Sputum Cellularity and Immune Biomarkers During Acute Exacerbations of Asthma

Abstract Rationale: Acute asthma exacerbations are frequently triggered by viral infections and the airway epithelium is the principal target. Various pro-inflammatory molecules are released including the alarmins (interleukin (IL)-33, thymic stromal lymphopoietin and IL-25). The sputum is frequently used to phenotype asthma but is subject to contamination from the upper airways. The aim of the present study was to assess the markers of innate immune responses during an acute exacerbation of asthma by the analysis of sputum and to relate the levels of pro-inflammatory molecules to the quality of the sputum as reflected in the percentage of squamous cells.Methods: We studied asthmatic subjects (n= 45) presenting to the Montreal Chest Institute with a physician diagnosed asthma exacerbation. Clinical data, spirometry, Asthma Control Questionnaires (ACQ), complete blood counts and expectorated sputum were acquired on admission to the study (day 1) and on days 10 and 28. Sputum was processed for differential cell counts and sputum supernatants from a subset of subjects (n=25) were analysed by Bioplex for 25 inflammatory markers. Cysteinyl leukotrienes (CysLTs) in the sputum supernatants were analyzed by enzyme immunoassay. Results: On day 1, subjects had an ACQ score of 3.56 ± 0.84 and an FEV1 was 69.1 ± 21.4 (% predicted). Blood eosinophil counts >150/µl were present in 26/45. Expectorated sputum had squamous cell counts that ranged from 0 to 90%, with a median value of 16.81%. Mediator levels (interleukin (IL)-4, IL-5, IL-8, and TNF-α) were significantly lower in samples for which squamous cells exceeded 16 (%). Similarly, CysLTs were significantly higher in sputum squamous cell counts < 16% (median 697 pg/ml) than when sputum squamous cells were > 16% (median 364 pg/ml; p=0.006). Alarmins were an exception to this finding and levels of IL-33 were 5.29 ± 1.12pg/ml for samples below 16% squamous cells and 8.13 ±2.74 pg/ml for samples above 16%. Similarly, TSLP levels were not statistically significantly different for sputum with < 16% squamous cells to > 16% squamous cells (1.39pg ± 0.43/ml vs 2.15 ± 2.23 pg/ml) and for IL-25 (4.16 ± 2.87 pg/ml and 6.19 ± 1.17 pg/ml, respectively). Alarmin levels were not significantly different between subjects with peripheral blood eosinophils above and below 150/µl.Conclusions: Selected cytokines/chemokines and CysLTs in expectorated sputum are likely under-estimated in sputum samples in which squamous cells are elevated. Alarmin levels are not affected by the squamous cell counts, suggesting a significant contribution to these mediators from the oropharynx.