Nitrosative events in atopic asthma pathogenesis

O. O. Parilova, T. T. Volodina, S. G. Shandrenko © 2015 O. O. Parilova et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 616.248


Introduction
Asthma is a chronic inflammation of the airways that involves airflow obstruction and increased airway responsiveness to a variety of stimuli, which leads to the symptoms of recurrent coughing and dyspnea [1].Albeit the understanding of disease pathogenesis has progressed enormously in the last several decades [1][2][3], asthma is still a major public health problem, affecting 300 million people worldwide and rapidly increasing in all age groups [4,5].Clinically, several distinct phenotypes are recognized, but the therapy and research have been basically focused on allergic (atopic) asthma [1][2][3].
Currently, the routine asthma treatment relies on suppressing the inflammation (glucocorticoids, leukotriene inhibitors as additional medication) as well as removal of bronchoconstriction (β agonists, anticholinergics) [6,7].The symptom-based guidance of treatment could predispose to an increased num-ber of exacerbations.Thus, asthmatics might benefit from the inflammatory marker-addressed therapy [1,8].Although many physiologic processes have been implicated in asthma pathogenesis, the range of evidence supports the key roles of endogenous nitric oxide (NO) and NO-derived reactive nitrogen species (RNS) in modulating either a normal airway function [8,9] or inflammation in asthma [10,11].Therefore, NO has received a tremendous amount of attention in the medical community as a selective pulmonary disorder marker [8,12,13].
NO is an ubiquitous messenger that regulates various biological functions -either at low concentrations as a signal molecule in many physiological processes, or at high concentrations as cytotoxic and cytostatic defensive mechanisms against tumors and pathogens [9,11].Also it is thought to be a marker of lung inflammation.NO is detected in exhaled breath [8,12,13].Fractional exhaled NO (FeNO) levels were shown to correlate with sputum eosinophil Reviews ISSN 1993-6842 (on-line); ISSN 0233-7657 (print) Biopolymers and Cell. 2015. Vol. 31. N 6. P 405-416 doi: http://dx.doi.org/10.7124/bc.0008FDcount, airway hyperresponsiveness, bronchodilator response, serum IgE levels, allergen skin prick testing, asthma symptoms, and lung function.This led to the general belief that atopy is associated with higher levels of exhaled NO (eNO) [11].
From this background, the current article deals with the consideration of the source of NO formation and its triggers in asthma pathogenesis.We aim to discuss the cases, when NO becomes a marker of allergic inflammation.Simultaneously, we present our data that revealed the contribution of immune cells settling down the airways to the respiratory NO generation.
nNOS and eNOS are constitutively expressed isoforms (cNOS) in cells as the preformed proteins that are activated in response to the cell-specific stimuli through the elevation of intracellular Ca 2+ concentrations and the binding of calmodulin.iNOS form is generally associated with the immune system and produces NO for prolonged periods of time in a calcium-independent manner [9][10][11][14][15][16][17][18][19][20][21][22].iNOS is activated by bacterial lipopolysaccharides and proinflammatory cytokines such as inteleukin-1, TNF-α, IFN-γ [15].However, IFN-γ is the most potent and prevailing inducer of iNOS in vivo [23].As T-lymphocytes and NK-cells are the main source of IFN-γ production, the iNOS induction is mediated by them.The endothelial cell derived factor, endothelin-1 (ET-1), has been shown to promote iNOS expression [24,25].Ultimately, iNOS incorporates a calmodulin binding site to which the calmodulin is tightly bound independently of a calcium signal -this is thought to be responsible for the continuous activity of the enzyme [15][16][17].Up to 1000 times higher levels of NO can be generated by iNOS compared to cNOS [11,19].
The NOS enzyme functions as a dimer consisting of two identical monomers, which can be functionally and structurally divided into two major domains: a C-terminal reductase domain and an N-terminal oxygenase (catalytic) domain.All NOS isoforms are members of the cytochrome P450 enzyme group owing to the presence of a haem complex [20].Thus the mammalian NOS share similar domain architecture, with an N-terminal catalytic domain containing a haem active site, a nearby cofactor site for BH4 and a C-terminal reductase domain consisting of FMN, FAD and NADPH binding sites.We visualized catalytic domain of human NOS enzymes in three-dimensional form using PDB files via Cn3D viewer from MMDB (Fig. 1).The macromolecular 3D structures display the evidence, that eNOS and iNOS isoforms are very similar in the overall molecular shape, relative orientation of cofactors and stereochemistry within the catalytic centre.As the catalytic sites of iNOS and eNOS are so similar it is not obvious from a structural starting point how the selective inhibitors may be designed [26].

Localization of nitric oxide synthase isoforms in respiratory tract and its biological role
In the respiratory tract all isoforms have been identified [20,21].cNOS-derived NO along with other NO adduct molecules are involved in maintaining normal metabolic functions, such as airway and pulmonary vascular tone, intracellular signaling, immunity (platelet aggregation, leukocyte adhesion) and neurotransmission [7,22].By contrast to this, iNOSderived NO seems to play a pro-inflammatory primary role in the cytokine dependent processes [19].Albeit, agonists such as sheer stress, bradykinin, acetylcholine and histamine still may activate cNOS, resulting in the release of NO within seconds [20].
eNOS is constitutively expressed in both the endothelial cells of the bronchial and pulmonary circu-lation and the alveolar endothelial cells, and airway epithelial cells throughout the respiratory tract [27].The traces of NO from eNOS activity have been isolated in the airway epithelium and found to participate in ciliary movement and mucus ejection [28,29].Although eNOS is present in airway epithelium, iNOS is considered to be a predominant form in it [19].The recent evidence has demonstrated a link between certain eNOS gene polymorphisms and increased serum IgE levels in patients with allergic asthma [30].Functionally, nNOS mediates neuronal bronchodilation in the skeletal muscles and cholinergic airway nerves [27].When allergic asthma was being launched, the increased arginase activity attenuates the nNOS-catalyzed synthesis of NO and induces neural bronchoconstriction [31].In this way cNOS isoforms reveal an indirect involvement in asthma pathophysiology.
In general, iNOS regulates approximately 200 genes that are relevant to inflammation, infection or apoptosis [21].In the airways iNOS is expressed by epithelial and endothelial cells [23,32], smooth muscle cells [18,32,33], lung fibroblasts [34].In contrast to other cell types that do not express NOS2 unless induced by cytokines, the NOS2 mRNA expression and NOS2 protein were detected in epithelial cells of normal, noninflamed upper and lower airways and in pulmonary myocytes of large hilar veins by a variety of techniques, including Northern blotting, in situ hybridization, Western blotting, and immunohistochemistry [23,35].Additionally, iNOS-derived NO is produced by the genuine immune-system cells including monocytes and macrophages [16,17,36], eosinophils [37,38], neutrophils [39,40], dendritic cells [41].The reports about the NOS expression in the mast cell population appear to be ambiguous.The data presented in numerous papers clearly indicate distinct differences in diversity and localization of NOS in a variety of mast cell populations expressed in human and rodents [42][43][44].Notably, the iNOS expression and NO generation are up-regulated by the antigen stimulation FcɛRI engagement in the different subsets of mast cells [44].Additionally, the NO production prevents mitochondrial integrity collapse, thereby protecting mast cells against antigen-induced apoptosis [45].Previously several reports have confirmed that the rat mast cells produce iNOS mRNA and the protein upon treatment [46][47][48].However subsequent studies have demonstrated that some types of human mast cell were positive for eNOS but showed variable expression of nNOS and no detectable iNOS [42,43].Together these findings suggest the important differences between the rat and human mast cells in the NO production and NO-mediated regulation of mast cell function [42].NK cells express constitutive eNOS mRNA and protein, however the levels of iNOS are not detectable.The endogenous NO production is involved in protection of NK cells from activation of apoptosis induced by CD16 cross-linking, thereby maintaining NK activity [49].This is the further evidence that, unlike NO produced by iNOS, cNOS-derived NO is critical for normal physiology.

Nitric oxide formation as a marker of Th2-mediated infllammation
There is rising evidence for an important immunoregulatory role of NO in the development of the adaptive immune responses associated with autoimmune and allergic diseases.Increased levels of exhaled NO [53][54][55][56] along with an up-regulation of iNOS in asthmatic airways [57] confirm that this radical should be involved in asthma.It has been demonstrated, that, in humans, a higher than normal NO concentration in exhaled breath is closely associated with the enlarged transcriptional activation of NOS2 gene [58].
Nevertheless, the previous studies, using murine and guinea pig models of asthma and iNOS inhibitor treatment or iNOS-deficient animals, have generated controversial results.In two different studies, iNOS deficiency [59] or iNOS inhibitor treatment [60] had no effect on the lung inflammation, whereas in other study the airway inflammation was inhibited in the iNOS deficient mice [61].Such a discrepancy was attributed to significant differences in the immunization and challenge protocols [59].
Further investigations have shown that inhibited iNOS activity correlates with a decrease of nitrotyrosine and, more importantly, ameliorates FEV1 and airway responsiveness to histamine [14].However, the different subgroups analysis of atopic individuals now suggests that it is the inflammation in atopic individuals with clinical manifestations of airway disease, rather than atopy itself, that accounts for the increased production of NO by iNOS [62].eNO was commonly regarded qua a marker of eosinophilic inflammation [63][64][65].In addition, the 2011 American Thoracic Society (ATS) guideline for the use of FeNO in clinical practice characterized FeNO as an indicator of eosinophilic airway inflammation [12].However, the recent studies have indicated that FeNO is more representative of a Th2-driven local inflammation, specifically of the bronchial mucosa, rather than general eosinophilic inflammation, as measured by blood or induced sputum [66].The disconnect between FeNO and eosinophilic inflammation has been highlighted by two separate studies with monoclonal antibodies (mAb) against IL-5 and IL-13, which indicate that treatment with mepolizumab, an anti-IL-5 mAb, significantly reduces blood and sputum eosinophils without affecting FeNO levels [67] whereas treatment with lebrikizumab, an anti-IL-13 mAb, significantly reduces FeNO levels without reducing blood eosinophils [68].Another research has shown that, in asthmatic children but not in controls, eNO levels and BAL fluid eosinophil percentages correlate well regardless of the methods used to measure eNO [69].Thus, eNO levels interconnect better with bronchial eosinophils than with sputum eosinophils.
Based on the findings of the bronchoalveolar lavage fluid (BALF) studies and the lung tissue histological analysis, the mechanisms for late allergic responsiveness (LAR) are considered to be causally related to the infiltration of eosinophils and other inflammatory cells into the bronchial mucosa [70].It has been suggested that NO play an important role in LAR [10,71].In the airways of asthmatic patients [57] or in rodent lung after allergen challenge [61,72], iNOS expression and/or enzymatic activity are increased.
To give insight into the role of nitrosative stress established by immune cells infiltrating the airways we examined the NO formation in BAL cells under the guinea pig model of acute ovalbumin (OVA)induced asthma with repeated challenges following 32 weeks after sensitization [73].This model mimics those seen in humans exposed to an allergen a long period after acquired hypersensitivity.A suggested protocol reflects the allergen-driven pathway of asthma reproducing several characteristic features, such as airways infiltration by inflammatory cells, early allergic responsiveness (EAR) and LAR, AHR.Flow cytometry analysis using DAF-2DA dye showed, that multiple allergen challenge exposures of sensitized guinea pigs were associated with an excessive level of the intracellular NO generation in BAL cells (Figure 2).Hence, OVA aerosol provocations of guinea pigs with sensitization resulted in a substantial growth of BAL cells containing NO in comparison with sensitized group (33.50[27.80-45.80]% versus 6.30[2.20-7.82]%; p=0.0001).Our results are in line with the data confirming that allergic inflammation is accompanied by NO.Moreover, there is apparent evidence that iNOS inhibition, during the challenge period, markedly reduced the development of the inflammatory process in the OVA-induced murine model after allergen challenge through down-regulation of chemokine expression [74].
Taking into account that BAL explores large areas of the alveolar compartment providing cellular constituents from the lower respiratory tract, we identified the alterations in cellular composition of BAL as indicated in FACS density graphs (Fig. 3).OVA provocations in sensitized animals were shown to cause a strong inflammation in lungs.This led to the redistribution of immune subpopula-  In addition to quantitative assessment of NO formation, the allocation of intracellular NO content in BAL suspension was analyzed depending on the size and granularity (Fig. 4, Fig. 5) [73].
In view of these findings we infer that in asthma, due to increased iNOS protein expression and activity, the excessive NO generation is activated in lung tissue cells (mainly airway epithelial cells), but also in multi-cellular airway infiltrate recruited to trigger organ during allergic inflammation.High BAL levels of NO may reflect circulating nitrosative stress in respiratory tract, when exacerbation in asthma is present.Indeed inhibition of iNOS-derived NO attenuates antigen-induced airway constriction, inflammatory, and reduces collagen and elastic fiber deposition in a guinea pig model of allergic asthma [37].

Immunoregulatory cross-talk of nitric oxide in asthma pathogenesis
The network of molecules involved in the allergic inflammatory processes becomes more complex since NO derivatives have been demonstrated to play a role in these reactions.The allergic disorder pathogenesis is modulated by NO at the level of immune system [20,32].In turn, iNOS transcription is regulated by a number of pathways, including the JNK, JAK-STAT, and p38 MAPK pathways, which are largely activated through the cytokine induction  and OVA sensitized animals treated with OVA aerosol exposures (II column).DAF-2DA stained cells are colored in green (adopted from [73] and supplemented here) Fig. 5. NO generation in accordance with the complexity of BALF cell subpopulations of OVA sensitized guinea pigs (I column) and of OVA sensitized animals treated with OVA aerosol exposures (II column).DAF-2DA stained cells are colored in green (adopted from [73] and supplemented here) [75].In allergic asthma the immune response to inhaled antigens results from the activation of mast cells and antigen-specific Th2 cells, followed by the production of cytokines, including interleukin IL-4, IL-5 and IL-13 [3].Further, IL-4 and IL-13 cause lung epithelial expression of iNOS to be upregulated via STAT-6, a process which is corticosteroid sensitive [76].Moreover, the group of researchers provided evidence, that IL-13 robustly induces the expression of an active dimeric iNOS enzyme in primary HAEC (human airway epithelial cells) maintained in air-liquid interface culture, consistent with its expression in relation to Th2 inflammation [77].Therefore, eNO is a direct signal of the Th2mediated, pro-inflammatory cytokine mechanisms of central importance in the pathophysiology of allergic airway inflammation.At present, there is open debate about whether NO exacerbates or reduces autoimmune and allergic chronic inflammation.
The detrimental impact of NO is implemented due to its ability to launch a hyperinflammatory response, leading to tissue damage.Two mechanisms are primarily responsible for inducing NO-mediated damage: action of NO-induced inflammatory mediators and apoptosis of NO-targeted cells.NO modulates the production and function of cytokines released by immune cells, chemokines, and growth factors [78].NO-mediated apoptosis represents one of the key factors contributing to enhanced inflammation and tissue damage during noninfectious respiratory diseases, such as asthma [79].The recent study has revealed that NO induces eosinophil apoptosis in a mechanism mediated via ROS, JNK, and later mitochondrial permeability transition [80].
iNOS-derived NO plays an immunomodulatory role, as it may shift the Th1/Th2 balance in favor of Th2, thus promoting IgE-mediated allergy [75,81].Th1 cells secrete IFN-γ, TNF-α, and IL-2 and promote cellular immune responses against intracellular antigens, whereas Th2 cells secrete IL-4, IL-5, IL-10, and IL-13, induce IgG1-and IgE-mediated humoral responses, and are important for the elimination of large extracellular parasites such as helminthes and nematodes [82].The molecular mechanisms of Th1 versus Th2 regulation rely on the counterbalance between expression of the IL-12 receptor β 2chain (IL-12Rβ 2 ) 3 on Th1 cells and GATA-3 transcription in Th2 cells.IL-4 induced GATA-3 allows the stable commitment to the Th2 phenotype through promotion of Th2 cytokine production, such as IL-4, IL-5, IL-10 and IL-13 [83,84].IL-10 produced by Th2 cells further suppresses the Th1 development by inhibiting the secretion of IFN-γ and IL-12 and may also directly inhibit the induction of iNOS [81].Moreover, NO markedly inhibits the induction of IL-2 promoter, which could account for most of the reduction in IL-2 production, and increases the production of IL-4 by effector T cells [85].To conclude, NO can inhibit Th1 lymphokines but has no direct impact on the Th2 lymphokines production [78,81].As the continuous expression of iNOS in the normal epithelium is maintained mainly by IFN-γ [19,21,23], the present observations indicated that NO selectively inhibits the expansion of Th1 cells by a negative feedback mechanism.In contrast, low concentrations of NO stimulate T cells to express IL-12R and promote Th1 differentiation [86].
The counter-evidence of the absence of a direct effect of NO on Th2 cells was found since the NO donors inhibit the proliferation of Th1 and Th2 populations.The NO donors SIN-1 (3-morpholino-sydnonimine) and SNAP (S-nitroso-N-acetyl-DLpenicillamine) suppress the proliferation of anti-CD3 or mitogen activated human peripheral Th1 and Th2 cell clones, and prevent the release of IFN-γ, IL-2, IL-4, IL-5 and IL-10 [87].Additionally it has also been reported that NO inclusion to human bronchial epithelial cells reversibly inhibits the proliferation of activated Th1 and Th2 CD4+ T cells in atopic asthma [88].
IgE-dependent synthesis of NO by various cell populations bearing low-affinity IgE receptors (FceRII; CD23) implicates in local allergic and inflammatory reactions, including macrophages, eosinophils and keratinocytes [89].NO is also an important agent in eosinophil migration and infiltration.Several reports demonstrated that the acute treatment with the non-selective inhibitors of NO reduced allergen-induced eosinophilia, showing that NO is involved in the inflammatory cell recruitment [44,90,91].Furthermore, it has been found that the l-NAME (N ω-nitro-l-arginine methyl ester) treatment decreases the number of eosinophils positive for both nNOS and iNOS, whereas the treatment with 1400W, a highly selective iNOS inhibitor, decreases only the iNOS-positive eosinophils, reducing the eosinophil density in alveolar septa of allergen-sensitized animals [92].In addition to these findings, NO is crucial to the numerous mast cell functions, including degranulation, adhesion, and protease release, as well as the production and secretion of chemokines and cytokines [42].NO also controls the processes in immune system through regulation of the chemokine expression.Among the asthma pathophysiology processes, involving NO, are modulation of the co-stimulatory and adhesion molecules, such as VCAM-1 (vascular cell adhesion molecule-1), ICAM-1 (intercellular adhesion molecule-1), CD62E (E-selectin) and CD62P (P-selectin), synthesis and deposition of the extracellular material components via TGF-β [93].
The anti-inflammatory effects of NO may be mediated by several mechanisms including the inhibition of gene expression and the secretion of proinflammatory cytokines [94,95], reduction of the proinflammatory effects of cytokines such as IL-8 through the covalent modification by nitration of tyrosine [96], or by protection against the programmed cell death through inactivation of the proteolytic enzymes responsible for apoptosis [97,98].Thus, the endogenous NO synthesis in asthma, may be an important early physiological defense mechanism against the injury and inflammation.Nevertheless, persistently elevated levels of the NO formation can deepen the tissue injury over time.

Conclusions
The presented evidence posits the nitrosative stress as a relevant pathogenic mechanism underlying atopic asthma.The diversity of NOS enzymes produces different profiles of NO activities as well as of its derivatives facilitating and broadening the func-tional versatility of nitrosative events.Many studies and clinical observations are bringing to light a multitude of different mechanisms through which NO operates in respiratory tract and mediates dysfunction in the disease.Mounting evidence of a link between the NO synthesis and the allergic inflammation raises the possibilities for therapeutic intervention.Two major challenges currently hinder the progress towards this aim; on one hand the multiplicity of asthma nature with a complex immunogenetic basis and a strong environmental contribution; on the other hand the likelihood of mimicking the full spectrum of responses to the allergen exposures and the tested pharmaceuticals by in vivo and in vitro models seems fragile.Hence, despite the significant advances in our understanding, the effective markeraddressed therapy strategies and reliable approaches for the precise diagnostics and monitoring are currently still limited.

Fig. 1 .
Fig. 1.The structures of oxygenase domain of human NOS isoforms.A -dimeric unit of eNOS haem domain with L-arginine bound (PBD ID:4D1O).B -tetrameric unit of nNOS haem domain with L-arginine bound (PBD ID: 4D1N).C -tetrameric unit of iNOS with Zn-bound and L-arginine complex (PBD ID: 1NSI).The enzyme structures show a ball and stick backbone in a trace shape, no side chains to alignment protein views, and solid objects -condensed lines and massive cylinders with arrows -to represent strands and helices.The arrows on helix cylinders point in the N-to-C direction.The extent of each monomer element in macromolecule is designated by color (blue, violet, green and brown).Haem, BH4 and arginine, glycerol are shown in a tube representation.The association into a dimer involves a large interface, which includes the binding site for BH4 and helps to structure the active-site pocket containing the haem and the L-arginine binding site

Fig. 2 .
Fig. 2. NO content in BAL cells 18-20 h after induction of allergic lung inflammation: morphological flow cytometric parameters of cell types concerning size (FS) and granularity (SS).Panel A: BAL cells derived from sensitized guinea pigs with allergen challenges (II coumn) display augment of NO generation compared to sensitized animals (I column).Panel B: fractions of BAL cells incorporating NO from two compared groups (I and II columns) are also demonstrated in the individual dot plots.Green-colored cells include NO molecules (adopted from [73] and supplemented in this paper)

Fig. 3 .
Fig. 3. Distinction of BAL cellular compostion of sensitized guinea pigs (I column) compared to sensitized animals, that received allergen challenges (II column).Subpopulation content and ratio in BAL suspensions based on the morphological flow cytometric parameters side scatter (SS) versus forward scatter (FS) are illustrated in density plots

Fig. 4 .
Fig. 4. NO formation as a function of cellular size in BALF subpopulations derived from OVA sensitized guinea pigs (I column) and OVA sensitized animals treated with OVA aerosol exposures (II column).DAF-2DA stained cells are colored in green (adopted from [73] and supplemented here)