Stacey A. Bass

Dectin-1 and IL-17A Suppress Murine Asthma Induced by Aspergillus versicolor but Not Cladosporium cladosporioides Due to Differences in β-Glucan Surface Exposure

There is considerable evidence supporting a role for mold exposure in the pathogenesis and expression of childhood asthma. Aspergillus versicolor and Cladosporium cladosporioides are common molds that have been implicated in asthma. In a model of mold-induced asthma, mice were repeatedly exposed to either A. versicolor or C. cladosporioides spores. The two molds induced distinct phenotypes, and this effect was observed in both BALB/c and C57BL/6 strains. C. cladosporioides induced robust airway hyperresponsiveness (AHR), eosinophilia, and a predominately Th2 response, whereas A. versicolor induced a strong Th17 response and neutrophilic inflammation, but very mild AHR. Neutralization of IL-17A resulted in strong AHR and eosinophilic inflammation following A. versicolor exposure. In Dectin-1–deficient mice, A. versicolor exposure resulted in markedly attenuated IL-17A and robust AHR compared with wild-type mice. In contrast, C. cladosporioides induced AHR and eosinophilic inflammation independent of IL-17A and Dectin-1. A. versicolor, but not C. cladosporioides, spores had increased exposure of β-glucans on their surface and were able to bind Dectin-1. Thus, the host response to C. cladosporioides was IL-17A– and Dectin-1–independent, whereas Dectin-1– and IL-17A–dependent pathways were protective against the development of asthma after exposure to A. versicolor.

Downregulation of glutathione S-transferase pi in asthma contributes to enhanced oxidative stress

BACKGROUND Glutathione S-transferase pi (GSTPi) is the predominant redox regulator in the lung. Although evidence implicates an important role for GSTPi in asthma, the mechanism for this has remained elusive. OBJECTIVES We sought to determine how GSTPi is regulated in asthma and to elucidate its role in maintaining redox homeostasis. METHODS We elucidated the regulation of GSTPi in children with asthma and used murine models of asthma to determine the role of GSTPi in redox homeostasis. RESULTS Our findings demonstrate that GSTPi transcript levels are markedly downregulated in allergen- and IL-13-treated murine models of asthma through signal transducer and activator of transcription 6-dependent and independent pathways. Nuclear factor erythroid 2-related factor 2 was also downregulated in these models. The decrease in GSTPi expression was associated with decreased total glutathione S-transferase activity in the lungs of mice. Examination of cystine intermediates uncovered a functional role for GSTPi in regulating cysteine oxidation, whereby GSTPi-deficient mice exhibited increased oxidative stress (increase in percentage cystine) compared with wild-type mice after allergen challenge. GSTPi expression was similarly downregulated in children with asthma. CONCLUSIONS These data collectively suggest that downregulation of GSTPi after allergen challenge might contribute to the asthma phenotype because of disruption of redox homeostasis and increased oxidative stress. Furthermore, GSTPi might be an important therapeutic target for asthma, and evaluation of GSTPi expression might prove beneficial in identifying patients who would benefit from therapy targeting this pathway.

IL-13Rα2 Has a Protective Role in a Mouse Model of Cutaneous Inflammation

IL-13 is expressed in lesions of atopic dermatitis (AD) and has been associated with increased disease severity. IL-13 has two cognate receptors: IL-13Rα1 and IL-13Rα2. Although IL-13Rα2 expression is known to be induced in response to IL-13 in keratinocytes, its function in AD has never been evaluated. We characterized the loss of skin barrier function and the development of cutaneous inflammation in IL-13Rα2–null versus wild-type BALB/c mice following an epicutaneous allergen-sensitization/challenge model that shares similarities with human AD. Mice lacking IL-13Rα2 had significantly increased transepidermal water loss, cutaneous inflammation, peripheral eosinophilia, and IgG1 and IgE levels compared with wild-type mice. The rate of resolution of the cutaneous inflammation was not significantly altered in the IL-13Rα2–null mice. IL-13 induced expression of IL-13Rα2 in keratinocyte cell lines and primary human keratinocytes. Depletion of IL-13Rα2 in a keratinocyte cell line resulted in increased STAT6 signaling in response to IL-13. In conclusion, IL-13Rα2 serves a protective role in the pathogenesis of allergic inflammation and loss of skin barrier function in a mouse model of AD, suggesting that it may be an important endogenous regulator of IL-13–induced cutaneous inflammation in humans.

A nonredundant role for mouse Serpinb3a in the induction of mucus production in asthma

BACKGROUND Asthma is a major public health burden worldwide. Studies from our group and others have demonstrated that SERPINB3 and SERPINB4 are induced in patients with asthma; however, their mechanistic role in asthma has yet to be determined. OBJECTIVE To evaluate the role of Serpin3a, the murine homolog of SERPINB3 and SERPINB4, in asthma. METHODS We studied wild-type Balb/c and Serpinb3a-null mice in house dust mite or IL-13-induced asthma models and evaluated airway hyperresponsiveness, inflammation, and goblet cell hyperplasia. RESULTS Airway hyperresponsiveness and goblet cell hyperplasia were markedly attenuated in the Serpinb3a-null mice compared with the wild-type mice after allergen challenge, with minimal effects on inflammation. Expression of sterile alpha motif pointed domain containing v-ets avian erythroblastosis virus E26 oncogene homolog transcription factor (SPDEF), a transcription factor that mediates goblet cell hyperplasia, was decreased in the absence of Serpinb3a. IL-13-treated Serpinb3a-null mice showed attenuated airway hyperresponsiveness, inflammation, and mucus production. CONCLUSION Excessive mucus production and mucus plugging are key pathologic features of asthma, yet the mechanisms responsible for mucus production are not well understood. Our data reveal a novel nonredundant role for Serpinb3a in mediating mucus production through regulation of SPDEF expression. This pathway may be used to target mucus hypersecretion effectively.

Exacerbation of Allergen-Induced Eczema in TLR4- and TRIF-Deficient Mice

Despite its presence on resident skin cells, the role of TLR4 in skin diseases remains poorly understood. This is highly significant because the skin biome is rich with potential TLR4 agonists. We aimed to establish the contribution of TLR4 to atopic dermatitis and determine the mechanism by which TLR4 acts in an experimental model of atopic dermatitis. MyD88, TLR4, or Toll–IL-1R domain-containing adapter-inducing IFN-β (TRIF)–deficient and wild-type mice were epicutaneously exposed to Aspergillus fumigatus allergen over 3 wk. Impaired skin barrier function was assessed by measuring transepidermal water loss (TEWL). Skin levels of innate and adaptive genes were quantified. In an experimental model of atopic dermatitis, TEWL, allergic sensitization, and epidermal thickness were increased following cutaneous allergen exposure, and these were further enhanced in the absence of TLR4. Increased allergen-induced skin levels of innate (S100A8/A9, IL-1β, TNF-α, and CXCL2) and Th17 genes (IL-17A and IL-17F) were observed in TLR4-deficient mice compared with wild-type mice. The absence of MyD88 alleviated disease (decreased TEWL, skin thickness, proinflammatory cytokines), whereas TRIF deficiency exacerbated disease. In conclusion, signaling through the TLR4 and TRIF pathways limits skin barrier dysfunction, cutaneous allergic sensitization, and proinflammatory cytokine production.

SERPINB3/B4 Contributes to Early Inflammation and Barrier Dysfunction in an Experimental Murine Model of Atopic Dermatitis

Serine proteases are critical for epidermal barrier homeostasis and their aberrant expression and/or activity is associated with chronic skin diseases. Elevated levels of the serine protease inhibitors SERPINB3 and SERPINB4 are seen in patients with atopic dermatitis and psoriasis. However their mechanistic role in the skin is unknown. To evaluate the contribution of Serpinb3a (mouse homolog of SERPINB3 and SERPINB4) in atopic dermatitis, we examined the effect of topical Aspergillus fumigatus extract exposure in wild-type and Serpinb3a null mice on transepidermal water loss (TEWL), sensitization and inflammation. Allergen exposure induced Serpinb3a expression in the skin, along with increased TEWL, epidermal thickness, and skin inflammation, all of which were attenuated in the absence of Serpinb3a. Attenuated TEWL correlated with decreased expression of the pro-inflammatory marker S100A8. Silencing of SERPINB3/B4 in human keratinocytes decreased S100A8 expression supporting a role for SERPINB3/B4 in initiation of the acute inflammatory response. RNA-Seq analysis following allergen exposure identified a network of pro-inflammatory genes induced in the wild type mice that was absent in the Serpinb3a null mice. In conclusion, Serpinb3a deficiency attenuates barrier dysfunction and the early inflammatory response following cutaneous allergen exposure, supporting a role for Serpinb3a (mice) and SERPINB3/B4 (humans) early in atopic dermatitis.

IL-13 receptor α2 contributes to development of experimental allergic asthma

BACKGROUND IL-13 receptor α2 (IL-13Rα2) binds IL-13 with high affinity and modulates IL-13 responses. There are soluble and membrane forms of IL-13Rα2 generated by alternative splicing in mice, but human subjects express only the membrane form of IL-13Rα2 (memIL-13Rα2). OBJECTIVE We determined the role of memIL-13Rα2 in the development of allergic inflammation in mouse models of asthma. METHODS IL-13Rα2-deficient and memIL-13Rα2 lung epithelium-specific transgenic mice were challenged with house dust mite (HDM). Airway hyperresponsiveness (AHR) and inflammation were assessed based on the airway pressure-time index, bronchoalveolar lavage (BAL) cell counts, and lung histology. Mucus production was determined by means of periodic acid-Schiff staining of lung sections, Western blot analysis of chloride channel calcium activated 3 (CLCA3) expression in lung homogenates, and ELISA of Muc5ac in BAL fluid. The expression of cytokines and chemokines was determined by using RT-quantitative PCR. RESULTS In IL-13Rα2-deficient mice AHR and airway inflammation were attenuated compared with levels seen in wild-type mice after HDM challenge. Lung epithelial overexpression of memIL-13Rα2 in the IL-13Rα2-deficient mice reconstituted AHR and inflammation to levels similar to those observed in HDM-challenged wild-type mice. Mucus production was attenuated in lungs from HDM-treated IL-13Rα2-deficient mice, whereas lung epithelial overexpression of memIL-13Rα2 increased mucus production. Lung epithelial overexpression of memIL-13Rα2 had no effect on levels of the soluble form of IL-13Rα2 in serum or BAL fluid and did not affect IL-13-dependent signal transducer and activator of transcription 6 activation in the lungs. CONCLUSION These data collectively support a distinct role for memIL-13Rα2 in the lung and suggest that memIL-13Rα2 might contribute to allergic inflammation.

EGFR Signaling Blunts Allergen-Induced IL-6 Production and Th17 Responses in the Skin and Attenuates Development and Relapse of Atopic Dermatitis

Despite the important role for epidermal growth factor (EGF) in epithelial homeostasis and wound healing, it has not been investigated in atopic dermatitis (AD). We used AD animal models to explore the role of EGF in AD. In an acute AD model, skin transepidermal water loss was significantly attenuated in EGF-treated mice. Blockade of EGFR signaling genetically or pharmacologically confirms a protective role for EGFR signaling in AD. In a chronic/relapsing AD model, EGF treatment of mice with established AD resulted in an attenuation of AD exacerbation (skin epithelial thickness, cutaneous inflammation, and total and allergen specific IgE) following cutaneous allergen rechallenge. EGF treatment did not alter expression of skin barrier junction proteins or antimicrobial peptides in the AD model. However, EGF treatment attenuated allergen-induced expression of IL-17A, CXCL1, and CXCL2 and neutrophil accumulation in AD skin following cutaneous allergen exposure. IL-17A production was decreased in the in vitro restimulated skin-draining lymph node cells from the EGF-treated mice. Similarly, IL-17A was increased in waved-2 mice skin following allergen exposure. Whereas IL-6 and IL-1β expression was attenuated in the skin of EGF-treated mice, EGF treatment also suppressed allergen-induced IL-6 production by keratinocytes. Given the central role of IL-6 in priming Th17 differentiation in the skin, this effect of EGF on keratinocytes may contribute to the protective roles for EGFR in AD pathogenesis. In conclusion, our study provides evidence for a previously unrecognized protective role for EGF in AD and a new role for EGF in modulating IL-17 responses in the skin.

β-Glucan exacerbates allergic asthma independent of fungal sensitization and promotes steroid-resistant TH2/TH17 responses

Background: Allergic sensitization to fungi has been associated with asthma severity. As a result, it has been largely assumed that the contribution of fungi to allergic disease is mediated through their potent antigenicity. Objective: We sought to determine the mechanism by which fungi affect asthma development and severity. Methods: We integrated epidemiologic and experimental asthma models to explore the effect of fungal exposure on asthma development and severity. Results: We report that fungal exposure enhances allergen‐driven TH2 responses, promoting severe allergic asthma. This effect is independent of fungal sensitization and can be reconstituted with &bgr;‐glucan and abrogated by neutralization of IL‐17A. Furthermore, this severe asthma is resistant to steroids and characterized by mixed TH2 and TH17 responses, including IL‐13+IL‐17+CD4+ double‐producing effector T cells. Steroid resistance is dependent on fungus‐induced TH17 responses because steroid sensitivity was restored in IL‐17rc−/− mice. Similarly, in children with asthma, fungal exposure was associated with increased serum IL‐17A levels and asthma severity. Conclusion: Our data demonstrate that fungi are potent immunomodulators and have powerful effects on asthma independent of their potential to act as antigens. Furthermore, our results provide a strong rationale for combination treatment strategies targeting IL‐17A for this subgroup of fungus‐exposed patients with difficult‐to‐treat asthma.

Surface availability of beta-glucans is critical determinant of host immune response to Cladosporium cladosporioides

BACKGROUND It is well accepted that mold exposure is a major contributor to the development of asthma, and beta-glucans are often used as a surrogate for mold exposure in the environment. Beta-glucans are an important component of mold spores and are recognized by the immune system by their receptor, Dectin-1. Cladosporium cladosporioides spores have a high beta-glucan content, but the beta-glucans are not available on the surface of live spores. OBJECTIVE We sought to determine whether altering the exposure of beta-glucans in C cladosporioides through heat killing could alter the immune response through binding to Dectin-1. METHODS In a murine model of mold-induced asthma, mice were repeatedly exposed to either live or heat-killed C cladosporioides and the phenotype was determined by the measurement of airway hyperresponsiveness, airway inflammation, and cytokine production. Pro-inflammatory cytokines from dendritic cells were measured by using quantitative PCR and ELISA. RESULTS Live C cladosporioides induced robust airway hyperresponsiveness, eosinophilia, and a predominately TH2 response, while heat-killed C cladosporioides induced a strong TH17 response and neutrophilic inflammation, but very mild airway hyperresponsiveness. Heat killing of C cladosporioides spores effectively exposed beta-glucans on the surface of the spores and increased binding to Dectin-1. In the absence of Dectin-1, heat-killed spores induced a predominantly TH2 response analogous to live spores. Furthermore, the production of TH17-skewing IL-6, IL-23, and TNF-α by dendritic cells in response to heat-killed C cladosporioides was dependent on Dectin-1. CONCLUSIONS The host immune response to C cladosporioides is dependent on the surface availability of beta-glucans rather than the total beta-glucan content.