Edna A. Leick

Eosinophilic inflammation in allergic asthma.

Eosinophils are circulating granulocytes involved in pathogenesis of asthma. A cascade of processes directed by Th2 cytokine producing T-cells influence the recruitment of eosinophils into the lungs. Furthermore, multiple elements including interleukin (IL)-5, IL-13, chemoattractants such as eotaxin, Clara cells, and CC chemokine receptor (CCR)3 are already directly involved in recruiting eosinophils to the lung during allergic inflammation. Once recruited, eosinophils participate in the modulation of immune response, induction of airway hyperresponsiveness and remodeling, characteristic features of asthma. Various types of promising treatments for reducing asthmatic response are related to reduction in eosinophil counts both in human and experimental models of pulmonary allergic inflammation, showing that the recruitment of these cells really plays an important role in the pathophysiology of allergic diseases such asthma.

Effects of Rho-kinase inhibition in lung tissue with chronic inflammation

We evaluated whether Rho-kinase inhibition (Y-27632) modulated distal lung responsiveness, inflammation, extracellular matrix remodeling and oxidative stress activation in guinea pigs (GPs) with chronic allergic inflammation. GPs were submitted to inhalation of ovalbumin (OVA-2×/week/4 weeks). From the 5th inhalation on, the Rho-kinase inhibitor group animals were submitted to Y-27632 inhalation 10min before each inhalation of OVA. Seventy-two hours after the seventh inhalation, the oscillatory mechanics of the distal lung strips were assessed under the baseline condition and after the ovalbumin challenge. Subsequently, the lung slices were submitted to morphometry. Rho-kinase inhibition in the ovalbumin-exposed animals attenuated distal lung elastance and resistance, eosinophils, IL-2, IL-4, IL-5, IL-13, TIMP-1, MMP-9, TGF-β, IFN-γ, NF-κB and iNOS-positive cells and the volume fraction of 8-iso-PGF2α, elastic, collagen and actin in alveolar walls compared with the OVA group (P<0.05). Rho-kinase inhibition contributed to the control of distal lung responsiveness, eosinophilic and Th1/Th2 responses and extracellular matrix remodeling in an animal model of chronic allergic inflammation.

Effects of corticosteroid, montelukast and iNOS inhibition on distal lung with chronic inflammation

UNLABELLED We evaluated the effects of anti-iNOS (1400W - W) associated with leukotriene antagonist (montelukast - M) or corticosteroid (dexamethasone - D) on distal lung of guinea pigs (GP) with chronic pulmonary inflammation. METHODS GP were inhaled with ovalbumin (OVA-2×/week/4 weeks), treated with M (OVAM), D (OVAD) and/or W (OVAW, OVADW, OVAMW) and distal lungs were evaluated by morphometry. RESULTS Isolated treatments were not sufficient to reduce all parameters. In OVADW, all parameters were reduced with greater reduction in elastic fibers, TIMP-1, IL-4, IL-5, IFN-gamma and PGF2-alpha compared with OVAD (p<0.05). OVAMW potentiated the reduction of actin, elastic fibers, TIMP-1, IL-4, IL-5, TGF-beta, IFN-gamma, iNOS, and PGF2-alpha to a greater extent than OVAM (p<0.05). A reduction of TIMP-1, IL-4, IL-5, TGF-beta, IFN-gamma and iNOS was observed in OVADW compared with OVAMW (p<0.05). CONCLUSIONS Although anti-iNOS paired with montelukast or dexamethasone yields better results than isolated treatments, the most effective pairing for controlling inflammation, oxidative stress and remodeling in this asthma model was found to be corticosteroids and anti-iNOS.

Inducible Nitric Oxide Synthase Inhibition Attenuates Physical Stress-Induced Lung Hyper-Responsiveness and Oxidative Stress in Animals with Lung Inflammation

Mechanisms involved in stress-induced asthmatic alterations have been poorly characterised. We assessed whether inducible nitric oxide synthase (iNOS) inhibition modulates the stress-amplified lung parenchyma responsiveness, oxidative stress and extracellular matrix remodelling that was previously increased by chronic lung inflammation. Guinea pigs were subjected to 7 exposures to ovalbumin (1–5 mg/ml) or saline (OVA and SAL groups) over 4 weeks. To induce behavioural stress, animals were subjected to a forced swimming protocol (5 times/week, over 2 weeks; SAL-Stress and OVA-Stress groups) 24 h after the 4th inhalation. 1400W (iNOS-specific inhibitor) was administered intraperitoneally in the last 4 days of the protocol (SAL-1400W, OVA-1400W, SAL-Stress+1400W and OVA-Stress+1400W groups). Seventy-two hours after the last inhalation, animals were anaesthetised and exsanguinated, and adrenal glands were removed. Lung tissue resistance and elastance were evaluated by oscillatory mechanics and submitted for histopathological evaluation. Stressed animals had higher adrenal weights compared to non-stressed groups, which were reduced by 1400W treatment. Behavioural stress in sensitised animals amplified the resistance and elastance responses after antigen challenge, numbers of eosinophils and iNOS+ cells, actin content and 8-iso-PGF2α density in the distal lung compared to the OVA group. 1400W treatment in ovalbumin-exposed and stressed animals reduced lung mechanics, iNOS+ cell numbers and 8-iso-PGF2α density compared to sensitised and stressed animals that received vehicle treatment. We concluded that stress amplifies the distal lung constriction, eosinophilic inflammation, iNOS expression, actin content and oxidative stress previously induced by chronic lung inflammation. iNOS-derived NO contributes to stress-augmented lung tissue functional alterations in this animal model and is at least partially due to activation of the oxidative stress pathway.

Modulation of the oscillatory mechanics of lung tissue and the oxidative stress response induced by arginase inhibition in a chronic allergic inflammation model

BackgroundThe importance of the lung parenchyma in the pathophysiology of asthma has previously been demonstrated. Considering that nitric oxide synthases (NOS) and arginases compete for the same substrate, it is worthwhile to elucidate the effects of complex NOS-arginase dysfunction in the pathophysiology of asthma, particularly, related to distal lung tissue. We evaluated the effects of arginase and iNOS inhibition on distal lung mechanics and oxidative stress pathway activation in a model of chronic pulmonary allergic inflammation in guinea pigs.MethodsGuinea pigs were exposed to repeated ovalbumin inhalations (twice a week for 4 weeks). The animals received 1400 W (an iNOS-specific inhibitor) for 4 days beginning at the last inhalation. Afterwards, the animals were anesthetized and exsanguinated; then, a slice of the distal lung was evaluated by oscillatory mechanics, and an arginase inhibitor (nor-NOHA) or vehicle was infused in a Krebs solution bath. Tissue resistance (Rt) and elastance (Et) were assessed before and after ovalbumin challenge (0.1%), and lung strips were submitted to histopathological studies.ResultsOvalbumin-exposed animals presented an increase in the maximal Rt and Et responses after antigen challenge (p<0.001), in the number of iNOS positive cells (p<0.001) and in the expression of arginase 2, 8-isoprostane and NF-kB (p<0.001) in distal lung tissue. The 1400 W administration reduced all these responses (p<0.001) in alveolar septa. Ovalbumin-exposed animals that received nor-NOHA had a reduction of Rt, Et after antigen challenge, iNOS positive cells and 8-isoprostane and NF-kB (p<0.001) in lung tissue. The activity of arginase 2 was reduced only in the groups treated with nor-NOHA (p <0.05). There was a reduction of 8-isoprostane expression in OVA-NOR-W compared to OVA-NOR (p<0.001).ConclusionsIn this experimental model, increased arginase content and iNOS-positive cells were associated with the constriction of distal lung parenchyma. This functional alteration may be due to a high expression of 8-isoprostane, which had a procontractile effect. The mechanism involved in this response is likely related to the modulation of NF-kB expression, which contributed to the activation of the arginase and iNOS pathways. The association of both inhibitors potentiated the reduction of 8-isoprostane expression in this animal model.

The Plant-Derived Bauhinia bauhinioides Kallikrein Proteinase Inhibitor (rBbKI) Attenuates Elastase-Induced Emphysema in Mice

Background. Elastase mediates important oxidative actions during the development of chronic obstructive pulmonary disease (COPD). However, few resources for the inhibition of elastase have been investigated. Our study evaluated the ability of the recombinant plant derived Bauhinia bauhinioides Kallikrein proteinase Inhibitor (rBbKI) to modulate elastase-induced pulmonary inflammation. Methods. C57Bl/6 mice were given intratracheal elastase (ELA group) or saline (SAL group) and were treated intraperitoneally with rBbKI (ELA-rBbKI and SAL-rBbKI groups). At day 28, the following analyses were performed: (I) lung mechanics, (II) exhaled nitric oxide (ENO), (III) bronchoalveolar lavage fluid (BALF), and (IV) lung immunohistochemical staining. Results. In addition to decreasing mechanical alterations and alveolar septum disruption, rBbKI reduced the number of cells in the BALF and decreased the cellular expression of TNF-α, MMP-9, MMP-12, TIMP-1, eNOS, and iNOS in airways and alveolar walls compared with the ELA group. rBbKI decreased the volume proportion of 8-iso-PGF2α, collagen, and elastic fibers in the airways and alveolar walls compared with the ELA group. A reduction in the number of MUC-5-positive cells in the airway walls was also observed. Conclusion. rBbKI reduced elastase-induced pulmonary inflammation and extracellular matrix remodeling. rBbKI may be a potential pharmacological tool for COPD treatment.

A Treatment with a Protease Inhibitor Recombinant from the Cattle Tick (Rhipicephalus Boophilus microplus) Ameliorates Emphysema in Mice

Aims To determine whether a serine protease inhibitor treatment can prevent or minimize emphysema in mice. Methods C57BL/6 mice were subjected to porcine pancreatic elastase (PPE) nasal instillation to induce emphysema and were treated with a serine protease inhibitor (rBmTI-A) before (Protocol 1) and after (Protocol 2) emphysema development. In both protocols, we evaluated lung function to evaluate the airway resistance (Raw), tissue damping (Gtis) and tissue elastance (Htis). The inflammatory profile was analyzed in the bronchoalveolar lavage (BALF) and through the use of morphometry; we measured the mean linear intercept (Lm) (to verify alveolar enlargement), the volume proportion of collagen and elastic fibers, and the numbers of macrophages and metalloprotease 12 (MMP-12) positive cells in the parenchyma. We showed that at both time points, even after the emphysema was established, the rBmTI-A treatment was sufficient to reverse the loss of elastic recoil measured by Htis, the alveolar enlargement and the increase in the total number of cells in the BALF, with a primary decrease in the number of macrophages. Although, the treatment did not control the increase in macrophages in the lung parenchyma, it was sufficient to decrease the number of positive cells for MMP-12 and reduce the volume of collagen fibers, which was increased in PPE groups. These findings attest to the importance of MMP-12 in PPE-induced emphysema and suggest that this metalloprotease could be an effective therapeutic target.

Effects of repeated stress on distal airway inflammation, remodeling and mechanics in an animal model of chronic airway inflammation.

Background/Aims: Epidemiological studies suggest that stress has an impact on asthmatic exacerbations. We evaluated if repeated stress, induced by forced swimming, modulates lung mechanics, distal airway inflammation and extracellular matrix remodeling in guinea pigs with chronic allergic inflammation. Methods: Guinea pigs were submitted to 7 ovalbumin or saline aerosols (1–5 mg/ml during 4 weeks; OVA and SAL groups). Twenty-four hours after the 4th inhalation, guinea pigs were submitted to the stress protocol 5 times a week during 2 weeks (SAL-S and OVA-S groups). Seventy-two hours after the 7th inhalation, guinea pigs were anesthetized and mechanically ventilated. Resistance and elastance of the respiratory system were obtained at baseline and after ovalbumin challenge. Lungs were removed, and inflammatory and extracellular matrix remodeling of distal airways was assessed by morphometry. Adrenals were removed and weighed. Results: The relative adrenal weight was greater in stressed guinea pigs compared to non-stressed animals (p < 0.001). Repeated stress increased the percent elastance of the respiratory system after antigen challenge and eosinophils and lymphocytes in the OVA-S compared to the OVA group (p < 0.001, p = 0.003 and p < 0.001). Neither collagen nor elastic fiber contents were modified by stress in sensitized animals. Conclusions: In this animal model, repeated stress amplified bronchoconstriction and inflammatory response in distal airways without interfering with extracellular matrix remodeling.

New Pharmacological Targets for Asthma Drug Development

Asthma is an inflammatory disorder characterized by airway hyperresponsiveness, followed by inflammation, remodeling and oxidative stress in the respiratory system and lung tissue. While glucocorticosteroids remain the gold-standard of asthma therapy, they have limitations because of their potentially severe adverse effects and the presence of corticosteroid resistance in some patients. In the present review we will focus in four main groups of experimental pharmacological approaches for future asthma and hyperresponsiveness treatment: proteinase inhibitors and flavonoids, arginase and iNOS inhibition, Rho-kinase inhibitors, cholinergic anti-inflammatory system and nicotinic receptors.

Effects of Anti-IL-17 on Inflammation, Remodeling, and Oxidative Stress in an Experimental Model of Asthma Exacerbated by LPS

Inflammation plays a central role in the development of asthma, which is considered an allergic disease with a classic Th2 inflammatory profile. However, cytokine IL-17 has been examined to better understand the pathophysiology of this disease. Severe asthmatic patients experience frequent exacerbations, leading to infection, and subsequently show altered levels of inflammation that are unlikely to be due to the Th2 immune response alone. This study estimates the effects of anti-IL-17 therapy in the pulmonary parenchyma in a murine asthma model exacerbated by LPS. BALB/c mice were sensitized with intraperitoneal ovalbumin and repeatedly exposed to inhalation with ovalbumin, followed by treatment with or without anti-IL-17. Twenty-four hours prior to the end of the 29-day experimental protocol, the two groups received LPS (0.1 mg/ml intratracheal OVA-LPS and OVA-LPS IL-17). We subsequently evaluated bronchoalveolar lavage fluid, performed a lung tissue morphometric analysis, and measured IL-6 gene expression. OVA-LPS-treated animals treated with anti-IL-17 showed decreased pulmonary inflammation, edema, oxidative stress, and extracellular matrix remodeling compared to the non-treated OVA and OVA-LPS groups (p < 0.05). The anti-IL-17 treatment also decreased the numbers of dendritic cells, FOXP3, NF-κB, and Rho kinase 1- and 2-positive cells compared to the non-treated OVA and OVA-LPS groups (p < 0.05). In conclusion, these data suggest that inhibition of IL-17 is a promising therapeutic avenue, even in exacerbated asthmatic patients, and significantly contributes to the control of Th1/Th2/Th17 inflammation, chemokine expression, extracellular matrix remodeling, and oxidative stress in a murine experimental asthma model exacerbated by LPS.