Honing in on the effectors of oxidative stress in the asthmatic lung: oxidised phosphatidylcholines

Oxidative stress is a major pathogenic driver in many diseases, and evidence to date suggests the obstructive lung disease asthma is no exception. Various lung insults, including air pollution, smoking and allergens can induce reactive oxygen species (ROS) in the lung [1].

The presence of multiple types of ROS associates with asthma severity and cardinal features of the disease, including airway hyperresponsiveness (AHR) and inflammation. However, very limited insight currently exists into the mechanisms by which oxidative stress promotes the development of asthma.

Multiple effectors of ROS have the potential to initiate or exacerbate airway inflammation via oxidising effects on both resident and infiltrating lung cells. In this issue of the European Respiratory Journal, Pascoe et al. [2] hypothesised that the phosphatidylcholines are prime oxidising targets in the asthmatic airway based on their relative abundance, and that oxidised phosphatidylcholines (OxPC) are mechanistic effectors of oxidised stress in asthma, suggested by prior research demonstrating the ability of OxPC to perpetuate inflammation in ageing-related diseases, and their known causal role in both acute lung injury and acute respiratory distress syndrome [3–5].

The authors took advantage of banked bronchoalveolor lavage fluid (BALF) samples, from two distinct cohorts, linked to the Canadian Respiratory Research Network [6, 7]. One cohort contained 13 subjects who had no smoking history and lacked airway disease, yet five of the 13 exhibited AHR to methacholine challenge. A second cohort consisted of 10 mild atopic asthmatics subject to either diluent or allergen challenge, with BALF collected 24 h after challenge. The former cohort enabled comparison of the OxPC profile between subjects with or without AHR, whereas the latter cohort enabled assessment of changes in OxPC levels caused by allergen challenge. Clinical data available for each subject also allowed correlation analyses between specific OxPC levels and lung function data.

OxPC were extracted from BALF and specific OxPC species were identified and quantified by ultra-high performance liquid chromatography tandem mass spectrometry. Specific OxPCs were found to be differentially expressed between the subject groups with and without AHR. Moreover, three OxPCs were found to positively correlate with methacholine sensitivity, whereas one OxPC positively correlated with forced expiratory volume in 1 s (FEV₁) and negatively correlated with methacholine sensitivity.

Analysis of OxPC changes induced by allergen challenge in the cohort of mild atopic asthmatics revealed seven different OxPCs that discriminated the (diluent- versus allergen- challenge) groups and positively correlated with the (late phase) fall in FEV₁. Interestingly, there was no overlap between the OxPC profile that discriminated between the AHR/no AHR groups, and the diluent-/allergen- challenge groups.

The authors next turned their attention to mechanisms by which OxPCs might regulate airway inflammation and hyperresponsiveness. Because airway smooth muscle (ASM) is the principal cell determining airway contractile responsiveness, while also possessing immunomodulatory functions [8], human ASM cells in culture were stimulated with OxPAPC, a family of OxPCs containing several whose expression was shown to be increased in the (+AHR) and (allergen-challenged) samples. OxPAPC had somewhat selective effects, inducing the expression of three pro-inflammatory cytokines (interleukin (IL)-6, IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF)) but not 10 others that were also assayed in ASM cells. OxPAPC also induced expression of cyclo-oxygenase-2 (COX2), as well as 32 different oxylipins, including those generated in the cyclo-oxygenase/lipoxygenase pathways and capable of regulating either ASM contraction or airway inflammation. Moreover, a novel kinome analysis suggested OxPAPC increased the activity of 20 different kinases after 3 and 6 h treatment. Pre-treatment of cells with a cPKC inhibitor significantly inhibited OxPAPC-induced COX2, IL-6, IL-8 and GM-CSF, implicating PKC activity as a likely early event in the signalling induced by OxPAPC. Furthermore, the authors present evidence that among the panel of oxylipins induced by OxPAPC, several may have autocrine effects, as an EP receptor inhibitor was sufficient to dampen OxPAPC-induced GM-CSF. These data suggest that ROS-associated peroxidation of phosphatidylcholine may perpetuate a network that determines local airway inflammation, and perhaps could dictate the effectiveness of anti-inflammatory controller pharmacotherapy in asthmatics.

Extending the implications of these effects, to assess the effect of OxPC on airway contractility, murine lung slices were cultured and stimulated with OxPAPCs, which caused a dose-dependent narrowing of the airways within the lung slices. In combination with their ability to induce cytokines, COX2, and oxylipins that appear to have feedback effects, the potential for OxPCs to promote airway contractility and hyperresponsiveness may be an intriguing clue to understand the range in response to bronchodilator therapies that exists between asthma phenotypes. Furthermore, one might ask whether OxPCs could be relevant markers, not yet been considered, for asthma endotype analysis.

Collectively, these studies provide novel data that identify the specific OxPCs that associate with AHR and are induced by allergic lung challenge in humans. In addition, the sufficiency of OxPCs to cause ASM contraction, and to promote lung inflammation through regulation of ASM synthetic functions, are further demonstrated. These findings are an important starting point in unravelling the complex effects of oxidative stress in human asthma (figure 1). Future studies that expand the characterisation of OxPCs across the spectrum of asthma severity will contribute significantly with ongoing clinical research attempts to define patient endotypes to not only understand the pathogenesis of disease but also be able to tailor treatment to target specific key pathogenic effectors [9]. Anti-inflammatory therapies, such as inhaled corticosteroids, are an asthma management mainstay, but they do not specifically target oxidative stress, implying that the latter could be a contributor to persistent inflammation in steroid-refractory disease. However, the utility of general antioxidant treatments, such as N-acetylcysteine, to control asthma is questionable, as clinical trials have not revealed clinically significant effects, with primary impact chiefly linked to mucolytic effects [10]. Therefore, the new results presented here by Pascoe et al. [2] may open a door for defining more precise targeting of the bioactive effectors generated by ROS attack during oxidative stress. To uncover this potential, these new findings create momentum and a critical need to uncover the receptor and non-receptor mechanisms by which individual and panels of OxPCs may drive inflammation, and airway hyperresponsiveness.

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FIGURE 1

Oxidised phosphatidylcholine (OxPC) molecules are generated by reactive oxygen species (ROS) attack of phosphatidylcholine in cell membranes and the airway surface liquid. OxPCs accumulate in the airways after aeroallergen exposure, likely due to effects of ROS produced during the inflammatory response that recruits activated neutrophils and eosinophils. OxPCs stimulate airway smooth muscle cells to secrete pro-inflammatory molecules including interleukin (IL)-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), and a host of oxylipins. OxPCs can also cause airway narrowing by promoting airway smooth muscle contraction, which could be an unexplored cause of airway hyperresponsiveness, even prior to a clinical diagnosis of asthma. Because the profile of OxPCs spikes in the airways of atopic asthmatics following allergen challenge, they likely interact with other structural cells and recruited leukocytes, to sustain and to perpetuate airway inflammation, airway remodelling, and treatment resistance in asthma. This effect has the potential to reduce the effectiveness of inhaled corticosteroids (ICS) or short-acting β2-agonists (SABA), implicating OxPCs as important determinants of asthma phenotypes and endotypes.

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