in AD.

Defective lymphoid organogenesis underlies the immune deficiency caused by a heterozygous S32I mutation in IκBα.

Patients with ectodermal dysplasia with immunodeficiency (ED-ID) caused by mutations in the inhibitor of NF-κB α (IκBα) are susceptible to severe recurrent infections, despite normal T and B cell numbers and intact in vitro lymphocyte function. Moreover, the outcome of hematopoietic stem cell transplantation (HSCT) in these patients is poor despite good engraftment. Mice heterozygous for the IκBα S32I mutation found in patients exhibited typical features of ED-ID. Strikingly, the mice lacked lymph nodes, Peyer's patches, splenic marginal zones, and follicular dendritic cells and failed to develop contact hypersensitivity (CHS) or form germinal centers (GCs), all features not previously recognized in patients and typical of defective noncanonical NF-κB signaling. Lymphotoxin ß receptor (LTßR)-driven induction of chemokines and adhesion molecules mediated by both canonical and noncanonical NF-κB pathways was impaired, and levels of p100 were markedly diminished in the mutant. IκBα mutant → Rag2(-/-), but not WT→IκBα mutant, bone marrow chimeras formed proper lymphoid organs and developed CHS and GCs. Defective architectural cell function explains the immunodeficiency and poor outcome of HSCT in patients with IκBα deficiency and suggests that correction of this niche is critical for reconstituting their immune function.

CD66b regulates adhesion and activation of human eosinophils.

Eosinophils and their products are likely important in the pathophysiology of allergic diseases, such as bronchial asthma, and in host immunity to parasitic organisms. However, the mechanisms for proinflammatory mediator release by eosinophils are poorly understood. CD66b (CEACAM8, CGM6, NCA-95) is a single chain, GPI-anchored, highly glycosylated protein belonging to the carcinoembryonic Ag supergene family. CD66b is an activation marker for human granulocytes; however, its biological functions are largely unknown in eosinophils. We found that CD66b is highly expressed on the surface of human peripheral blood eosinophils isolated from healthy individuals. Engagement of CD66b, but not CD66a, by mAb or a natural ligand, galectin-3, activated a Src kinase family molecule, hemopoietic cell kinase (Hck), and induced cellular adhesion, superoxide production, and degranulation of eosinophils. CD66b molecules were localized in lipid rafts, and disruption of lipid rafts or removal of the GPI anchor inhibited the adhesion and activation of eosinophils. Importantly, CD66b was constitutively and physically associated with a beta2 integrin, CD11b, and cross-linking of CD66b induced a striking clustering of CD11b molecules. Thus, CD66b molecules are involved in regulating adhesion and activation of eosinophils, possibly through their localization in lipid rafts and interaction with other cell surface molecules, such as CD11b. Binding of exogenous or endogenous carbohydrate ligands(s) to CD66b may be important in the release of proinflammatory mediators by human eosinophils.

Selective activation of TACI by syndecan-2.

B-lymphocyte homeostasis and function are regulated by complementary actions of the TNFR family members TACI, BCMA, and BAFF-R, which are expressed by mature B cells. How these receptors are differentially activated is not entirely understood, because the primary ligand BAFF binds to all three. We searched for alternative ligands for TACI using recombinant TACI-Fc fusion protein as a probe and identified syndecan-2 as a new binding partner. TACI binding appears to require heparan sulfate posttranslational modifications of syndecan-2, because free heparin or pretreatment with heparitinase blocked the interaction. Syndecan-2 bound TACI but bound neither BAFF-R nor BCMA. Transfected cells expressing syndecan-2 activated signaling through TACI, as indicated by an NFAT-specific reporter. Syndecan-1 and syndecan-4 were also able to induce TACI signaling in a similar manner. This is the first identification of ligands that selectively activate TACI without simultaneously triggering BCMA or BAFF-R. This finding may help explain the alternative outcomes of signaling from this family of receptors in B cells.