Increased ornithine-derived polyamines cause airway hyperresponsiveness in a mouse model of asthma.

Up-regulation of arginase contributes to airways hyperresponsiveness (AHR) in asthma by reducing L-arginine bioavailability for the nitric oxide (NO) synthase isozymes. The product of arginase activity, L-ornithine, can be metabolized into polyamines by ornithine decarboxylase. We tested the hypothesis that increases in L-ornithine-derived polyamines contribute to AHR in mouse models of allergic airways inflammation. After measuring significantly increased polyamine levels in sputum samples from human subjects with asthma after allergen challenge, we used acute and subacute ovalbumin sensitization and challenge mouse models of allergic airways inflammation and naive mice to investigate the relationship of AHR to methacholine and polyamines in the lung. We found that spermine levels were elevated significantly in lungs from the acute model, which exhibits robust AHR, but not in the subacute murine model of asthma, which does not develop AHR. Intratracheal administration of spermine significantly augmented airways responsiveness to methacholine in both naive mice and mice with subacute airways inflammation, and reduced nitrite/nitrate levels in lung homogenates, suggesting that the AHR developed as a consequence of inhibition of constitutive NO production in the airways. Chronic inhibition of polyamine synthesis using an ornithine decarboxylase inhibitor significantly reduced polyamine levels, restored nitrite/nitrate levels to normal, and abrogated the AHR to methacholine in the acute model of allergic airways inflammation. We demonstrate that spermine increases airways responsiveness to methacholine, likely through inhibition of constitutive NO synthesis. Thus, inhibition of polyamine production may represent a new therapeutic target to treat airway obstruction in allergic asthma.

Spleen tyrosine kinase inhibition attenuates airway hyperresponsiveness and pollution-induced enhanced airway response in a chronic mouse model of asthma.

BACKGROUND: Asthma is a chronic inflammatory disease characterized by airways hyperresponsiveness (AHR), reversible airflow obstruction, airway remodeling, and episodic exacerbations caused by air pollutants, such as particulate matter (PM; PM <2.5 µm in diameter [PM(2.5)]) and ozone (O(3)). Spleen tyrosine kinase (Syk), an immunoregulatory kinase, has been implicated in the pathogenesis of asthma. OBJECTIVE: We sought to evaluate the effect of Syk inhibition on AHR in a chronic mouse model of allergic airways inflammation and pollutant exposure. METHODS: We used a 12-week chronic ovalbumin (OVA) sensitization and challenge mouse model of airways inflammation followed by exposure to PM(2.5) plus O(3). Respiratory mechanics and methacholine (MCh) responsiveness were assessed by using the flexiVent system. The Syk inhibitor NVP-QAB-205 was nebulized intratracheally by using a treatment-based protocol 15 minutes before assessment of MCh responsiveness. RESULTS: Syk expression increased significantly in the airway epithelia of OVA-sensitized and OVA-challenged (OVA/OVA) mice compared with OVA-sensitized but PBS-challenged (OVA/PBS) control mice. OVA/OVA mice exhibited AHR to MCh, which was attenuated by a single administration of NVP-QAB-205 (0.3 and 3 mg/kg). PM(2.5) plus O(3) significantly augmented AHR to MCh in the OVA/OVA mice, which was abrogated by NVP-QAB-205. Total inflammatory cell counts were significantly higher in the bronchoalveolar lavage fluid from OVA/OVA than OVA/PBS mice and were unaffected by PM(2.5) plus O(3) or NVP-QAB-205. CONCLUSION: NVP-QAB-205 reduced AHR and the enhanced response to PM(2.5) plus O(3) to normal levels in an established model of chronic allergic airways inflammation, suggesting that Syk inhibitors have promise as a therapy for asthma.

Hypertrophic airway smooth muscle mass correlates with increased airway responsiveness in a murine model of asthma.

The increase of airway smooth muscle (ASM) mass in asthma results from hypertrophic and hyperplastic stimuli, and leads to an increase in cellular contractile proteins. However, little evidence correlates the relative contributions of hypertrophic and hyperplastic muscle with functional effects on airway resistance. We performed a ventilator-based assessment of respiratory mechanics and responsiveness to methacholine in a murine model of acute (3-week) ovalbumin (OVA)-induced airway inflammation, compared with a chronic (12-week) model. We correlated functional changes in airways Newtonian resistance (RN), peripheral tissue damping (G), and elastance (H) with the relative contributions of proliferation, hypertrophy, and apoptosis to increased ASM mass. Immunohistochemical analyses of treated (OVA-sensitized and OVA-challenged; OVA/OVA) and control (OVA-sensitized and saline-challenged; OVA/PBS) murine lungs showed an increase in ASM area in chronic, but not acute, OVA/OVA-treated mice that correlated positively with increased airway resistance to methacholine. Acute OVA/OVA-treated ASM exhibited an increase in proliferation with diminished apoptosis, which resolved in the chronic OVA/OVA model. Chronic OVA/OVA-treated ASM exhibited hypertrophy. Distinct temporal differences exist in the response of murine airways to antigenic challenge. We report that ASM proliferation and diminished apoptosis occur during the acute phase, followed by the development of smooth muscle hypertrophy and an increased muscle mass with chronic challenge, that correlate strongly with increased airway Newtonian resistance. The identification of a functionally relevant hypertrophic bronchial muscle mass highlights the possibility of regulating airway muscle hypertrophy as a novel therapeutic target in asthma.

Augmentation of arginase 1 expression by exposure to air pollution exacerbates the airways hyperresponsiveness in murine models of asthma.

BACKGROUND: Arginase overexpression contributes to airways hyperresponsiveness (AHR) in asthma. Arginase expression is further augmented in cigarette smoking asthmatics, suggesting that it may be upregulated by environmental pollution. Thus, we hypothesize that arginase contributes to the exacerbation of respiratory symptoms following exposure to air pollution, and that pharmacologic inhibition of arginase would abrogate the pollution-induced AHR. METHODS: To investigate the role of arginase in the air pollution-induced exacerbation of airways responsiveness, we employed two murine models of allergic airways inflammation. Mice were sensitized to ovalbumin (OVA) and challenged with nebulized PBS (OVA/PBS) or OVA (OVA/OVA) for three consecutive days (sub-acute model) or 12 weeks (chronic model), which exhibit inflammatory cell influx and remodeling/AHR, respectively. Twenty-four hours after the final challenge, mice were exposed to concentrated ambient fine particles plus ozone (CAP+O3), or HEPA-filtered air (FA), for 4 hours. After the CAP+O3 exposures, mice underwent tracheal cannulation and were treated with an aerosolized arginase inhibitor (S-boronoethyl-L-cysteine; BEC) or vehicle, immediately before determination of respiratory function and methacholine-responsiveness using the flexiVent®. Lungs were then collected for comparison of arginase activity, protein expression, and immunohistochemical localization. RESULTS: Compared to FA, arginase activity was significantly augmented in the lungs of CAP+O3-exposed OVA/OVA mice in both the sub-acute and chronic models. Western blotting and immunohistochemical staining revealed that the increased activity was due to arginase 1 expression in the area surrounding the airways in both models. Arginase inhibition significantly reduced the CAP+O3-induced increase in AHR in both models. CONCLUSIONS: This study demonstrates that arginase is upregulated following environmental exposures in murine models of asthma, and contributes to the pollution-induced exacerbation of airways responsiveness. Thus arginase may be a therapeutic target to protect susceptible populations against the adverse health effects of air pollution, such as fine particles and ozone, which are two of the major contributors to smog.

Functionally important role for arginase 1 in the airway hyperresponsiveness of asthma.

l-Arginine metabolism by the arginase and nitric oxide (NO) synthase (NOS) families of enzymes is important in NO production, and imbalances between these pathways contribute to airway hyperresponsiveness (AHR) in asthma. To investigate the role of arginase isozymes (ARG1 and ARG2) in AHR, we determined the protein expression of ARG1, ARG2, the NOS isozymes, and other proteins involved in l-arginine metabolism in lung tissues from asthma patients and in acute (3-wk) and chronic (12-wk) murine models of ovalbumin-induced airway inflammation. Expression of ARG1 was increased in human asthma, whereas ARG2, NOS isoforms, and the other l-arginine-related proteins (i.e., cationic amino acid transporters 1 and 2, agmatinase, and ornithine decarboxylase) were unchanged. In the acute murine model of allergic airway inflammation, augmentation of ARG1 expression was similarly the most dramatic change in protein expression. However, ARG2, NOS1, NOS2, and agmatinase were also increased, whereas NOS3 expression was decreased. Arginase inhibition in vivo with nebulized S-(2-boronoethyl)-l-cysteine attenuated the methacholine responsiveness of the central airways in mice from the acute model. Further investigations in the chronic murine model revealed an expression profile that more closely paralleled the human asthma samples: only ARG1 expression was significantly increased. Interestingly, in the chronic mouse model, which generates a remodeling phenotype, arginase inhibition attenuated methacholine responsiveness of the central and peripheral airways. The similarity in arginase expression between human asthma and the chronic model and the attenuation of AHR after in vivo treatment with an arginase inhibitor suggest the potential for therapeutic modification of arginase activity in asthma.

Virus-specific and bystander CD8 T cells recruited during virus-induced encephalomyelitis.

Neurotropic coronavirus-induced encephalitis was used to evaluate recruitment, functional activation, and retention of peripheral bystander memory CD8+ T cells. Mice were first infected with recombinant vaccinia virus expressing a non-cross-reactive human immunodeficiency virus (HIV) epitope, designated p18. Following establishment of an endogenous p18-specific memory CD8+ T-cell population, mice were challenged with coronavirus to directly compare recruitment, longevity, and activation characteristics of both primary coronavirus-specific and bystander memory populations trafficking into the central nervous system (CNS). HIV-specific memory CD8+ T cells were recruited early into the CNS as components of the innate immune response, preceding CD8+ T cells specific for the dominant coronavirus epitope, designated pN. Although pN-specific T-cell numbers gradually exceeded bystander p18-specific CD8+ T-cell numbers, both populations peaked concurrently within the CNS. Nevertheless, coronavirus-specific CD8+ T cells were preferentially retained. By contrast, bystander CD8+ T-cell numbers declined to background numbers following control of CNS virus replication. Furthermore, in contrast to highly activated pN-specific CD8+ T cells, bystander p18-specific CD8+ T cells recruited to the site of inflammation maintained a nonactivated memory phenotype and did not express ex vivo cytolytic activity. Therefore, analysis of host CD8+ T-cell responses to unrelated infections demonstrates that bystander memory CD8+ T cells can comprise a significant proportion of CNS inflammatory cells during virus-induced encephalitis. However, transient CNS retention and the absence of activation suggest that memory bystander CD8+ T cells may not overtly contribute to pathology in the absence of antigen recognition.