Biomolecular markers in assessment and treatment of asthma
- (Associate Editor: Darryl Knight).
Asthma is a chronic and heterogeneous inflammatory disorder with several different phenotypes. Whereas clinical features of asthma are non-specific and pulmonary function tests are often insensitive, further development is needed for efficient treatment or even early diagnosis. Recently, several airway inflammatory biomarkers have emerged as valuable tools in diagnosis and management of asthma. The analysis of molecular markers of airways inflammation has provided promising and non-invasive techniques that facilitate the detection of disease phenotypes as well as measurement of therapeutic efficacy. Although conventional treatments remain the preferred therapy, they do not adequately control some severe cases of asthma. Novel therapeutic agents have been developed to target various biomarkers involved in the inflammatory responses and have been investigated in patients with asthma. In this article, we summarized the most studied asthma biomarkers, derived from a variety of biological sources including exhaled gases, induced sputum, serum and urine. Likewise, the effects of current anti-inflammatory asthma treatments on inflammatory biomarkers and some promising biomarkers for developing new targeted therapies are also discussed.
C-C chemokine receptor type 3
exhaled breath condensate
exhaled breath temperature
eosinophil cationic proteins
eosinophil protein X
fraction of exhaled nitric oxide
forced expiratory volume in 1 s
matrix metalloprotease 9
nitric oxide synthase
regulated on activation, normal T cell expressed and secreted
transforming growth factor
tissue inhibitors of metalloproteinases
tumour necrosis factor
Asthma is a global public health problem. People of all ages may be affected by this chronic respiratory disease. When not successfully controlled, it causes many restrictions on daily life and even leads to death. During the last 40 years, urbanization and modern lifestyle have increased the prevalence of asthma in most countries.
Asthma is a heterogeneous inflammatory disorder with diversity in pathology, symptoms and response to treatment. Asthma phenotypes can be defined according to the number and type of inflammatory cells, for example eosinophils and neutrophils. Eosinophilic asthma is a phenotype with allergen-mediated bronchial inflammation and associated pathologically by subepithelial basement thickening and pharmacologically by steroid responsiveness. Non-eosinophilic asthma phenotype is characterized by non-allergic mediated inflammation, normal basement membrane thickness and partial resistance to corticosteroid. Less than 50% of patients have asthma due to eosinophilia and the remaining proportion of asthma is related to non-eosinophilic airway inflammation which may involve neutrophils. Identification of such inflammatory phenotypes may help to control asthma.
Heterogeneity makes asthma a challenge to manage therapeutically. Current therapies for asthma are effective in most people, and evidence indicates that clinical manifestation of asthma can be controlled with appropriate treatment. Therefore, making improvement in diagnosis and monitoring is important to identify susceptible individual and to start early treatment. Traditional diagnostic techniques rely on clinical features, spirometry examinations or peak flow rate measurement. However, asthma symptoms are non-specific and pulmonary function tests are insensitive and often normal or unchanging in patients.
The analysis of biomarkers of airways inflammation provides a promising and non-invasive technique that facilitates the detection of inflammation and measurement of therapeutic efficacy. In this article, we review a number of asthma biomarkers which are derived from a variety of physiological sources, including exhaled gases, exhaled breath condensate, sputum, serum and urine. We also report the effects of corticosteroid medications on inflammatory biomarkers, and the identification of some promising biomarkers as a suitable target for specific therapy.
Biomarkers in Asthma
Recently, several airway inflammatory biomarkers have emerged as potential new valuable tools in the diagnosis and management of asthma. The analysis of biomarkers obtained non-invasively from a variety of sources such as exhaled breath, urine or blood have great advantages in asthma diagnosis over conventional techniques. Bronchial biopsies have become the ‘gold standard’ for investigating airway inflammation, but they are invasive, costly, complex to perform and not readily accessible in clinics and research centres. An optimal biomarker should be safer, cheaper and technically easier to use than the ‘gold standard’. This biomarker might be a mediator in a biochemical pathway involved in the development and progression of asthma disease, and hence, it could act as a beneficial target for specific treatment. To be suitable, the biomarker's level should be stable and low in healthy individuals and significantly increased among asthmatic patients. Moreover, methods used for molecular markers measurement should be fast, sensitive and inexpensive. The most well-known biomarkers of airway inflammation for diagnosis and management of asthma are discussed in detail below.
Biomarkers in exhaled breath
Exhaled nitric oxide
The breath analysis in clinical practice has been given great consideration because of the potential application of exhaled breath biomarkers for diagnosing pulmonary diseases. The most common use of breath analysis is the measurement of the fraction of exhaled nitric oxide (FeNO) for asthma. Nitric Oxide (NO) is a gaseous molecule that was first discovered in the exhaled breath of mammals in 1991. It is produced by airway epithelial cells, airway and circulatory endothelial cells, and trafficking inflammatory cells. Endogenous NO is generated by three nitric oxide synthase (NOS) isoenzymes: two constitutive and one inducible. Constitutive NOS isoenzymes include neuronal NOS (NOS1) and endothelial NOS (NOS3). They are activated by calcium ions, and produce small amount of NO and have a local regulatory role. Inducible NOS (NOS2) is not dependent on calcium ion and produces large amount of NO. All NOS isoenzymes catalyse the conversion of L-arginine to L-citrulline with concomitant NO release. The overexpression of NOS2 has been observed in the airway epithelium of asthmatic patients and is decreased by inhaled corticosteroids (ICS) therapy. Moreover, selective NOS2 inhibitors may reduce FeNO in asthmatics and even normal individual.
However, NO could be produced from other sources than NOS. NO reacts with sulfhydryl residues of cysteine and glutathione to form S-nitroso proteins and S-nitrosothiols. Approximately 70–90% of exhaled NO is released by S-nitrosothiols serving as the main source of NO for tissues. NO in exhaled air may also be derived from nitrate protonation to form nitrous acid, which releases NO during acidification.
The level of exhaled NO can be measured by chemiluminescence and electrochemical analysers. The American Thoracic Society and the European Respiratory Society have standardized a method to measure FeNO and provided a simple, fast, non-invasive and reproducible technique in asthma management. FeNO value can be affected by several demographic characteristics, biological and lifestyle factors such as age, height, weight, gender, race, smoking and alcohol consumption.
FeNO level correlates with eosinophilic airway inflammation and is significantly higher in severe asthmatics versus patients with moderate asthma and normal individual. As FeNO level decreased significantly in response to ICS therapy, FeNO can be used to determine the likelihood of response to steroid therapy. Low FeNO levels (less than 25 parts per billion) indicate eosinophilic asthma and responsiveness to steroids are less likely. A high FeNO level of more than 50 parts per billion is associated with airway eosinophilia and steroid responsiveness. Additionally, evaluation of FeNO concentration in pregnant women and treatment decisions based on FeNO levels can reduce asthma exacerbations during pregnancy. Nevertheless, Petsky et al. reported negative outcomes on the use of FeNO to modulate the dose of ICS. FeNO levels have not shown superiority in tailoring asthma treatment compared with clinical symptoms and spirometry/peak flow and cannot be routinely recommended for clinical practice. FeNO may also be used to distinguish atopic and non-atopic asthma. Elevated levels of FeNO have been reported in atopic adults and children. FeNO is thus a promising biomarker for the diagnosis of atopic/allergic asthma.[16, 17]
Exhaled breath condensate
Exhaled breath condensate (EBC) measurement is a relatively recent development that helps in the understanding of the pathophysiology of asthma. In principle, numerous biomarkers could be identified in EBC and the technique is easy, non-invasive, reproducible and can be used in children. EBC is obtained as the warm breath of subject is exhaled into a cooled (0°C) collecting tube, allowing the collection of both volatile and non-volatile molecules of the respiratory tract and then the analysis of inflammatory biomarkers and mediators in the breath condensate. However, the procedure of EBC collection and biomarker detection are not standardized. Currently, several biomarkers have been found in EBC of asthmatics such as pH, hydrogen peroxide (H2O2), nitric oxides, leukotrienes, isoprostanes, cytokines and others.
The mean value of EBC pH is lower in asthmatics and returns to normal after anti-inflammatory therapy. EBC pH was found to reflect acute exacerbations of asthma and correlated with sputum eosinophilia, total nitrite/nitrate and oxidative stress. Total nitrite/nitrate levels were increased in patients with asthma compared with control subjects and decreased after the use of ICS. The volatilization of acids in the airways of asthma patients can lead to low pH value in EBC. Non-volatile basic compounds become protonated in the airway lining fluid and thereby convert to volatile acids.
H2O2 is a form of reactive oxygen species that contributes to oxidative stress in the airways. H2O2 levels in EBC are elevated in adults and children with severe asthma compared with healthy subjects. Cigarette smoking and disease severity can affect H2O2 levels, thus, smoker asthmatics have a fivefold higher level of H2O2 than non-smokers. These elevated levels in asthmatics approach normal value by corticosteroid therapy. Therefore, H2O2 in EBC might be a potential biomarker for monitoring asthma control.
Pinkerton et al. reported for the first time that micro-ribonucleic acid (miRNA) can be isolated from EBC and differences between normal and inflamed asthmatic lungs can be identified via miRNA expression in EBC. A number of T helper (Th)-2 mediators may be regulated by miRNA, for example miR-1248 is predicted to regulate interleukin (IL)-13, IL-5 and high-affinity immunoglobulin receptor (FcεRI), whereas miR-21 is predicted to regulate the IL-13 receptor. Future studies will determine the role of miRNA repression in asthma pathogenesis.
Prostaglandin E2 (PGE2), thromboxane B2 (TXB2), leukotriene B4 (LTB4) and cysteinyl leukotrienes (Cys-LT) have been found in EBC samples. No significant difference in PGE2 levels were found between asthmatics and healthy controls; moreover, PGE2 in EBC of asthmatic children was not reduced by ICS and leukotriene receptor antagonists (montelukast). But PGE2 levels in EBC among smoker asthmatics were higher than non-smokers and control subjects. Therefore, PGE2 may be a more reliable marker for investigating environmental effects on airways inflammation in asthma. TXB2, LTB4 and Cys-LT levels were also increased in the EBC of patients with asthma.[26, 27] Anti-inflammatory therapy with corticosteroids and montelukast can decrease the elevated Cys-LT to the normal levels, suggesting that the level of Cys-LT may be a useful marker in monitoring response to treatment among asthmatic patients.
Isoprostanes are a series of prostaglandin-like compounds that accurately predict oxidative stress in airways. 8-isoprostane is a well-known isoprostane in EBC of asthmatics. The concentrations of 8-isoprostane have increased by twofolds in mild asthma and further increased in moderate to severe asthma.
Asthma is associated with eosinophilic airway inflammation and expression of Th-2 lymphocyte-related cytokines. Several cytokines, chemokines and growth factors have been found in asthmatic airways including IL-4, IL-8, IL-17, tumour necrosis factor (TNF)-α, regulated on activation, normal T cell expressed and secreted (RANTES), interferon (IFN)-γ inducible protein 10, transforming growth factor (TGF)-β, macrophage-derived chemokine, eotaxin, macrophage inflammatory protein 1α and 1β. The concentrations of cytokines in EBC are below the limit of detection, and more sensitive techniques must be used to detect these biomarkers. In future, measurement of cytokine levels in EBC may be a promising method in clinics for assessing airway inflammation in asthma and monitoring response to current treatments.
Additionally, other inflammatory mediators have been measured in EBC of patients with asthma. Endothelin-1 (ET-1) is a pro-inflammatory mediator that is involved in severe bronchial hyperactivity and airway remodelling in asthma. Prolonged exercise can cause the release of ET-1 from bronchial epithelium and increase the concentration of ET-1 in EBC of asthmatics. Similarly, adenosine, a purine nucleoside that is found in EBC and bronchoalveolar lavage (BAL), increases in asthmatic patients during physical exercise. Eotaxin is the strongest chemoattractant that can affect the function of eosinophil and plays an important role in development of allergic asthma. The levels of eotaxin can be measured in EBC of adults and children with asthma and an increased levels of eotaxin have been shown in asthmatic patients. Matrix metalloprotease 9 (MMP-9) is a major mediator in the pathogenesis and the severity of asthma. Karakoc et al. have shown that patients with asthma have elevated levels of MMP-9 and EBC MMP-9 levels correlated with pulmonary functions and other inflammatory markers in exhaled breath such as IL-4/IL-10. These findings suggest that the measurements of ET-1, adenosine, eotaxin and MMP-9 in EBC of asthmatic patients may provide another useful approach for asthma management.
Exhaled breath temperature (EBT) has been suggested by many studies as a new biomarker to assess airway inflammation in a non-invasive manner. Patients with asthma have higher EBT during exacerbations compared with control subjects. Elevated levels of NO in asthmatic patients as a result of airway inflammation may cause an increased bronchial blood flow and heat exchange. The clinical control of asthma with ICS is correlated with reduced levels of NO, bronchial blood flow and conversely with an increase in forced expiratory volume in 1 s (FEV1). Additionally, airway remodelling may be responsible for increased bronchial blood flow and EBT in asthmatic patients, since expression of MMP-9, which is an important marker of remodelling, is considered to be associated with EBT gradients.
Biomarkers in induced sputum
Sputum induction is a relatively safe and non-invasive procedure that can be successfully performed in adults and children with asthma and provide a rich source of biomarkers. Induced sputum is usually collected by inhalation of hypertonic saline in increasing concentrations (3, 4 and 5%) via an ultrasonic nebulizer for 12–15 min. Although this procedure is safe, FEV1 needs to be monitored after each concentration and saline inhalation must be abandoned if the FEV1 decreases by 15%. Induced sputum contains the cell phase (e.g. eosinophils and neutrophils) and the supernatant (e.g. cytokines) which can be used to predict asthma severity and exacerbation.
Sputum eosinophil count acts as a key marker of asthma severity and steroid responsiveness. The number of eosinophils in sputum from asthmatic patients is significantly raised compared with healthy subjects and correlates with severe exacerbations and airway hyperresponsiveness (AHR). The severity of airway obstruction is also related to the sputum eosinophils elevation during an exacerbation. Treatment with corticosteroids consistently decreases sputum eosinophils. However, withdrawal of steroid therapy leads to a rapid increase in the number of sputum eosinophils. The neutrophils, particularly in non-eosinophilic severe asthma, play an important role in airway inflammation. Some studies have reported the raise of sputum neutrophilia in severe asthma. Sputum neutrophil counts are associated with post bronchodilator FEV1 suggesting that neutrophilic inflammation has a role in persistent airway obstruction in asthma.
Several soluble mediators have been examined in the sputum supernatant including eosinophil-derived proteins, NO derivates and cytokines. Induced sputum from patients with severe asthma have elevated levels of eosinophil cationic proteins (ECP), IL-4, IL-5, IL-13, TNF-α, IL-6, granulocyte macrophage colony stimulator factor and IL-12 compared with normal individuals. Cyst-LT have been found in sputum supernatant and montelukast (a leukotriene antagonist) markedly inhibited the eosinophilic chemotaxis among asthmatics. Urokinase plasminogen activator and plasminogen activator inhibitor-1 are increased in asthmatics and neurokinin A levels significantly elevated during exacerbation. Patients with asthma have lower pH levels in induced sputum than healthy subjects that may be related to different pathophysiological features.
Airway remodelling, the structural changes in airway wall in response to long-term inflammation, is a specific and distinctive feature of asthma. These structural changes constitute subepithelial fibrosis, airway smooth muscle hyperplasia, goblet cell hyperplasia and angiogenesis. Several important mediators in the sputum supernatant are associated with the remodelling process including procollagen synthesis peptides, MMPs, tissue inhibitors of metalloproteinase (TIMP) and TGF-β. The level of MMP-9 in induced sputum is elevated in asthmatics and is not affected by ICS. Moreover, imbalances in the MMP-9/TIMP-1 ratio in induced sputum have been observed that may be the cause of degradation of extracellular matrix proteins. Levels of TGF-β in induced sputum are increased despite ICS treatment and associated with airway wall thickening and airflow limitation. Also, collagen type I synthesis is increased during exacerbation of asthma, while collagen degradation is not, and leading to airway remodelling. Despite the lack of ICS efficacy in controlling remodelling mediators, montelukast treatment can reduce eosinophilic infiltration, goblet cell metaplasia and epithelial desquamation, as well as improve pulmonary functions. Moreover, tyrosine kinase inhibitors may have a beneficial effect in the management of airway remodelling. Some studies have found evidence that erlotinib, which is an epidermal growth factor receptor tyrosine kinase inhibitor, can decrease subepithelial collagen deposition, smooth muscle thickening and the level of inflammatory cytokines including IL-4, IL-5, IL-13, TGF-β and TNF-α.
Biomarkers in blood
The levels of total IgE and allergen-specific IgE in serum are both biomarkers to define phenotype of asthmatic patients. Total serum IgE levels are raised in asthmatics and associated with disease severity. The presence of IgE against specific allergen is a biomarker for atopic asthma that can be detected by skin prick test. Serum IgE level is a weak biomarker of asthma and cannot predict the response to treatment; however, some studies have reported a significant reduction in IL-4 levels after corticosteroid administration that results in the reduction of total and allergen-specific IgE levels. The anti-IgE antibody omalizumab is also associated with a modest reduction in exacerbations of severe asthma.
Blood eosinophils count correlates with asthma and can be used as a biomarker to monitor pharmacologic effects in asthmatics. In both adult and children with asthma, the numbers of circulating eosinophils are increased and an inverse relationship was found between blood eosinophils and pulmonary function (FEV1). The blood eosinophil count reflects the levels of proinflammatory mediators in blood and tissue, released by activated eosinophils and may cause tissue damage and remodelling. The serum levels of eosinophil granular proteins such as ECP and eosinophil-derived neurotoxin (EDN) are also increased in children with severe asthma. Treatment of asthmatic patients with corticosteroids inhibits the eosinophil development and leads to a fall in blood eosinophil counts. Furthermore, treatment with anti-IL-5 antibody, anti-IgE antibody, leukotriene antagonists and 5-lipoxygenase inhibitors reduced the numbers of eosinophils in peripheral blood.
Verrills et al. proposed that a panel of peripheral blood biomarkers can discriminate between healthy subjects and asthmatic patients. They have identified a panel of four acute-phase proteins (ceruloplasmin, haptoglobin, hemopexin and α2-macroglobulin) that can have important anti-inflammatory activity. Proteomic analysis, using two dimensional gel electrophoresis, indicated that serum levels of these proteins were elevated among asthmatic patients. Therefore, the combination of protein biomarkers can improve their diagnostic predictive power and this panel has the potential as a useful tool in clinical practice.
Yamamoto et al. evaluated changes in the miRNA expression in the peripheral blood of asthmatic patients after allergen inhalation challenge. Among them, the alteration of miR-192 level may be involved in asthma. The level of miR-192 in asthmatic patients after allergen inhalation challenge was significantly lower than pre-challenge asthmatics and normal individuals.
Other circulating markers have been found to be raised in asthmatics. The serum levels of C3 and C4 complement components were elevated in patients with atopic asthma compared with healthy individuals. The levels of cytokines and growth factors, except IFN-γ, were raised in serum and associated with clinical control of asthma. Particularly, IL-3, IL-18, fibroblast growth factor, hepatocyte growth factor and stem cell growth factor-β were significantly higher among poorly controlled asthmatic patients than healthy subjects or well-controlled patients. Recently, Th-17 cells have been suggested to play a pathogenic role in allergic asthma and IL-23 may be involved in modulating Th-17 response. The levels of IL-23 were increased among asthmatic children and correlated negatively with pulmonary function. Osteopontin (OPN) is a multifunctional cytokine that can be expressed by eosinophils and implicated in allergic airway responses. The levels of OPN were elevated in serum of asthmatic patients, but its concentration did not correlated with disease severity. The chemokine RANTES, a key chemotaxis element in allergic airway inflammation, was significantly increased in patients with severe asthma. The serum level of RANTES was associated with eosinophil count and total serum IgE level and displayed inverse relationships with FEV1.
Periostin is a systemic marker of Th-2 derived asthma that induces the recruitment of eosinophils into the airway. The expression of periostin is upregulated by IL-13 in bronchial epithelial cells of asthmatics. Serum periostin levels may be more accurate than serum IgE levels and blood eosinophil numbers in predicting airway eosinophilia. Corren et al. reported that lebrikizumab, a monoclonal antibody to IL-13, exerted an anti-asthmatic effect especially in patients with high pretreatment levels of serum periostin. The chitinase-like protein YKL-40 is implicated in inflammatory diseases such as asthma. The serum YKL-40 levels were elevated in patients with asthma; however, the relationships between YKL-40 and total serum IgE, asthma severity and exacerbation attacks remain controversial.[64, 65] The costimulatory molecule OX40 and its ligands are involved in eosinophilic airway inflammation and hyperresponsiveness. The serum OX40L levels were significantly increased in asthmatic children during exacerbations and correlated with total eosinophil count. Serum leptin levels and body mass index have also related to the severity of inflammation in childhood asthma. However, further investigations are needed to clarify the role of these systemic biomarkers in various aspects of asthma.
Biomarkers in urine
Measurement of urinary metabolites is possibly the least invasive way to investigate airway allergic responses in patients with atopic asthma. Urine samples can be easily obtained from those who cannot produce sputum and is therefore appropriate for assessment of asthma in young children.
Cys-LT (LTC4, LTD4 and LTE4) are synthesized from arachidonic acid through a 5-lipoxygenase pathway by mast cells, eosinophils, basophils and monocytes. LTC4, the dominant arachidonic acid metabolite in lung tissues, is very unstable and rapidly metabolizes to LTD4 and then to LTE4, which is the stable end product of this pathway and excretes in the urine. Levels of urinary LTE4 are increased in children and adults with asthma compared with healthy subjects and associated with disease severity, exacerbations, aspirin and allergen challenges. The aspirin intolerant asthma group exhibits a significantly higher urinary LTE4 excretion level than the aspirin-tolerant asthma group. Although ICS are effective regimens for most asthmatic patients, they do not alter urinary LTE4 excretion. The 5-lipoxygenase inhibitors block the synthetic pathway of cys-LT metabolism and significantly reduce urinary LTE4 levels.
Eosinophil protein X (EPX) or EDN is released from eosinophil granulocytes and found in the urine of patients with asthma. In childhood asthma, the urinary EPX (uEPX) concentrations are increased during exacerbations and significantly decreased after 3 months anti-inflammatory treatment. Nuijsink et al. assessed the relationship between uEPX and other markers of eosinophilic airway inflammation. They found that uEPX is associated with lung function (FEV1) and percentage eosinophils in induced sputum.
Prostaglandin D2 (PGD2) is the major product of cyclooxygenase pathway that causes induction of bronchoconstriction and vasodilation in the airway of asthmatic patients. PGD2 is released by activated human lung mast cells and metabolized to 9α, 11β-PGF2 which is rapidly excreted in the urine. A number of studies have demonstrated increased urinary 9α, 11β-PGF2 levels in asthmatic patients undergoing allergen or aspirin challenges. Similarly, children with acute asthma and exercise-induced asthma have elevated urinary concentrations of this compound. The urinary 9α, 11β-PGF2 levels are of limited value in distinguishing between asthma severity levels and negatively correlated with lung function. Corticosteroids reduce the number of eosinophils and mast cells in lung tissues of patients with asthma and may indirectly reduce the urinary PGD2 metabolites.
Bromotyrosine (BrTyr) is the major product of protein oxidation by eosinophils. Activated eosinophils release granular proteins and H2O2, and then eosinophil peroxidase catalyses H2O2 to form brominating oxidant from bromide. Brominating oxidants damage tissue proteins and cause bromination of tyrosine residues to form 3-BrTyr and 3,5-dibromotyrosine. The oxidized amino acids excrete in the urine and can serve as a non-invasive marker for monitoring asthma control and predicting subsequent exacerbations particularly in children. Urinary BrTyr levels are significantly higher in patients with asthma and associated with exacerbations and spirometric parameters of airway obstruction.
Targeting Biomarkers of Airway Inflammation
Although ICS remain the preferred medicine for asthma, they do not adequately control some severe cases and the increasing global prevalence of asthma necessitates the search for better treatment modalities. Novel therapeutic agents have been developed that target various biomarkers involved in the inflammatory cascade and have been investigated in patients with asthma.
Omalizumab (Xolair; Novartis Pharmaceuticals UK Ltd, North East Lincolnshire, UK) is a recombinant humanized anti-IgE monoclonal antibody that was approved in 2003 by the Food and Drug Administration for treatment of patients with moderate to severe allergic asthma. Omalizumab specifically prevents IgE from interacting with high affinity IgE receptor (FcεRI). It inhibits the degranulation of allergen-bound mast cells and release of inflammatory cytokines associated with airway remodelling including IL-4, IL-5 and IL-13. Omalizumab decreases the level of ET-1 in EBC, which is a key molecule in airway remodelling. Proinflammatory cytokines and ET-1 are growth factors for bronchial subepithelial myofibroblasts that may contribute to structural changes in the airways. Moreover, treatment with omalizumab significantly reduces the rate of asthma exacerbations, decreases requirement for oral and ICS, and improves lung function and quality of life of asthmatic patients. Clinical studies have shown that omalizumab is safe and rarely causes anaphylaxis. The association between omalizumab treatment and risk of malignancy has been investigated, and no relationship was observed between anti-IgE therapy and cancer development. Recent concerns have been raised about possible induction of Churg–Strauss syndrome (CSS) or allergic granulomatosis in patients receiving omalizumab. A retrospective review has indicated that CSS may develop in patients having eosinophilic disorder due to withdrawal of corticosteroids, which is common among patients treated with omalizumab.
Mepolizumab (Bosatria; GlaxoSmithKline, London, UK) is a fully humanized anti-IL-5 monoclonal antibody that blocks the binding of IL-5 to the alpha chain of IL-5 receptor complex on the surface of eosinophils. Eosinophil products such as TGF-β, MMP-9, TIMP-1 and IL-13 contribute to airway remodelling. Blocking IL-5 with mepolizumab decreases the eosinophils number in blood, sputum and bronchial tissues of patients with asthma and improves the feature of airway remodelling. Mepolizumab can decrease the risk of exacerbation in patients with severe eosinophilic asthma and reduce patients' need for corticosteroid use. Although use of mepolizumab significantly decreased peripheral eosinophil counts, there was only weak correlations observed between mepolizumab use and clinical symptoms. Reslizumab and benralizumab are other anti-IL-5 antibodies which are currently put under phase III clinical trials.[85, 86]
Altrakincept (Nuvance; Immunex (Amgen), Thousand Oaks, CA, USA) is a recombinant soluble IL-4 receptor antagonist that inactivates IL-4 and decreases IL-13 production by activated Th-2 lymphocytes. Altrakincept demonstrated promising efficacy for moderate asthma with declined FEV1 and improved symptom scores in phase I and II clinical trials. However, a phase III trial did not confirm the early findings. Pitrakinra (Aerovant; Aerovance Inc., Berkeley, CA, USA) is a recombinant form of IL-4 that blocks IL-4 Rα complex and inhibits binding of IL-4 and IL-13 to the receptor. Targeting of IL-4 and IL-13 at the same time showed some efficacy. Treatment with pitrakinra in a phase IIa study in asthmatics reduced asthma symptoms and improved pulmonary function.
Lebrikizumab is a humanized anti-IL-13 monoclonal antibody that has successfully passed through phase II clinical study. Lebrikizumab blocks the proinflammatory effects of IL-13 and significantly reduces bronchial hyperresponsiveness and prevents airway remodelling specially among asthmatic patients with higher levels of serum periostin. Musculoskeletal adverse effects, such as back and joint pain, happened slightly more frequent with lebrikizumab treatment. CAT-354 is a therapeutic human monoclonal antibody against IL-13. This antibody prevents expression of FcεRI, IL-13-dependent proliferation of mast cells and release of histamine. A phase I study represented an admissible safety profile with few mild to moderate adverse effects. CAT-354 suppresses eosinophil recruitment and reduces serum IgE levels in human, thereby preventing the development of AHR.
Etanercept (Enbrel; Amgen, Thousand Oaks, CA, USA) is a recombinant soluble fusion protein of TNF-α type II receptor that blocks interaction of TNF-α with cellular TNF receptors. This recombinant protein is a well-tolerated treatment, significantly reduces AHR, number of eosinophils and neutrophils, and improves lung function parameters. Infliximab (Remicade; Janssen Biotech, Inc., PA, USA) is a human-murine chimeric anti-TNF-α monoclonal antibody. Anti-TNF-α therapy significantly reduced the incidence of asthma exacerbations and was associated with decreased levels of TNF-α, IL-6, IL-8 and eotaxin in sputum supernatant. However, infliximab had no significant effects on the number of eosinophils in blood and sputum. Because of the immunosuppressive properties of TNF-α blockers, this class of drugs may cause serious adverse events including new infection or reactivation of previous infections (e.g. hepatitis B), nervous system diseases such as multiple sclerosis, lymphoma, congestive heart failure, allergic reactions and lupus-like syndrome. Moreover, there is substantial evidence that anti-TNF-α regimens slightly increase the risk of malignant melanoma. Due to concerns regarding the safety of TNF-α blockers, etanercept has been withdrawn from asthma clinical trials.
Chemokine receptors blocking strategies are alternative approaches to decrease recruitment of inflammatory cells to the lung. C-C chemokine receptor type 3 (CCR3) receptor is the most studied target involved in asthma pathogenesis. Anti-CCR3 monoclonal antibody can eliminate influx of eosinophils into the lung tissues, resulting in reduction of allergic airway inflammation and AHR. Therefore, CCR3 receptor antagonists are not only associated with decreased eosinophil numbers in BAL fluid and lung, but also prevent the development of airway remodelling.
Sitaxentan (Thelin) is an ET-1 receptor antagonist that is in clinical trial for treating acute asthma. ET-1 is overexpressed in airway epithelium and induced bronchoconstriction. However, hepatotoxicity is a known class effect of endothelin receptor antagonists and several recently published reports have confirmed the potential of sitaxentan to cause acute liver failure.[97, 98]
Other promising cytokines are currently under investigation as a new therapeutic target for asthma. Anti-IL-33 antibody is a novel approach to treat allergic airway inflammation that can suppress Th-2-mediated responses and significantly decreases the number of eosinophils in lung, IgE serum concentration, levels of IL-4, IL-5 and IL-13 in BAL. A new IL-9 specific monoclonal antibody (MEDI-528) is also being studied in phase II clinical trial for the treatment of moderate to severe asthma. This antibody may only be effective in a subgroup of asthmatics in which IL-9 plays a dominant role in disease pathogenesis. Blocking the effect of IL-9 by MEDI-528 decreased airway inflammation and AHR. Further, therapeutic agents targeting IL-17A and IL-25 might inhibit acute exacerbations of asthma.
In recent years, most studies have emphasized the importance of new techniques for the assessment of airway inflammation. Lung tissue biopsies and BAL fluid contain a large number of inflammatory cells but the procedures used for specimen collection are invasive, expensive and not suitable for clinical use. Therefore, biomarkers that could be obtained in a non-invasive manner are promising markers for asthma management. In addition, the heterogeneity of asthma phenotype necessitates the development of a biomarker panel to improve disease diagnosis.
At present, FeNO is the most reliable diagnostic biomarker of airway inflammation. However, the FeNO analyser has not been approved as a medical device and different analysers have shown variations in FeNO values. Moreover, normal FeNO values were observed in some patients with asthmatics symptoms while some individuals have shown elevated levels of FeNO without asthmatic symptoms. EBC provides a rich source of different mediators but the procedure of EBC collection and biomarker detection are not standardized. Since various EBC biomarkers have originated from different parts of the airway, one technique cannot accurately measure all biomarkers. The origin of biomarkers found in EBC must be identified prior to standardization of techniques and reference values should be created for each biomarker. Sputum induction can be effectively performed in adults. However, in children under 8 years, there might be difficulty in collecting sufficient sputum. Therefore, uncomfortable sputum collection process, need of skilled personnel and reproducibility problems have limited the use of sputum biomarkers in clinics. Measurement of biomarkers in biological fluids (blood and urine) is a minimally invasive way to evaluate airway inflammation and may find clinical applications in the future.
The different airway inflammatory phenotypes in asthma may make it possible to target various molecular markers especially different cytokines involved in asthma pathogenesis. Development of new targeted therapies for asthma shows a promising future in the management of severe asthma exacerbations and their clinical use will hopefully control the progression of disease and improve the quality of life for asthmatic patients.
In the future, biomarkers will be widely required to identify patient's response to a given treatment because asthmatic patients present different clinical, inflammatory and immunological phenotypes. In addition, progress in providing sensitive and high throughput detection techniques will increase hopes to achieve an array of biomarkers that can detect different phenotypes in clinical practice.