Sputum Fluid Phase Research Articles

Sputum proteomics in lung disorders.

Lung diseases affect pulmonary and respiratory function and are caused by bacterial viral and fungal infection as well as environmental factors. Unfortunately, symptom overlap between various pulmonary diseases often prevents clear differentiation and uncertain diagnosis. Accordingly, identification of specific markers of inflammatory activity in early disease stage could potential unveil the intrinsic molecular mechanisms of the underlying pathology. Proteomic studies aimed at understanding the genetic/environmental contributions to the development and progression of lung diseases represent a promising approach for diagnosis and treatment. The fluid phase of sputum represents a rich protein source and is frequently used in these studies. This chapter addresses causes of lung disorders, sputum composition, collection and processing as well as the clinical significance and challenges associated with the presence of interfering factors. Basics of proteomics and mass spectrometry are also described, together with the analytical approaches to investigate the sputum proteome. Finally, we explore the application of sputum proteomics in severe lung disorders including COVID-19 infection, chronic obstructive pulmonary disease, asthma, cystic fibrosis, lung cancer and tuberculosis.

Sputum induction and its diagnostic applications in inflammatory airway disorders: a review.

Sputum induction is a technique that covers the induction and the subsequent processing of the expectorate primarily for the analysis of cells and different inflammatory biomarkers present in the airways to further understand the pathophysiology of different inflammatory respiratory disorders such as asthma and chronic obstructive pulmonary disease (COPD) as well as the diagnosis of lung diseases such as lung cancer, tuberculosis, and Pneumocystis jirovecii pneumonia. It is a non-invasive, safe, cost-effective, and reliable technique reported to exhibit a high success rate. However, due to being technically demanding and time-consuming and having the need of employing trained staff, this technique is only used in restricted research centres and in limited centres of clinical use. When the sputum is collected after induction, the primary goal is to obtain a differential cell count and evaluate the molecular biomarkers of airway inflammation such as eosinophil cationic protein, eosinophil-derived neurotoxin, major basic protein, tryptase, cytokine production [e.g., interleukin (IL)-5], albumin, and fibrinogen. In addition, cytospins from the processed sputum are used for immunocytochemical staining of cellular products such as EG-2 reactive protein, granulocyte-macrophage colony-stimulating factor, tumour necrosis factor alpha, and IL-8 that play significant roles in understanding the pathophysiology of inflammatory airway diseases. Nowadays, this technique can be further used by performing an additional analysis such as flow cytometry and in situ hybridisation on the sputum supernatant to investigate more the immune response and pathophysiological process of such various respiratory diseases. In addition, the application of sputum fluid phase to assess the biomarkers could be used more routinely in pathological laboratories for diagnosing lung cancer, COPD, and asthma as well as for monitoring lung cancer progression and asthma and COPD treatment, allowing for early detection and a better treatment provided by the clinicians.

Proteomic Analysis of Human Sputum for the Diagnosis of Lung Disorders: Where Are We Today?

The identification of markers of inflammatory activity at the early stages of pulmonary diseases which share common characteristics that prevent their clear differentiation is of great significance to avoid misdiagnosis, and to understand the intrinsic molecular mechanism of the disorder. The combination of electrophoretic/chromatographic methods with mass spectrometry is currently a promising approach for the identification of candidate biomarkers of a disease. Since the fluid phase of sputum is a rich source of proteins which could provide an early diagnosis of specific lung disorders, it is frequently used in these studies. This report focuses on the state-of-the-art of the application, over the last ten years (2011–2021), of sputum proteomics in the investigation of severe lung disorders such as COPD; asthma; cystic fibrosis; lung cancer and those caused by COVID-19 infection. Analysis of the complete set of proteins found in sputum of patients affected by these disorders has allowed the identification of proteins whose levels change in response to the organism’s condition. Understanding proteome dynamism may help in associating these proteins with alterations in the physiology or progression of diseases investigated.

Sputum Protein Biomarkers in Airway Diseases: A Pilot Study.

BackgroundChronic mucous hypersecretion (CMH or chronic bronchitis) per se or when associated with chronic inflammatory airway diseases such as asthma or chronic obstructive pulmonary disease (COPD) has several adverse clinical consequences. The sputum fluid phase has several candidate proteins including mucins which have the potential of being therapeutic targets, but has not yet been explored in-depth. This study aimed at exploring the profile of sputum proteins in various airway diseases.MethodsSputum from thirty-one patients with various airway diseases was collected and the fluid phase analyzed by LC-MS/MS and subsequently by sequential window acquisition of all theoretical fragments ion spectra (SWATH) (n = 15) for protein quantitation. Hierarchical clustering and functional grouping were performed.ResultsA total of 185 proteins were quantitated by SWATH of which 21 proteins were identified which could distinguish between the clinical phenotypes by hierarchical clustering. Functional protein clustering revealed 4 groups: those that are inflammation related, oxidative stress related, mucin related and a cytoskeletal and calcium related group. The levels of eight proteins (Azurocidin1, Neutrophil defensin 3, Lactotransferrin, Calmodulin 3, Coronin1A, Mucin 5B, Mucin 5AC and BPI fold containing family B1) were significantly altered (relative to mean) in exacerbator prone subjects compared to nonexacerbators. Another simple but useful metric which emerged from this study was total protein concentration in sputum which was significantly higher in frequent exacerbators.ConclusionSputum proteins can detect the various airway disease clinical phenotypes. Total protein concentration and eight other proteins are biomarkers for frequent exacerbators. The clinical and therapeutic implications of the functional groups of proteins need further evaluation.

Influence of Cell Quality on Inflammatory Biomarkers in COPD Sputum Supernatant.

PurposeWe recently introduced a sputum cell quality score to rate how cell morphology, cellular debris and squamous cell contamination influence inflammatory cell identification during microscopic evaluation. However, sputum cell quality is generally not considered for the interpretation of sputum fluid phase biomarkers. Therefore, we compared the soluble protein concentrations between sputum samples with different cell quality. The impact of cell quality was compared to other factors potentially affecting soluble biomarker concentrations.MethodsA comprehensive sputum dataset from 154 clinically stable COPD patients was used to analyse the differences and the variability of sputum supernatant concentrations for 23 proteins between low, medium, and high sputum cell quality samples. A model was developed and tested to compare the impact of different factors on sputum supernatant protein levels.ResultsMean percentages of sputum macrophages, neutrophils, eosinophils, monocytes and lymphocytes showed no significant differences between low, medium and high cell quality levels. The mean percentage of squamous cells were lower, while total cell count/mL sputum and cell viability were significantly higher in sputum samples with higher cell quality. The concentrations of Interleukin-6, Interleukin-8 and Tumor Necrosis Factor Receptor 2 were significantly increased in sputum samples of higher cell quality. The variability of most protein concentrations declined with increasing cell quality levels. Sixteen proteins showed significantly negative correlations with the percentage of squamous cells. For 14 proteins we observed a positive correlation with cell number/mL sputum. Multiple regression analysis shows that generally less than 30% of the protein variability can be explained by the included factors.ConclusionSputum cell quality has a significant impact on some soluble biomarker concentrations in sputum supernatant. Sputum samples with low sputum cell quality show a higher variability of fluid phase proteins in comparison to medium and high sputum cell quality levels.

The C-C motif chemokine ligands CCL5, CCL11, and CCL24 induce the migration of circulating fibrocytes from patients with severe asthma.

The C-C motif chemokine ligand 5 (CCL5), CCL11, and CCL24 are involved in the pathogenesis of asthma, and their function is mainly associated with the airway recruitment of eosinophils. This study tested their ability to induce the migration of circulating fibrocytes, which may contribute to the development of irreversible airflow obstruction in severe asthma. The sputum fluid phase (SFP) from patients with severe/treatment-refractory asthma (PwSA) contained elevated concentrations of CCL5, CCL11, and CCL24 in comparison with the SFP from patients with non-severe/treatment-responsive asthma (PwNSA). The circulating fibrocytes from PwSA expressed the receptors for these chemokines at increased levels and migrated in response to recombinant CCL5, CCL11, and CCL24. The SFP from PwSA induced the migration of autologous fibrocytes, and its activity was significantly attenuated by neutralization of endogenous CCL5, CCL11, and CCL24. These findings suggest that CCL5, CCL11, and CCL24 may contribute to the airway recruitment of fibrocytes in severe asthma.

Repeatability of and Relationship between Potential COPD Biomarkers in Bronchoalveolar Lavage, Bronchial Biopsies, Serum, and Induced Sputum

Chronic Obstructive Pulmonary Disease (COPD) is a chronic inflammatory disease, primarily affecting the airways. Stable biomarkers characterizing the inflammatory phenotype of the disease, relevant for disease activity and suited to predict disease progression are needed to monitor the efficacy and safety of drug interventions. We therefore analyzed a large panel of markers in bronchoalveolar lavage, bronchial biopsies, serum and induced sputum of 23 healthy smokers and 24 smoking COPD patients (GOLD II) matched for age and gender. Sample collection was performed twice within a period of 6 weeks. Assays for over 100 different markers were validated for the respective matrices prior to analysis. In our study, we found 51 markers with a sufficient repeatability (intraclass correlation coefficient >0.6), most of these in serum. Differences between groups were observed for markers from all compartments, which extends (von-Willebrand-factor) and confirms (e.g. C-reactive-protein, interleukin-6) previous findings. No correlations between lung and serum markers were observed, including A1AT. Airway inflammation defined by sputum neutrophils showed only a moderate repeatability. This could be improved, when a combination of neutrophils and four sputum fluid phase markers was used to define the inflammatory phenotype.In summary, our study provides comprehensive information on the repeatability and interrelationship of pulmonary and systemic COPD-related markers. These results are relevant for ongoing large clinical trials and future COPD research. While serum markers can discriminate between smokers with and without COPD, they do not seem to sufficiently reflect the disease-associated inflammatory processes within the airways.

The study of the inflammation of the airway in bronchial asthma by cytological and chemical analysis in the fluid phase of samples taken by sputum induction

In recent years the study of samples of bronchial biopsies and bronchoalveolar lavage fluid has made it possible to show the existence of significant airway mucosal inflammation, even in cases of mild bronchial asthma. This has led to a renewed interest in the use of sputum to assess airway inflammation non-invasively. Sputum induction has recently been proposed as a non-invasive alternative to bronchoscopy for the collection of airway secretions from asthmatic subjects. Sputum induction yields satisfactory samples of sputum in the great majority of asthmatic subjects, and both differential cell counts and the measurement of molecular markers of inflammation in the sputum fluid phase are feasible. In this paper we review studies where these measurements have been taken from subjects who were asthmatic, affected by other respiratory pathologies or healthy, and we assess their validity. Finally, we consider possible future clinical applications of sputum measurement of airway inflammation in asthma.

Impaired Neutrophil Chemotaxis in Chronic Obstructive Pulmonary Disease

Neutrophilic airway inflammation is considered to be a major factor in the pathogenesis of chronic obstructive pulmonary disease (COPD), with sputum and bronchoalveolar lavage neutrophil counts broadly correlating with disease severity. The mechanisms responsible for neutrophil accumulation are poorly understood, but they could involve increased influx and/or survival of these cells. To investigate whether neutrophil chemotactic responsiveness and/or chemotactic activity in airway secretions are increased in subjects with COPD. Chemotaxis experiments were performed using induced sputum supernatants from subjects with and without COPD as a source of chemotactic activity, and neutrophils from healthy donors as responder cells. In addition, chemotactic responses to N-formyl-Met-Leu-Phe (fMLP) and interleukin-8 (IL-8/CXCL8) were studied using neutrophils from healthy subjects and subjects with COPD. As reported in the literature, sputum neutrophil counts were significantly increased in subjects with COPD compared with healthy subjects. However, this was associated with reduced chemotactic activity in sputum in COPD, as judged by reduced chemotaxis to the fluid phase of sputum from subjects with COPD compared with healthy subjects. Furthermore, whereas neutrophils from subjects with stage I COPD had normal responses to fMLP and IL-8, subjects with more severe stage II-IV COPD showed reduced levels of spontaneous migration and chemotaxis to fMLP and IL-8. Neither increased chemotactic activity in the airways nor increased chemotactic responsiveness of neutrophils explains the increased number of these cells in subjects with stable COPD. The implications of the observed reduction in neutrophil chemotactic activity remain to be established.

Airway neutrophilia in COPD is not associated with increased neutrophil survival

Neutrophilic airway inflammation is a prominent feature of chronic obstructive pulmonary disease (COPD) and correlates with disease severity. The mechanisms that determine the extent of neutrophilia could involve increased influx or prolonged survival of neutrophils. The aim of the study was to assess whether neutrophil pro-survival mechanisms are increased in the airways of subjects with COPD owing to the presence of anti-apoptotic factors in the bronchial lining fluid. Induced sputum samples were collected from 20 subjects with stable COPD, 14 healthy smokers and 14 healthy controls. Quantification of apoptotic neutrophils was based on typical morphological cell changes. Anti-apoptotic, pro-survival activity in the sputum was studied by culturing peripheral blood neutrophils with the fluid phase of induced sputum. Apoptosis was assessed both by morphology and flow cytometry using Annexin V/7-aminoactinomycin D staining. COPD patients and healthy smokers had significantly higher percentages of sputum neutrophils than healthy controls. However, there were no significant differences between the three subject groups in either the proportion of apoptotic neutrophils in sputum or the in vitro anti-apoptotic activity detected in the sputum fluid phase. In conclusion, prolonged survival of neutrophils in sputum is not a feature of chronic obstructive pulmonary disease and cannot explain the increased numbers of airway neutrophils in this disease.

Effect of saliva contamination on induced sputum cell counts, IL-8 and eosinophil cationic protein levels.

Excessive salivary contamination of induced sputum samples prevents the satisfactory examination of lower airway inflammation. The effects of salivary contamination on different sputum fluid phase measures and the levels of salivary contamination preventing analysis are not defined. The present study sought to examine this by investigating the effect of increasing salivary contamination on induced sputum samples. Sputum and saliva samples from subjects with asthma and healthy controls were collected, and treated with dithiothreitol (DTT). Saliva was then added to aliquots of dispersed sputum in increasing proportions (0% to 100%). The effect of increasing saliva contamination was assessed on sputum total cell count, viability, differential cell count and fluid phase levels of interleukin (IL)-8, eosinophil cationic protein (ECP) and total protein. The addition of saliva to induced sputum reduced total cell counts and absolute cell counts but did not change the differential cell count. Levels of fluid phase ECP and IL-8 were significantly reduced with increased salivary contamination. There was a progressive reduction in ECP and IL-8, which reached significance at 70% and 80% saliva contamination, respectively. IL-8 levels corrected for total protein showed no change with increasing saliva concentrations. Induced sputum differential cell counts expressed as the proportion of nonsquamous cells are robust measures that are not influenced by salivary contamination. Studies reporting total and absolute cell counts and fluid phase mediator levels require control for squamous contamination.

Analysis of fluid­phase mediators

Sputum cellular indices are valid, reliable and responsive to change 1–7. Increasingly, numerous inflammatory mediators are being measured in the fluid phase of sputum; these include cytokines, chemokines, granulocyte proteins, markers of vascular leakage, eicosanoids, proteases and others. Many of these mediators are included in table 1⇓, which gives details of methods used by investigators to process sputum and measure mediator levels. The table also provides the median/mean levels measured in studied subject groups to give an indication of the expected levels of these mediators in sputum. However, the reproducibility, precision and validity of many of these measurements in sputum have not been investigated and, therefore, their utility as a research and clinical tool remains uncertain and requires confirmation. The following issues are important in the analysis of mediators: 1) choice of methods for measuring fluid­phase mediators; 2) points to consider when planning an immunoassay; and 3) evaluation of the measurement of a soluble mediator in sputum, i.e. validation of the measurement method. The three main types of method used for the measurement of sputum fluid­phase mediators are bioassays, enzyme assays and immunoassays. ### Bioassays Bioassays rely on the retention of biological activity and the ability to exert a measurable effect, such as proliferation of cells, bone marrow colony formation or chemotaxis 3, 11, 58. Sputum processing with mucolytics such as dithiothreitol (DTT) or dithioerythritol, which are strong reducing agents, may decrease the biological activity of cytokines, many of which rely on disulphide bonds to provide a stable structure for bioactivity. Bioassays also have the disadvantage of being inconvenient, time­consuming and lacking in specificity 59. In addition, the presence of commonly occurring endogenous cytokine inhibitors, although allowing an estimate of net activity, may result in significant underestimation of total cytokine levels 58. ### Enzyme assays Many …

Analysis of Induced Sputum after Air and Ozone Exposures in Healthy Subjects

Exposure of healthy subjects to ozone is associated with increases in cellular and biochemical markers of inflammation in bronchoalveolar lavage fluid. To determine if analysis of induced sputum might similarly reveal the pulmonary inflammatory effects of ozone exposure, we performed cellular and biochemical analysis of induced sputum collected 4 hr after air and ozone (0.4 ppm for 2 hr) exposures from 10 healthy subjects (age 30.0 ± 5.0 years; 5 females) in a randomized crossover study in which exposures were separated by 2 weeks. We found that the total number of nonsquamous cells was significantly higher after ozone exposure than after air exposure (7.4 vs 3.9 × 105/ml, P < 0.05) as was the percentage of the nonsquamous cells that were neutrophils (80.0 ± 7.0% vs 51.0 ± 20.0%, P < 0.05) and the levels of myeloperoxidase in the sputum fluid phase (1.6 ± 0.6 vs 1.3 ± 0.6 μg/ml, P < 0.05). In addition, IL-6 and IL-8 levels were higher after ozone than after air exposures, but not significantly so (44.5 ± 32.4 pg/ml vs 26.8 ± 30.7 pg/ml, P = 0.11; 1.5 ± 0.5 ng/ml vs 1.1 ± 0.6 ng/ml, P = 0.09). Mucin-like glycoprotein levels were also not significantly different between exposures (1.6 ± 0.9 mg/ml vs 1.3 ± 1.0 mg/ml, P = 0.26). We conclude that analysis of induced sputum is a useful noninvasive method for studying the pulmonary response to ozone exposure in healthy subjects.