Effects of corticosteroid treatment on airway inflammation, mechanics, and hyperpolarized ³He magnetic resonance imaging in an allergic mouse model.

The purpose of this study was to assess the effects of corticosteroid therapy on a murine model of allergic asthma using hyperpolarized (3)He magnetic resonance imaging (MRI) and respiratory mechanics measurements before, during, and after methacholine (MCh) challenge. Three groups of mice were prepared, consisting of ovalbumin sensitized/ovalbumin challenged (Ova/Ova, n = 5), Ova/Ova challenged but treated with the corticosteroid dexamethasone (Ova/Ova+Dex, n = 3), and ovalbumin-sensitized/saline-challenged (Ova/PBS, n = 4) control animals. All mice underwent baseline 3D (3)He MRI, then received a MCh challenge while 10 2D (3)He MR images were acquired for 2 min, followed by post-MCh 3D (3)He MRI. Identically treated groups underwent respiratory mechanics evaluation (n = 4/group) and inflammatory cell counts (n = 4/group). Ova/Ova animals exhibited predominantly large whole lobar defects at baseline, with significantly higher ventilation defect percentage (VDP = 19 ± 4%) than Ova/PBS (+2 ± 1%, P = 0.01) animals. Such baseline defects were suppressed by dexamethasone (0%, P = 0.009). In the Ova/Ova group, MCh challenge increased VDP on both 2D (+30 ± 8%) and 3D MRI scans (+14 ± 2%). MCh-induced VDP changes were diminished in Ova/Ova+Dex animals on both 2D (+21 ± 9%, P = 0.63) and 3D scans (+7 ± 2%, P = 0.11) and also in Ova/PBS animals on 2D (+6 ± 3%, P = 0.07) and 3D (+4 ± 1%, P = 0.01) scans. Because MCh challenge caused near complete cessation of ventilation in four of five Ova/Ova animals, even as large airways remained patent, this implies that small airway (<188 µm) obstruction predominates in this model. This corresponds with respiratory mechanics observations that MCh challenge significantly increases elastance and tissue damping but only modestly affects Newtonian airway resistance.

Maternal diesel inhalation increases airway hyperreactivity in ozone-exposed offspring.

Air pollutant exposure is linked with childhood asthma incidence and exacerbations, and maternal exposure to airborne pollutants during pregnancy increases airway hyperreactivity (AHR) in offspring. To determine if exposure to diesel exhaust (DE) during pregnancy worsened postnatal ozone-induced AHR, timed pregnant C57BL/6 mice were exposed to DE (0.5 or 2.0 mg/m(3)) 4 hours daily from Gestation Day 9-17, or received twice-weekly oropharyngeal aspirations of the collected DE particles (DEPs). Placentas and fetal lungs were harvested on Gestation Day 18 for cytokine analysis. In other litters, pups born to dams exposed to air or DE, or to dams treated with aspirated diesel particles, were exposed to filtered air or 1 ppm ozone beginning the day after birth, for 3 hours per day, 3 days per week for 4 weeks. Additional pups were monitored after a 4-week recovery period. Diesel inhalation or aspiration during pregnancy increased levels of placental and fetal lung cytokines. There were no significant effects on airway leukocytes, but prenatal diesel augmented ozone-induced elevations of bronchoalveolar lavage cytokines at 4 weeks. Mice born to the high-concentration diesel-exposed dams had worse ozone-induced AHR, which persisted in the 4-week recovery animals. Prenatal diesel exposure combined with postnatal ozone exposure also worsened secondary alveolar crest development. We conclude that maternal inhalation of DE in pregnancy provokes a fetal inflammatory response that, combined with postnatal ozone exposure, impairs alveolar development, and causes a more severe and long-lasting AHR to ozone exposure.

Ozone inhalation promotes CX3CR1-dependent maturation of resident lung macrophages that limit oxidative stress and inflammation.

Inhalation of ambient ozone alters populations of lung macrophages. However, the impact of altered lung macrophage populations on the pathobiology of ozone is poorly understood. We hypothesized that subpopulations of macrophages modulate the response to ozone. We exposed C57BL/6 mice to ozone (2 ppm × 3 h) or filtered air. At 24 h after exposure, the lungs were harvested and digested and the cells underwent flow cytometry. Analysis revealed a novel macrophage subset present in ozone-exposed mice, which were distinct from resident alveolar macrophages and identified by enhanced Gr-1(+) expression [Gr-1 macrophages (Gr-1 Macs)]. Further analysis showed that Gr-1(+) Macs exhibited high expression of MARCO, CX3CR1, and NAD(P)H:quinone oxioreductase 1. Gr-1(+) Macs were present in the absence of CCR2, suggesting that they were not derived from a CCR2-dependent circulating intermediate. Using PKH26-PCL to label resident phagocytic cells, we demonstrated that Gr-1 Macs were derived from resident lung cells. This new subset was diminished in the absence of CX3CR1. Interestingly, CX3CR1-null mice exhibited enhanced responses to ozone, including increased airway hyperresponsiveness, exacerbated neutrophil influx, accumulation of 8-isoprostanes and protein carbonyls, and increased expression of cytokines (CXCL2, IL-1ß, IL-6, CCL2, and TNF-α). Our results identify a novel subset of lung macrophages, which are derived from a resident intermediate, are dependent upon CX3CR1, and appear to protect the host from the biological response to ozone.

S-nitrosoglutathione supplementation to ovalbumin-sensitized and -challenged mice ameliorates methacholine-induced bronchoconstriction.

S-nitrosoglutathione (GSNO) is an endogenous bronchodilator present in micromolar concentrations in airway lining fluid. Airway GSNO levels decrease in severe respiratory failure and asthma, which is attributable to increased metabolism by GSNO reductase (GSNOR). Indeed, we have found that GSNOR expression and activity correlate inversely with lung S-nitrosothiol (SNO) content and airway hyperresponsiveness (AHR) to methacholine (MCh) challenge in humans with asthmatic phenotypes (Que LG, Yang Z, Stamler JS, Lugogo NL, Kraft M. Am J Respir Crit Care Med 180: 226-231, 2009). Accordingly, we hypothesized that local aerosol delivery of GSNO could ameliorate AHR and inflammation in the ovalbumin-sensitized and -challenged (OVA) mouse model of allergic asthma. Anesthetized, paralyzed, and tracheotomized 6-wk-old male control and OVA C57BL/6 mice were administered a single 15-s treatment of 0-100 mM GSNO. Five minutes later, airway resistance to MCh was measured and SNOs were quantified in bronchoalveolar lavage (BAL). Duration of protection was evaluated following nose-only exposure to 10 mM GSNO for 10 min followed by measurements of airway resistance, inflammatory cells, and cytokines and chemokines at up to 4 h later. Acute delivery of GSNO aerosol protected OVA mice from MCh-induced AHR, with no benefit seen above 20 mM GSNO. The antibronchoconstrictive effects of GSNO aerosol delivered via nose cone were sustained for at least 4 h. However, administration of GSNO did not alter total BAL cell counts or cell differentials and had modest effects on cytokine and chemokine levels. In conclusion, in the OVA mouse model of allergic asthma, aerosolized GSNO has rapid and sustained antibronchoconstrictive effects but does not substantially alter airway inflammation.

NOS2 regulation of LPS-induced airway inflammation via S-nitrosylation of NF-{kappa}B p65.

Inducible nitric oxide synthase (NOS2) expression is increased in the airway epithelium in acute inflammatory disorders although the physiological impact remains unclear. We have previously shown that NOS2 inhibits NF-κB (p50-p65) activation in respiratory epithelial cells by inducing S-nitrosylation of the p65 monomer (SNO-p65). In addition, we have demonstrated that mouse lung SNO-p65 levels are acutely depleted in a lipopolysaccharide (LPS) model of lung injury and that augmenting SNO-p65 levels before LPS treatment results in decreased airway epithelial NF-κB activation, airway inflammation, and lung injury. We now show that aerosolized LPS induces NOS2 expression in the respiratory epithelium concomitant with an increase in lung SNO-p65 levels and a decrease in airway NF-κB activity. Genetic deletion of NOS2 results in an absence of SNO-p65 formation, persistent NF-κB activity in the respiratory epithelium, and prolonged airway inflammation. These results indicate that a primary function of LPS-induced NOS2 expression in the respiratory epithelium is to modulate the inflammatory response through deactivation of NF-κB via S-nitrosylation of p65, thereby counteracting the initial stimulus-coupled denitrosylation.

The role of the extracellular matrix protein mindin in airway response to environmental airways injury.

BACKGROUND: Our previous work demonstrated that the extracellular matrix protein mindin contributes to allergic airways disease. However, the role of mindin in nonallergic airways disease has not previously been explored. OBJECTIVES: We hypothesized that mindin would contribute to airways disease after inhalation of either lipopolysaccharide (LPS) or ozone. METHODS: We exposed C57BL/6J and mindin-deficient (-/-) mice to aerosolized LPS (0.9 µg/m3 for 2.5 hr), saline, ozone (1 ppm for 3 hr), or filtered air (FA). All mice were evaluated 4 hr after LPS/saline exposure or 24 hr after ozone/FA exposure. We characterized the physiological and biological responses by analysis of airway hyperresponsiveness (AHR) with a computer-controlled small-animal ventilator (FlexiVent), inflammatory cellular recruitment, total protein in bronchoalveolar lavage fluid (BALF), proinflammatory cytokine profiling, and ex vivo bronchial ring studies. RESULTS: After inhalation of LPS, mindin-/- mice demonstrated significantly reduced total cell and neutrophil recruitment into the airspace compared with their wild-type counterparts. Mindin-/- mice also exhibited reduced proinflammatory cytokine production and lower AHR to methacholine challenge by FlexiVent. After inhalation of ozone, mice had no detectible differences in cellular inflammation or total BALF protein dependent on mindin. However, mindin-/- mice were protected from increased proinflammatory cytokine production and AHR compared with their C57BL/6J counterparts. After ozone exposure, bronchial rings derived from mindin-/- mice demonstrated reduced constriction in response to carbachol. CONCLUSIONS: These data demonstrate that the extracellular matrix protein mindin modifies the airway response to both LPS and ozone. Our data support a conserved role of mindin in production of proinflammatory cytokines and the development of AHR in two divergent models of reactive airways disease, as well as a role of mindin in airway smooth muscle contractility after exposure to ozone.

c-Kit is essential for alveolar maintenance and protection from emphysema-like disease in mice.

RATIONALE: Previously, we demonstrated a candidate region for susceptibility to airspace enlargement on mouse chromosome 5. However, the specific candidate genes within this region accounting for emphysema-like changes remain unrecognized. c-Kit is a receptor tyrosine kinase within this candidate gene region that has previously been recognized to contribute to the survival, proliferation, and differentiation of hematopoietic stem cells. Increases in the percentage of cells expressing c-Kit have previously been associated with protection against injury-induced emphysema. OBJECTIVES: Determine whether genetic variants of c-Kit are associated with spontaneous airspace enlargement. METHODS: Perform single-nucleotide polymorphism association studies in the mouse strains at the extremes of airspace enlargement phenotype for variants in c-Kit tyrosine kinase. Characterize mice bearing functional variants of c-Kit compared with wild-type controls for the development of spontaneous airspace enlargement. Epithelial cell proliferation was measured in culture. MEASUREMENTS AND MAIN RESULTS: Upstream regulatory single-nucleotide polymorphisms in the divergent mouse strains were associated with the lung compliance difference observed between the extreme strains. c-Kit mutant mice (Kit(W-sh)/(W-sh)), when compared with genetic controls, developed altered lung histology, increased total lung capacity, increased residual volume, and increased lung compliance that persist into adulthood. c-Kit inhibition with imatinib attenuated in vitro proliferation of cells expressing epithelial cell adhesion molecule. CONCLUSIONS: Our findings indicate that c-Kit sustains and/or maintains normal alveolar architecture in the lungs of mice. In vitro data suggest that c-Kit can regulate epithelial cell clonal expansion. The precise mechanisms that c-Kit contributes to the development of airspace enlargement and increased lung compliance remain unclear and warrants further investigation.

ß-arrestin deficiency protects against pulmonary fibrosis in mice and prevents fibroblast invasion of extracellular matrix.

Idiopathic pulmonary fibrosis is a progressive disease that causes unremitting extracellular matrix deposition with resulting distortion of pulmonary architecture and impaired gas exchange. ß-Arrestins regulate G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors through receptor desensitization while also acting as signaling scaffolds to facilitate numerous effector pathways. Here, we examine the role of ß-arrestin1 and ß-arrestin2 in the pathobiology of pulmonary fibrosis. In the bleomycin-induced mouse lung fibrosis model, loss of either ß-arrestin1 or ß-arrestin2 resulted in protection from mortality, inhibition of matrix deposition, and protected lung function. Fibrosis was prevented despite preserved recruitment of inflammatory cells and fibroblast chemotaxis. However, isolated lung fibroblasts from bleomycin-treated ß-arrestin-null mice failed to invade extracellular matrix and displayed altered expression of genes involved in matrix production and degradation. Furthermore, knockdown of ß-arrestin2 in fibroblasts from patients with idiopathic pulmonary fibrosis attenuated the invasive phenotype. These data implicate ß-arrestins as mediators of fibroblast invasion and the development of pulmonary fibrosis, and as a potential target for therapeutic intervention in patients with idiopathic pulmonary fibrosis.

Particulate allergens potentiate allergic asthma in mice through sustained IgE-mediated mast cell activation.

Allergic asthma is characterized by airway hyperresponsiveness, inflammation, and a cellular infiltrate dominated by eosinophils. Numerous epidemiological studies have related the exacerbation of allergic asthma with an increase in ambient inhalable particulate matter from air pollutants. This is because inhalable particles efficiently deliver airborne allergens deep into the airways, where they can aggravate allergic asthma symptoms. However, the cellular mechanisms by which inhalable particulate allergens (pAgs) potentiate asthmatic symptoms remain unknown, in part because most in vivo and in vitro studies exploring the pathogenesis of allergic asthma use soluble allergens (sAgs). Using a mouse model of allergic asthma, we found that, compared with their sAg counterparts, pAgs triggered markedly heightened airway hyperresponsiveness and pulmonary eosinophilia in allergen-sensitized mice. Mast cells (MCs) were implicated in this divergent response, as the differences in airway inflammatory responses provoked by the physical nature of the allergens were attenuated in MC-deficient mice. The pAgs were found to mediate MC-dependent responses by enhancing retention of pAg/IgE/FcεRI complexes within lipid raft­enriched, CD63(+) endocytic compartments, which prolonged IgE/FcεRI-initiated signaling and resulted in heightened cytokine responses. These results reveal how the physical attributes of allergens can co-opt MC endocytic circuitry and signaling responses to aggravate pathological responses of allergic asthma in mice.

Gastrin-releasing peptide blockade as a broad-spectrum anti-inflammatory therapy for asthma.

Gastrin-releasing peptide (GRP) is synthesized by pulmonary neuroendocrine cells in inflammatory lung diseases, such as bronchopulmonary dysplasia (BPD). Many BPD infants develop asthma, a serious disorder of intermittent airway obstruction. Despite extensive research, early mechanisms of asthma remain controversial. The incidence of asthma is growing, now affecting >300 million people worldwide. To test the hypothesis that GRP mediates asthma, we used two murine models: ozone exposure for air pollution-induced airway hyperreactivity (AHR), and ovalbumin (OVA)-induced allergic airway disease. BALB/c mice were given small molecule GRP blocking agent 77427, or GRP blocking antibody 2A11, before exposure to ozone or OVA challenge. In both models, GRP blockade abrogated AHR and bronchoalveolar lavage (BAL) macrophages and granulocytes, and decreased BAL cytokines implicated in asthma, including those typically derived from Th1 (e.g., IL-2, TNFα), Th2 (e.g., IL-5, IL-13), Th17 (IL-17), macrophages (e.g., MCP-1, IL-1), and neutrophils (KC = IL-8). Dexamethasone generally had smaller effects on all parameters. Macrophages, T cells, and neutrophils express GRP receptor (GRPR). GRP blockade diminished serine phosphorylation of GRPR with ozone or OVA. Thus, GRP mediates AHR and airway inflammation in mice, suggesting that GRP blockade is promising as a broad-spectrum therapeutic approach to treat and/or prevent asthma in humans.

Extracellular Matrix Protein Mindin is Required for the Complete Allergic Response to Fungal-Associated Proteinase.

Asthma remains an important cause of morbidity and mortality with an incidence that continues to rise. Despite the importance of this disease, the mechanisms by which the host develops allergic airways disease remain poorly understood. The development of allergic airways disease appears to be contingent on activation of both the innate and adaptive immune system, but little is known about the cross-talk between these two systems. The extracellular matrix protein mindin (Spondin 2) has been previously demonstrated to have functional roles in both the innate and adaptive immunological responses. Previous work supports that pulmonary challenge with fungal-associated allergenic proteinase (FAP) induces an innate allergic response. We hypothesized that mindin would modify the biological response to FAP. Saline or FAP was administered by oropharyngeal aspiration to C57BL/6 wild type or mindin-null mice every 4 days for a total of five exposures. FAP exposed C57BL/6 mice developed enhanced airway hyperresponsiveness (AHR) to methacholine challenge and increased neutrophils and eosinophils in the bronchoalveolar lavage as compared to saline exposed controls. These responses were significantly reduced in mindin-null mice exposed to FAP. FAP challenge was associated with a broad induction of cytokines (IL-1ß, TNFα, Th1, Th2, and IL-17), chemokines, and growth factors, which were reduced in mindin-null mice exposed to FAP. RNA expression in lung monocytes for representative M1 and M2 activation markers were increased by FAP, but were independent of mindin. Our observations support that challenge with FAP results in activation of both innate and adaptive immune signaling pathways in a manner partially dependent on mindin. These findings suggest a potential role for the extracellular matrix protein mindin in cross-talk between the innate and adaptive immune systems.