A novel non-invasive method allowing for discovery of pathologically relevant proteins from small airways

Background There is a lack of early and precise biomarkers for personalized respiratory medicine. Breath contains an aerosol of droplet particles, which are formed from the epithelial lining fluid when the small airways close and re-open during inhalation succeeding a full expiration. These particles can be collected by impaction using the PExA method (Particles in Exhaled Air), and are derived from an area of high clinical interest previously difficult to access, making them a potential source of biomarkers reflecting pathological processes in the small airways. Research question Our aim was to investigate if PExA method is useful for discovery of biomarkers that reflect pathology of small airways. Methods and analysis Ten healthy controls and 20 subjects with asthma, of whom 10 with small airway involvement as indicated by a high lung clearance index (LCI ≥ 2.9 z-score), were examined in a cross-sectional design, using the PExA instrument. The samples were analysed with the SOMAscan proteomics platform (SomaLogic Inc.). Results Two hundred-seven proteins were detected in up to 80% of the samples. Nine proteins showed differential abundance in subjects with asthma and high LCI as compared to healthy controls. Two of these were less abundant (ALDOA4, C4), and seven more abundant (FIGF, SERPINA1, CD93, CCL18, F10, IgM, IL1RAP). sRAGE levels were lower in ex-smokers (n = 14) than in never smokers (n = 16). Gene Ontology (GO) annotation database analyses revealed that the PEx proteome is enriched in extracellular proteins associated with extracellular exosome-vesicles and innate immunity. Conclusion The applied analytical method was reproducible and allowed identification of pathologically interesting proteins in PEx samples from asthmatic subjects with high LCI. The results suggest that PEx based proteomics is a novel and promising approach to study respiratory diseases with small airway involvement. Supplementary Information The online version contains supplementary material available at 10.1186/s12014-022-09348-y.

Statistics for proteins found to be differentially abundant when comparing SOMAscan data for 207 proteins between the Asthma with high LCI (A-hLCI, n=10) and Non-asthma control group (NA, n=10), using a significance criteria including p-value below 0.05 and Benjamini-Hochberg corrected p-value (q) below 0.2, as described in Material and Methods. Statistics for comparison with asthma with normal LCI (A-nLCI) is also shown. Significance values above the threshold are indicated with n.s. Fold change was defined as 2 to the power of the difference difference being defined as the difference of the mean log2 of "group1" data and mean log2 of "group2" data. Fold change values >1 and < 1 indicate higher and lower levels in the first mentioned group, respectively. No significant difference were found for the A-nLCI vs. NA group comparison. Uniprot protein IDs and synonyms can be found in Table S1A  Table S3.
Result from manual reviewing the scientific literature on proteins found to be differentially abundant in A-hLCI as compared to NA group.

Protein name
Entrez symbol and synonyms (Uniprot ID) Abundance profile # Relevant literature findings, summary and reference NA L A-nLCI L A-hLCI H -Found to be the major inhibitor of neutrophil elastase in the lower respiratory system [1,2]. -Associated with an increased risk of pulmonary and extrapulmonary disease [3].
-Increased levels in circulation is associated with pathology localised to the Small airway and alveolar space [5].
-Elevated levels is associated with reduced FEV1/FVC. Suggested to be a marker and potential therapeutic target in patients with neutrophilic asthma and airflow obstruction [9]. -Elevated in patients with COPD [10].
-Is required for IL-33 signalling which is important in promoting and maintaining the asthma phenotype [11].

C-C motif chemokine 18 CCL18 (P55774)
NA L A-nLCI IM A-hLCI H -mRNA particularly abundant in lung (Protein Atlas) [12,13] -Levels in serum, BALF and Alveolar macrophage culture supernatant are markedly increased in various interstitial lung diseases [14]. -Suggested to play a predominant role in allergic asthma [15,16]. -Suggested to serve as a circulating biomarker in non-small cell lung cancer diagnosis [17].

Complement component C1q receptor
-mRNA and protein particularly abundant in lung (Protein Atlas) [12,13]. -Involved in e.g. macrophage activation and neutrophil degranulation (GO annotations) [6,7]. -Have been observed to be expressed at high levels in lung and at elevated levels in circulation of asthmatics.
-Have been suggested as a circulating biomarker with potential to aid in asthma diagnosis [18,19].
-Elevated levels have been observed in RTLF from asthmatic subjects. Suggested to play role in pathogenesis of asthma. [20,21].

Vascular endothelial growth factor D FIGF VEGF-D (O43915)
NA L A-nLCI IM A-hLCI H -Localized to the extracellular space, platelet alpha granule lumen. Involved in platelet degranulation (GO annotations) [6,7]. -Contributes to the small-airway remodelling in a rat model of COPD [22]. -Increased levels of vascular endothelial growth factor in induced sputum of asthmatics.. Suggested to play important role in the pathogenesis of bronchial asthma [23].
-Uncontrolled activation of the coagulation cascade contributes to the pathophysiology of several conditions, including acute and chronic lung diseases. Protease-activated receptors have been implicated as the molecular link between coagulation and allergic inflammation in asthma [24]. -In contradiction with our finding showing lower levels of FX in PEx from asthmatic subjects as compared healthy controls, some studies report increased levels of FX in BALF. However, other studies report similar result with significantly decreased levels in airways in severe asthma and intermediate levels in moderate asthma, as compared to healthy controls [25].

Fructose-bisphosphate aldolase A
NA H A-nLCI IM A-hLCI L -Localized to the extracellular region, platelet alpha granule lumen, extracellular exosome. Involved in platelet degranulation, neutrophil degranulation (GO annotations) [6,7]. -Patients with asthma have high presence of nitrotyrosine in both the airways and the lung parenchyma [25].
-Has been observed to undergo post-translational regulation by protein tyrosine nitration in mast cells. Suggested to be an important pathway that regulates mast cell phenotype and function [26][27][28]. -Nitrotyrosine formation in airway epithelial and inflammatory cells is elevated in asthma and COPD [37].
-The ability to detect post-translational modifications is an important advantage of aptamers as tools for identification and detection of biomarkers, as exemplified by Ray. P et al [29]. The lower levels of ALDOA in A-hLCI group may be explained by tyrosine nitration of ALDOA proteins in mast cells in the A-hLCI group, resulting in weaker binding of the SOMAscan aptamer to the nitrated isoform of ALDOA, giving rise to weaker signal in A-hLCI group.

C4A (P0C0L4)
NA H A-nLCI IM A-hLCI L -Localized to extracellular region, blood microparticle. Involved in innate immune response, inflammatory response, complement activation (GO annotations) [6,7]. -Pulmonary alveolar type II epithelial cells synthesize and secrete complement proteins C2, C3, C4, C5, and Factor B. Studies have demonstrated that complement may serve as a key link between innate and adaptive immunity in a variety of pulmonary conditions [30].