Functional role of glial-derived neurotrophic factor in a mixed allergen murine model of asthma.

Our previous study showed that glial-derived neurotrophic factor (GDNF) expression is upregulated in asthmatic human lungs, and GDNF regulates calcium responses through its receptor GDNF family receptor α1 (GFRα1) and RET receptor in human airway smooth muscle (ASM) cells. In this study, we tested the hypothesis that airway GDNF contributes to airway hyperreactivity (AHR) and remodeling using a mixed allergen mouse model. Adult C57BL/6J mice were intranasally exposed to mixed allergens (ovalbumin, Aspergillus, Alternaria, house dust mite) over 4 wk with concurrent exposure to recombinant GDNF, or extracellular GDNF chelator GFRα1-Fc. Airway resistance and compliance to methacholine were assessed using FlexiVent. Lung expression of GDNF, GFRα1, RET, collagen, and fibronectin was examined by RT-PCR and histology staining. Allergen exposure increased GDNF expression in bronchial airways including ASM and epithelium. Laser capture microdissection of the ASM layer showed increased mRNA for GDNF, GFRα1, and RET in allergen-treated mice. Allergen exposure increased protein expression of GDNF and RET, but not GFRα1, in ASM. Intranasal administration of GDNF enhanced baseline responses to methacholine but did not consistently potentiate allergen effects. GDNF also induced airway thickening, and collagen deposition in bronchial airways. Chelation of GDNF by GFRα1-Fc attenuated allergen-induced AHR and particularly remodeling. These data suggest that locally produced GDNF, potentially derived from epithelium and/or ASM, contributes to AHR and remodeling relevant to asthma.NEW & NOTEWORTHY Local production of growth factors within the airway with autocrine/paracrine effects can promote features of asthma. Here, we show that glial-derived neurotrophic factor (GDNF) is a procontractile and proremodeling factor that contributes to allergen-induced airway hyperreactivity and tissue remodeling in a mouse model of asthma. Blocking GDNF signaling attenuates allergen-induced airway hyperreactivity and remodeling, suggesting a novel approach to alleviating structural and functional changes in the asthmatic airway.

Moderate hyperoxia induces senescence in developing human lung fibroblasts.

Hyperoxia exposure in premature infants increases the risk of subsequent lung diseases, such as asthma and bronchopulmonary dysplasia. Fibroblasts help maintain bronchial and alveolar integrity. Thus, understanding mechanisms by which hyperoxia influences fibroblasts is critical. Cellular senescence is increasingly recognized as important to the pathophysiology of multiple diseases. We hypothesized that clinically relevant moderate hyperoxia (<50% O2) induces senescence in developing fibroblasts. Using primary human fetal lung fibroblasts, we investigated effects of 40% O2 on senescence, endoplasmic reticulum (ER) stress, and autophagy pathways. Fibroblasts were exposed to 21% or 40% O2 for 7 days with etoposide as a positive control to induce senescence, evaluated by morphological changes, ß-galactosidase activity, and DNA damage markers. Senescence-associated secretory phenotype (SASP) profile of inflammatory and profibrotic markers was further assessed. Hyperoxia decreased proliferation but increased cell size. SA-ß-gal activity and DNA damage response, cell cycle arrest in G2/M phase, and marked upregulation of phosphorylated p53 and p21 were noted. Reduced autophagy was noted with hyperoxia. mRNA expression of proinflammatory and profibrotic factors (TNF-α, IL-1, IL-8, MMP3) was elevated by hyperoxia or etoposide. Hyperoxia increased several SASP factors (PAI-1, IL1-α, IL1-ß, IL-6, LAP, TNF-α). The secretome of senescent fibroblasts promoted extracellular matrix formation by naïve fibroblasts. Overall, we demonstrate that moderate hyperoxia enhances senescence in primary human fetal lung fibroblasts with reduced autophagy but not enhanced ER stress. The resulting SASP is profibrotic and may contribute to abnormal repair in the lung following hyperoxia.

Ozone-induced eosinophil recruitment to airways is altered by antigen sensitization and tumor necrosis factor-α blockade.

Ozone is an atmospheric pollutant that causes lung inflammation and airway hyperresponsiveness. Ozone's effects occur in two distinct phases that are mediated by different populations of eosinophils. In the acute phase 1 day after exposure, mature airway-resident eosinophils alter parasympathetic nerve function that results in airway hyperresponsiveness. At this time point, the severity of hyperresponsiveness correlates with the number of eosinophils in close proximity to airway nerves, but not with eosinophils in bronchoalveolar lavage. Three days later, newly divided eosinophils are recruited to airways by a tumor necrosis factor-α-dependent mechanism. These new eosinophils paradoxically attenuate ozone-induced airway hyperresponsiveness. Ozone's effects on airway tissue eosinophils and nerve-associated eosinophils 3 days after exposure are unknown. Thus, we tested ozone's effects on eosinophils in airway subepithelium and around airway nerves 1 and 3 days after ozone in nonsensitized and ovalbumin-sensitized guinea pigs with or without the tumor necrosis factor-α antagonist, etanercept, and compared changes in eosinophils with ozone-induced airway hyperresponsiveness. More eosinophils were present in small, noncartilaginous airways and along small airway nerves compared to large cartilaginous airways in all treatment groups. The number of airway and nerve-associated eosinophils were unaffected 1 day after ozone exposure, whereas significantly fewer airway eosinophils were present 3 days later. Airway and nerve-associated eosinophils were also decreased in small airways 3 days after ozone in sensitized animals. These changes were blocked by etanercept. Airway eosinophils, but not nerve-associated or bronchoalveolar lavage eosinophils correlated with airway hyperresponsiveness 3 days after ozone. Our findings indicate ozone causes persistent alterations in airway eosinophils and reinforce the importance of characterizing eosinophils' effects within distinct airway compartments.

Newly divided eosinophils limit ozone-induced airway hyperreactivity in nonsensitized guinea pigs.

Ozone causes vagally mediated airway hyperreactivity and recruits inflammatory cells, including eosinophils, to lungs, where they mediate ozone-induced hyperreactivity 1 day after exposure but are paradoxically protective 3 days later. We aimed to test the role of newly divided eosinophils in ozone-induced airway hyperreactivity in sensitized and nonsensitized guinea pigs. Nonsensitized and sensitized guinea pigs were treated with 5-bromo-2-deoxyuridine (BrdU) to label newly divided cells and were exposed to air or ozone for 4 h. Later (1 or 3 days later), vagally induced bronchoconstriction was measured, and inflammatory cells were harvested from bone marrow, blood, and bronchoalveolar lavage. Ozone induced eosinophil hematopoiesis. One day after ozone, mature eosinophils dominate the inflammatory response and potentiate vagally induced bronchoconstriction. However, by 3 days, newly divided eosinophils have reached the lungs, where they inhibit ozone-induced airway hyperreactivity because depleting them with antibody to IL-5 or a TNF-α antagonist worsened vagally induced bronchoconstriction. In sensitized guinea pigs, both ozone-induced eosinophil hematopoiesis and subsequent recruitment of newly divided eosinophils to lungs 3 days later failed to occur. Thus mature eosinophils dominated the ozone-induced inflammatory response in sensitized guinea pigs. Depleting these mature eosinophils prevented ozone-induced airway hyperreactivity in sensitized animals. Ozone induces eosinophil hematopoiesis and recruitment to lungs, where 3 days later, newly divided eosinophils attenuate vagally mediated hyperreactivity. Ozone-induced hematopoiesis of beneficial eosinophils is blocked by a TNF-α antagonist or by prior sensitization. In these animals, mature eosinophils are associated with hyperreactivity. Thus interventions targeting eosinophils, although beneficial in atopic individuals, may delay resolution of airway hyperreactivity in nonatopic individuals.

Protective Role of Eosinophils and TNFa after Ozone Inhalation.

Introduction: Exposure to ozone induces deleterious responses in the airways that include shortness of breath, inflammation, and bronchoconstriction. People with asthma have increased airway sensitivity to ozone and other irritants. Dr. Allison Fryer and colleagues addressed how exposure to ozone affects the immune and physiological responses in guinea pigs. Guinea pigs are considered a useful animal model for studies of respiratory and physiological responses in humans; their response to airborne allergens is similar to that in humans and shares some features of allergic asthma. Fryer and colleagues had previously observed that within 24 hours of exposure, ozone not only induced bronchoconstriction but also stimulated the production of new cells in the bone marrow, where all white blood cells develop. As a result of ozone exposure, increased numbers of newly synthesized white blood cells, particularly eosinophils, moved into the blood and lungs. The central hypothesis of the current study was that newly synthesized eosinophils recruited to the lungs 3 days after ozone exposure were beneficial to the animals because they reduced ozoneinduced bronchoconstriction. The investigators also hypothesized that the beneficial effect seen in normal (nonsensitized) animals was lost in animals that had been injected with an allergen, ovalbumin (sensitized). They also planned to explore the effects of inhibitors of certain cytokines (cellsignaling molecules). Immune responses in sensitized animals are dominated by a Th2 pattern, which is characterized by the synthesis of cytokines (interleukin [IL]-4, IL-5, and IL-13) and the Th2 subset of CD4+ T lymphocytes and the cells they activate (predominantly eosinophils, and B lymphocytes that switch to making immunoglobulin E [IgE]). Thus, sensitized animals were used as a model of allergic humans, whose immune responses tend to be dominated by IgE. Approach: Fryer and colleagues exposed normal and sensitized (allergic) guinea pigs to 2 ppm ozone or filtered air for 4 hours and measured changes in cell numbers and airway responses 1 or 3 days later. They counted the numbers of eosinophils and other white blood cells (macrophages, neutrophils, and lymphocytes) in bone marrow, blood, and bronchoalveolar lung lavage fluid. The investigators also measured important physiological responses, including bronchoconstriction. Some animals were pretreated with etanercept and monoclonal anti-IL-5, which block tumor necrosis factor-a (TNFa) and IL-5, respectively. TNFa and IL-5 blockers have been used to treat patients with asthma. A key feature of the study was a technique to distinguish which white blood cells were synthesized after exposure from those that already existed, by injecting animals with bromodeoxyuridine (BrdU). BrdU is a thymidine analogue that is incorporated into the DNA of dividing cells, serving as a marker of newly produced cells. Therefore, a snapshot can be obtained of the proportion of newly synthesized (BrdU-positive) versus pre-existing (BrdU-negative) cell types. Key results: 1. Allergic and normal animals differed in the time course of bronchoconstriction and changes in cell types after ozone exposure. In normal animals, bronchoconstriction increased substantially at day 1 but decreased by day 3 after ozone exposure. In contrast, in allergic animals bronchoconstriction remained high at day 3. Ozone also increased the percentage of newly formed, BrdU2 positive eosinophils in the bone marrow and lungs of normal but not allergic animals. 2. Pretreatment with the TNFa blocker etanercept had complex effects, which differed between normal and allergic animals. In normal animals, etanercept decreased ozone-induced new synthesis of eosinophils in the bone marrow and blocked eosinophil migration to the lung; it also increased bronchoconstriction at day 3 (relative to day 1 without etanercept). In allergic animals, etanercept had no effect on any cell type in the bone marrow or lung after exposure to ozone and did not change bronchoconstriction compared with allergic animals not treated with etanercept. Etanercept tended to increase the numbers of blood monocytes and lymphocytes in air- and ozone-exposed normal and allergic animals at day 3, but had no effect on eosinophils in blood at this time point. This was one of the few statistically significant findings in the blood of exposed animals in the study. 3. Anti-IL-5 reduced bronchoconstriction at day 3 after exposure of allergic animals to ozone. In contrast, bronchoconstriction was greatly increased in normal animals treated with anti-IL-5. Conclusions: Fryer and colleagues explored the airway and cellular responses in guinea pigs exposed to ozone. The HEI Review Committee, which conducted an independent review of the study, agreed that the findings supported the authors' hypothesis (1) that exposure to ozone stimulates production of eosinophils in bone marrow, (2) that these newly formed eosinophils migrate to the lungs, and (3) that those eosinophils play a delayed but potentially beneficial role in reducing ozone-induced inflammation in the airways of healthy normal animals, but not in allergen-sensitized animals. The Committee also agreed that guinea pigs were a good model for studying responses to an allergen, because a major subtype of asthma (the high Th2 or allergic type) is associated with high levels of eosinophils in the blood. A novel finding was that the TNFa blocker etanercept decreased ozone-induced formation of eosinophils in the bone marrow and blocked eosinophil migration to the lung in normal animals. However, because injecting etanercept had little effect on eosinophils and did not decrease bronchoconstriction in allergic guinea pigs, the potential for treating patients with allergic asthma with TNFa blockers is uncertain. This is consistent with the poor performance of TNFa blockers in clinical studies of asthma treatment. Blocking the cytokine IL-5 with an anti-IL-5 antibody substantially decreased bronchoconstriction in sensitized animals. This suggests that therapies targeting IL-5 and eosinophils would be promising in at least some types of asthma. The Committee expressed caution toward experiments with cytokine blockers, both in animal models and humans, because such blockers are often not specific to a particular cell type and may differ at different sites in the body. Without further detailed confirmation of the effects of the blockers, interpreting these experiments can be challenging. The Committee concluded that the study by Fryer and colleagues raises several intriguing directions for future research, including exploring ways in which newly formed eosinophils differ from pre-existing ones, and how such findings apply to humans with allergy or asthma.