The anaphylatoxin C3a downregulates the Th2 response to epicutaneously introduced antigen

Mechanical injury to the skin results in activation of the complement component C3 and release of the anaphylatoxin C3a. C3a binds to a seven-transmembrane G protein-coupled receptor, C3aR. We used C3aR(-/-) mice to examine the role of C3a in a mouse model of allergic inflammation induced by epicutaneous sensitization with OVA. C3aR(-/-) mice exhibited an exaggerated Th2 response to epicutaneous but not to intraperitoneal sensitization with OVA, as evidenced by significantly elevated levels of serum OVA-specific IgG1 and significantly increased secretion of the Th2 cytokines IL-4, IL-5, and IL-10 by antigen-stimulated splenocytes. Presentation of OVA peptide by C3aR(-/-) APCs caused significantly more IL-4 and IL-5 secretion by T cells from OVA-T cell receptor (OVA-TCR) transgenic mice compared with presentation by WT APCs. C3a inhibited the ability of splenocytes, but not of highly purified T cells, to secrete Th2 cytokines in response to TCR ligation. This inhibition was mediated by IL-12 secreted by APCs in response to C3a. These results suggest that C3a-C3aR interactions inhibit the ability of APCs to drive Th2 cell differentiation in response to epicutaneously introduced antigen and may have important implications for allergic skin diseases.     

Epicutaneous challenge of orally immunized mice redirects antigen-specific gut-homing T cells to the skin

Patients with atopic dermatitis (AD) often suffer from food allergy and develop flares upon skin contact with food allergens. However, it is unclear whether T cells sensitized to allergens in the gut promote this skin inflammation. To address this question, we orally immunized WT mice and mice lacking the skin-homing chemokine receptor Ccr4 (Ccr4-/- mice) with OVA and then challenged them epicutaneously with antigen. Allergic skin inflammation developed in the WT mice but not in the mutants and was characterized by epidermal thickening, dermal infiltration by eosinophils and CD4+ T cells, and upregulation of Th2 cytokines. T cells purified from mesenteric lymph nodes (MLNs) of orally immunized WT mice transferred allergic skin inflammation to naive recipients cutaneously challenged with antigen, but this effect was lost in T cells purified from Ccr4-/- mice. In addition, the ability of adoptively transferred OVA-activated T cells to home to the skin following cutaneous OVA challenge was ablated in mice that lacked lymph nodes. These results indicate that cutaneous exposure to food antigens can reprogram gut-homing effector T cells in LNs to express skin-homing receptors, eliciting skin lesions upon food allergen contact in orally sensitized AD patients.     

Myeloid dendritic cells induce Th2 responses to inhaled antigen, leading to eosinophilic airway inflammation

The aim of this study was to investigate whether dendritic cells (DCs) can induce sensitization to aeroallergen in a mouse model of allergic asthma. Ovalbumin-pulsed (OVA-pulsed) or unpulsed myeloid DCs that were injected into the airways of naive mice migrated into the mediastinal lymph nodes. When challenged 2 weeks later with an aerosol of OVA, activated CD4 and CD8 lymphocytes, eosinophils, and neutrophils were recruited to the lungs of actively immunized mice. These CD4(+) lymphocytes produced predominantly IL-4 and IL-5 but also IFN-gamma, whereas CD8(+) lymphocytes produced predominantly IFN-gamma. Histological analysis revealed perivascular and peribronchial eosinophilic infiltrates and goblet cell hyperplasia. Studies in IL-4(-/-) and CD28(-/-) mice revealed that production of IL-4 by host cells and provision of costimulation to T cells by DCs were critical for inducing the response. Lung CD4(+) T cells strongly expressed the Th2 marker T1/ST2, and signaling through this molecule via a ligand expressed on DCs was essential for the establishment of airway eosinophilia. These data demonstrate that DCs in the airways induce sensitization to inhaled antigen and that molecules expressed on the surface of these cells are critical for the development of Th2-dependent airway eosinophilia.     

CCR3 is essential for skin eosinophilia and airway hyperresponsiveness in a murine model of allergic skin inflammation

The CC chemokine receptor 3 (CCR3) is expressed by eosinophils, mast cells, and Th2 cells. We used CCR3(-/-) mice to assess the role of CCR3 in a murine model of allergic skin inflammation induced by repeated epicutaneous sensitization with ovalbumin (OVA), and characterized by eosinophil skin infiltration, local expression of Th2 cytokines, and airway hyperresponsiveness (AHR) to inhaled antigen. Eosinophils and the eosinophil product major basic protein were absent from the skin of sham and OVA-sensitized CCR3(-/-) mice. Mast cell numbers and expression of IL-4 mRNA were normal in skin of CCR3(-/-) mice, suggesting that CCR3 is not important for infiltration of the skin by mast cells and Th2 cells. CCR3(-/-) mice produced normal levels of OVA-specific IgE, and their splenocytes secreted normal amounts of IL-4 and IL-5 following in vitro stimulation with OVA, indicating effective generation of systemic Th2 helper responses. Recruitment of eosinophils to lung parenchyma and bronchoalveolar lavage (BAL) fluid was severely impaired in CCR3(-/-) mice, which failed to develop AHR to methacholine following antigen inhalation. These results suggest that CCR3 plays an essential role in eosinophil recruitment to the skin and the lung and in the development of AHR.     

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.     

Resting Respiratory Tract Dendritic Cells Preferentially Stimulate T Helper Cell Type 2 (Th2) Responses and Require Obligatory Cytokine Signals for Induction of ?Th1 Immunity

Consistent with their role in host defense, mature dendritic cells (DCs) from central lymphoid organs preferentially prime for T helper cell type 1 (Th1)-polarized immunity. However, the “default” T helper response at mucosal surfaces demonstrates Th2 polarity, which is reflected in the cytokine profiles of activated T cells from mucosal lymph nodes. This study on rat respiratory tract DCs (RTDCs) provides an explanation for this paradox. We demonstrate that freshly isolated RTDCs are functionally immature as defined in vitro, being surface major histocompatibility complex (MHC) II ^lo, endocytosis^hi, and mixed lymphocyte reaction^lo, and these cells produce mRNA encoding interleukin (IL)-10. After ovalbumin (OVA)-pulsing and adoptive transfer, freshly isolated RTDCs preferentially stimulated Th2-dependent OVA-specific immunoglobulin (Ig)G₁ responses, and antigen-stimulated splenocytes from recipient animals produced IL-4 in vitro. However, preculture with granulocyte/macrophage colony stimulating factor increased their in vivo IgG priming capacity by 2–3 logs, inducing production of both Th1- and Th2-dependent IgG subclasses and high levels of IFN-γ by antigen-stimulated splenocytes. Associated phenotypic changes included upregulation of surface MHC II and B7 expression and IL-12 p35 mRNA, and downregulation of endocytosis, MHC II processing– associated genes, and IL-10 mRNA expression. Full expression of IL-12 p40 required additional signals, such as tumor necrosis factor α or CD40 ligand. These results suggest that the observed Th2 polarity of the resting mucosal immune system may be an inherent property of the resident DC population, and furthermore that mobilization of Th1 immunity relies absolutely on the provision of appropriate microenvironmental costimuli.     

Beta 2-microglobulin-dependent T cells are dispensable for allergen- induced T helper 2 responses

CD4+ and CD8+ alpha/beta+ T cells of the T helper cell (Th)2 phenotype produce the cytokines IL-4, IL-5, and IL-13 that promote IgE production and eosinophilic inflammation. IL-4 may play an important role in mediating the differentiation of antigenically naive alpha/beta+ T cells into Th2 cells. Murine NK1.1+ (CD4+ or CD4-CD8-) alpha/beta+ T cells comprise a beta 2-microglobulin (beta 2m)-dependent cell population that rapidly produces IL-4 after cell activation in vitro and in vivo and has been proposed as a source of IL-4 for Th2 cell differentiation. alpha/beta+ CD8+ T cells, most of which require beta 2m for their development, have also been proposed as positive regulators of allergen-induced Th2 responses. We tested whether beta 2m- dependent T cells were essential for Th2 cell-mediated allergic reactions by treating wild-type, beta 2m-deficient (beta 2m -/-), and IL-4-deficient (IL-4 -/-) mice of the C57BL/6 genetic background with ovalbumin (OVA), using a protocol that induces robust allergic pulmonary disease in wild-type mice. OVA-treated beta 2m -/- mice had circulating levels of total and OVA-specific IgE, pulmonary eosinophilia, and expression of IL-4, IL-5, and IL-13 mRNA in bronchial lymph node tissue similar to that of OVA-treated wild-type mice. In contrast, these responses in OVA-treated IL-4 -/- mice were all either undetectable or markedly reduced compared with wild-type mice, confirming that IL-4 was required in this allergic model. These results indicate that the NK1.1+ alpha/beta+ T cell population, as well as other beta 2m-dependent populations, such as most peripheral alpha/beta+ CD8+ T cells, are dispensable for the Th2 pulmonary response to protein allergens.     

The protective role of TLR6 in a mouse model of asthma is mediated by IL-23 and IL-17A

TLRs are a family of receptors that mediate immune system pathogen recognition. In the respiratory system, TLR activation has both beneficial and deleterious effects in asthma. For example, clinical data indicate that TLR6 activation exerts protective effects in asthma. Here, we explored the mechanism or mechanisms through which TLR6 mediates this effect using mouse models of Aspergillus fumigatus-induced and house dust mite antigen-induced (HDM antigen-induced) chronic asthma. Tlr6-/- mice with fungal- or HDM antigen-induced asthma exhibited substantially increased airway hyperresponsiveness, inflammation, and remodeling compared with WT asthmatic groups. Surprisingly, whole-lung levels of IL-23 and IL-17 were markedly lower in Tlr6-/- versus WT asthmatic mice. Tlr6-/- DCs generated less IL-23 upon activation with lipopolysaccharide, zymosan, or curdlan. Impaired IL-23 generation in Tlr6-/- mice also corresponded with lower levels of expression of the pathogen-recognition receptor dectin-1 and expansion of Th17 cells both in vivo and in vitro. Exogenous IL-23 treatment of asthmatic Tlr6-/- mice restored IL-17A production and substantially reduced airway hyperresponsiveness, inflammation, and lung fungal burden compared with that in untreated asthmatic Tlr6-/- mice. Together, our data demonstrate that TLR6 activation is critical for IL-23 production and Th17 responses, which both regulate the allergic inflammatory response in chronic fungal-induced asthma. Thus, therapeutics targeting TLR6 activity might prove efficacious in the treatment of clinical asthma.     

The H1 histamine receptor regulates allergic lung responses

Histamine, signaling via the type 1 receptor (H1R), has been shown to suppress Th2 cytokine production by in vitro cultured T cells. We examined the role of H1R in allergic inflammation in vivo using a murine asthma model. Allergen-stimulated splenic T cells from sensitized H1R-/- mice exhibited enhanced Th2 cytokine production. Despite this Th2 bias, allergen-challenged H1R-/- mice exhibited diminished lung Th2 cytokine mRNA levels, airway inflammation, goblet cell metaplasia, and airway hyperresponsiveness (AHR). Restoration of pulmonary Th2 cytokines in H1R-/- mice by intranasal IL-4 or IL-13 restored inflammatory lung responses and AHR. Further investigation revealed that histamine acts as a T cell chemotactic factor and defective T cell trafficking was responsible for the absence of lung inflammation. Cultured T cells migrated in response to histamine in vitro, but this was ablated by blockade of H1R but not H2R. In vivo, allergen-specific WT but not H1R-/- CD4+ T cells were recruited to the lungs of naive recipients following inhaled allergen challenge. H1R-/- T cells failed to confer airway inflammation or AHR observed after transfer of WT T cells. Our data establish a role for histamine and H1R in promoting the migration of Th2 cells into sites of allergen exposure.     

CCR6 is required for IL-23–induced psoriasis-like inflammation in mice

Psoriasis is a common immune-mediated chronic inflammatory skin disorder, but the mechanisms of pathogenesis are still poorly understood. IL-23 is expressed in psoriatic skin, and IL-23 injection produces IL-22–dependent psoriasiform changes in mouse skin. Th17 cells produce IL-22 and display CCR6, the CCL20 receptor; CCR6⁺ T cells and CCL20 are abundant in psoriatic skin. We investigated a possible role for CCR6 in recruiting Th17 cells and producing psoriasiform pathology by injecting IL-23 into the skin of WT and Ccr6^–/– mice. Unlike for WT mice, IL-23–injected ears of Ccr6^–/– mice showed neither substantial epidermal/dermal changes nor increased Il22 mRNA expression. However, injection of IL-22 yielded equivalent psoriasiform changes in WT and Ccr6^–/– mice. Surprisingly, IL-23–injected ears of WT and Ccr6^–/– mice contained similar numbers of Th cells able to make IL-17A and/or IL-22. Furthermore, in ears of Rag1^–/– mice, IL-23 initially induced skin changes and levels of Il22 mRNA that were indistinguishable from WT mice, revealing at least one non–T cell source for IL-22. We conclude that CCR6 is essential in a model of IL-23–induced, IL-22–mediated dermatitis, which develops in sequential T cell–independent and T cell–dependent phases. These findings reveal an expanded role for CCR6 in IL-23–related responses and identify CCR6 as a potential therapeutic target in psoriasis.     

Interleukin-17 is a negative regulator of established allergic asthma

T helper (Th)17 cells producing interleukin (IL)-17 play a role in autoimmune and allergic inflammation. Here, we show that IL-23 induces IL-17 in the lung and IL-17 is required during antigen sensitization to develop allergic asthma, as shown in IL-17R-deficient mice. Since IL-17 expression increased further upon antigen challenge, we addressed its function in the effector phase. Most strikingly, neutralization of IL-17 augmented the allergic response in sensitized mice. Conversely, exogenous IL-17 reduced pulmonary eosinophil recruitment and bronchial hyperreactivity, demonstrating a novel regulatory role of IL-17. Mechanistically, IL-17 down modulated eosinophil-chemokine eotaxin (CCL11) and thymus- and activation-regulated chemokine/CCL17 (TARC) in lungs in vivo and ex vivo upon antigen restimulation. In vitro, IL-17 reduced TARC production in dendritic cells (DCs)-the major source of TARC-and antigen uptake by DCs and IL-5 and IL-13 production in regional lymph nodes. Furthermore, IL-17 is regulated in an IL-4-dependent manner since mice deficient for IL-4Ralpha signaling showed a marked increase in IL-17 concentration with inhibited eosinophil recruitment. Therefore, endogenous IL-17 is controlled by IL-4 and has a dual role. Although it is essential during antigen sensitization to establish allergic asthma, in sensitized mice IL-17 attenuates the allergic response by inhibiting DCs and chemokine synthesis.     

In vivo depletion of lung CD11c+ dendritic cells during allergen challenge abrogates the characteristic features of asthma

Although dendritic cells (DCs) play an important role in sensitization to inhaled allergens, their function in ongoing T helper (Th)2 cell-mediated eosinophilic airway inflammation underlying bronchial asthma is currently unknown. Here, we show in an ovalbumin (OVA)-driven murine asthma model that airway DCs acquire a mature phenotype and interact with CD4(+) T cells within sites of peribronchial and perivascular inflammation. To study whether DCs contributed to inflammation, we depleted DCs from the airways of CD11c-diphtheria toxin (DT) receptor transgenic mice during the OVA aerosol challenge. Airway administration of DT depleted CD11c(+) DCs and alveolar macrophages and abolished the characteristic features of asthma, including eosinophilic inflammation, goblet cell hyperplasia, and bronchial hyperreactivity. In the absence of CD11c(+) cells, endogenous or adoptively transferred CD4(+) Th2 cells did not produce interleukin (IL)-4, IL-5, and IL-13 in response to OVA aerosol. In CD11c-depleted mice, eosinophilic inflammation and Th2 cytokine secretion were restored by adoptive transfer of CD11c(+) DCs, but not alveolar macrophages. These findings identify lung DCs as key proinflammatory cells that are necessary and sufficient for Th2 cell stimulation during ongoing airway inflammation.     

Defective IL-10 signaling in hyper-IgE syndrome results in impaired generation of tolerogenic dendritic cells and induced regulatory T cells

Hyper-IgE syndrome (HIES) is a primary immunodeficiency characterized by recurrent staphylococcal infections and atopic dermatitis associated with elevated serum IgE levels. Although defective differentiation of IL-17-producing CD4(+) T cells (Th17) partly accounts for the susceptibility to staphylococcal skin abscesses and pneumonia, the pathogenesis of atopic manifestations in HIES still remains an enigma. In this study, we examined the differentiation and function of Th1, Th2, regulatory T cells (T(reg) cells), and dendritic cells (DCs) in HIES patients carrying either STAT3 or TYK2 mutations. Although the in vitro differentiation of Th1 and Th2 cells and the number and function of T(reg) cells in the peripheral blood were normal in HIES patients with STAT3 mutations, primary and monocyte-derived DCs showed defective responses to IL-10 and thus failed to become tolerogenic. When treated with IL-10, patient DCs showed impaired up-regulation of inhibitory molecules on their surface, including PD-L1 and ILT-4, compared with control DCs. Moreover, IL-10-treated DCs from patients displayed impaired ability to induce the differentiation of naive CD4(+) T cells to FOXP3(+) induced T(reg) cells (iT(reg) cells). These results suggest that the defective generation of IL-10-induced tolerogenic DCs and iT(reg) cells may contribute to inflammatory changes in HIES.     

Altered cutaneous immune parameters in transgenic mice overexpressing viral IL-10 in the epidermis

IL-10 is a pleiotropic cytokine that inhibits several immune parameters, including Th1 cell-mediated immune responses, antigen presentation, and antigen-specific T cell proliferation. Recent data implicate IL-10 as a mediator of suppression of cell-mediated immunity induced by exposure to UVB radiation (280-320 nm). To investigate the effects of IL-10 on the cutaneous immune system, we engineered transgenic mice that overexpress viral IL-10 (vIL-10) in the epidermis. vIL-10 transgenic mice demonstrated a reduced number of I-A(+) epidermal and dermal cells and fewer I-A(+) hapten-bearing cells in regional lymph nodes after hapten painting of the skin. Reduced CD80 and CD86 expression by I-A(+) epidermal cells was also observed. vIL-10 transgenic mice demonstrated a smaller delayed-type hypersensitivity response to allogeneic cells upon challenge but had normal contact hypersensitivity to an epicutaneously applied hapten. Fresh epidermal cells from vIL-10 transgenic mice showed a decreased ability to stimulate allogeneic T cell proliferation, as did splenocytes. Additionally, chronic exposure of mice to UVB radiation led to the development of fewer skin tumors in vIL-10 mice than in WT controls, and vIL-10 transgenic mice had increased splenic NK cell activity against YAC-1targets. These findings support the concept that IL-10 is an important regulator of cutaneous immune function.     

Eosinophil-derived neurotoxin acts as an alarmin to activate the TLR2-MyD88 signal pathway in dendritic cells and enhances Th2 immune responses

Eosinophil-derived neurotoxin (EDN) is an eosinophil granule-derived secretory protein with ribonuclease and antiviral activity. We have previously shown that EDN can induce the migration and maturation of dendritic cells (DCs). Here, we report that EDN can activate myeloid DCs by triggering the Toll-like receptor (TLR)2-myeloid differentiation factor 88 signaling pathway, thus establishing EDN as an endogenous ligand of TLR2. EDN activates TLR2 independently of TLR1 or TLR6. When mice were immunized with ovalbumin (OVA) together with EDN or with EDN-treated OVA-loaded DCs, EDN enhanced OVA-specific T helper (Th)2-biased immune responses as indicated by predominant production of OVA-specific interleukin (IL)-5, IL-6, IL-10, and IL-13, as well as higher levels of immunoglobulin (Ig)G1 than IgG2a. Based on its ability to serve as a chemoattractant and activator of DCs, as well as the capacity to enhance antigen-specific immune responses, we consider EDN to have the properties of an endogenous alarmin that alerts the adaptive immune system for preferential enhancement of antigen-specific Th2 immune responses.     

Changes in immune responses to antigen applied to tape-stripped skin with CpG-oligodeoxynucleotide in mice.

CpG-oligodeoxynucleotide (CpG-ODN) plays a critical role in immunity via the augmentation of Th1 and suppression of Th2 responses. We examined here the effect of CpG-ODN on the immune response to an antigen applied to tape-stripped mouse skin by evaluating the production of cytokines and Ig isotypes. Confocal laser scanning microscopy revealed that the model antigen, OVA, and CpG-ODN easily penetrated the tape-stripped skin. Co-administration of CpG-ODN and OVA to the disrupted skin elicited an antigen-specific Th1-predominant immune response and enhanced the production of Th1-type cytokines, IL-12 and IFN-gamma. On the other hand, the production of a Th2-type cytokine, IL-4, was drastically suppressed. Cytokine production was supported by the expression of mRNA in the draining lymph node. In terms of antigen-specific antibody production, the level of IgG2a which is regulated by IFN-gamma was increased by CpG-ODN, but IgE production regulated by IL-4 was suppressed. Furthermore, administration of CpG-ODN via the skin drastically attenuated the production of IgE in mice undergoing IgE-type immune response. Administration of CpG-ODN through the skin may shift the immune response from Th2 to Th1-like response. These results suggested that administration of CpG-ODN via skin is a simple strategy for patients with diseases like AD, which is characterized by Th2-dominated inflammation.     

B lymphocytes regulate dendritic cell (DC) function in vivo: increased interleukin 12 production by DCs from B cell-deficient mice results in T helper cell type 1 deviation

Increasing evidence indicates that dendritic cells (DCs) are the antigen-presenting cells of the primary immune response. However, several reports suggest that B lymphocytes could be required for optimal T cell sensitization. We compared the immune responses of wild-type and B cell-deficient (muMT) mice, induced by antigen emulsified in adjuvant or pulsed on splenic dendritic cells. Our data show that lymph node cells from both control and muMT animals were primed, but each released distinct cytokine profiles. Lymph node T cells from control animals secreted interferon (IFN)-gamma, interleukin (IL)-2, and IL-4, whereas those from muMT mice produced IFN-gamma and IL-2 but no IL-4. To test whether B cells may influence the T helper cell type 1 (Th1)/Th2 balance by affecting the function of DCs, we immunized mice by transferring antigen-pulsed DCs from wild-type or mutant mice. Injection of control DCs induced the secretion of IL-4, IFN-gamma, and IL-2, whereas administration of DCs from muMT animals failed to sensitize cells to produce IL-4. Analysis of IL-12 production revealed that DCs from muMT mice produce higher levels of IL-12p70 than do DCs from wild-type animals. These data suggest that B lymphocytes regulate the capacity of DCs to promote IL-4 secretion, possibly by downregulating their secretion of IL-12, thereby favoring the induction of a nonpolarized immune response.