DISCUSSION
Based on the analysis of select soluble immune mediators as well as leukocyte subset frequency and phenotype, findings of this pilot study suggest that collection of IS in 2-year-olds with CF is achievable and yield inflammatory biomarkers that are broadly comparable to BAL. As highly effective CFTR modulator therapy is expected to be introduced earlier and earlier in the course of early CF airway disease in coming years, ongoing changes are expected in the start and rate of progression of structural damage and development of bronchiectasis. This further emphasizes the need for identifying and validating non-invasive markers of lung disease that can be used to monitor the presence of lower airway inflammation in young children with CF. It is also important to recognize the regional heterogeneity that occurs in the initial stages of CF lung disease and performing BAL in one bronchopulmonary segment of a lobe may not accurately represent the changes that are occurring in other lung segments. IS has the advantage that it represents an aggregate sample that is reflective of global changes while likely overrepresenting diseased regions (with ongoing mucus impaction). In addition, IS is not affected by the dilution effect of serial lavages that are performed during the collection of a BAL sample. It is important to recognize intrinsic differences between IS and BAL as we investigate inflammatory markers best suited for disease monitoring via each sampling technique.
Out of the 20 soluble immune mediators selected for analysis, GM-CSF was the only one below the detection limit in the majority of IS samples, and IL-18 was the only one with lower levels in IS than in BAL. The other 18 were at equal or greater abundance in IS compared to BAL. This may be explained by the convergence of secretions from distal airways (reflected in BAL) to large airways (reflected in IS) and will benefit IS-based studies by enabling detection of mediators that may be below detection limits in BAL. Importantly, this pilot study shows that established markers of early inflammation in CF airways are measured robustly in IS. Among these, CXCL8 (IL-8) is one of the most potent neutrophil chemoattractants in CF lung disease [28], along with IL-6 and TNF-α. VEGF-A contributes to vascular permeability [29] and IL-1RA serves as a counter mechanism to IL-1α- and IL-1β-mediated pro-inflammatory signaling [30]. IL-1α itself was comparable between IS and BAL and is one of the first pro-inflammatory mediators present in early lung disease [31], which makes its measurement particularly critical.
By flow cytometry, IS neutrophils exhibited significant exocytosis of the secondary granules compared to visit-matched blood cells, reflecting a similar shift from blood as for BAL neutrophils. We previously described significant exocytosis of neutrophil primary granules in early CF lung disease, as evidenced by high CD63 and low CD16 expression characteristic of CF airway GRIM neutrophils [11]. Here, we observed similarly high proportion of GRIM neutrophils in IS and BAL, demonstrating that this key functional shift in neutrophils can be captured from distinct regions of the airways. As a result of primary granule exocytosis, NE is released into the extracellular environment, which we and others have shown to be associated with symptoms and severity of lung disease [7, 11]. Measurements of surface and soluble NE demonstrated some differences between IS and BAL in this study. Surface NE trended lower on IS than BAL neutrophils but was greater on IS monocytes/macrophages. In addition, soluble NE activity in IS was significantly reduced in IS compared to BAL. Taken together, these findings suggest that NE may be differentially compartmentalized in IS and BAL and that although sufficient NE is present in the IS to be captured on the surface of scavenger cells, its activity in IS may be inhibited by the antiprotease shield, as we previously suggested [11].
Besides phenotypic data of specific cell populations, comparing the presence of each major leukocyte class in the airways can provide important insights on status of lung disease. Early in disease, the CF airway lumen is dominated by monocytes/macrophages with occasional T cells. As disease progresses, neutrophils infiltrate the lumen with T cells largely excluded (reviewed in [32]). As expected in this 2-year-old cohort, monocytes/macrophages were dominant in BAL, and present in equal proportions in IS samples. Neutrophils were present to a lesser degree, which is also typical of early CF airway disease, and were equally represented in BAL and IS samples, too. T cells, however, while representing between 1-10% of total live leukocytes in BAL samples, were present at less than 1% in all IS samples. Thus, T cell numbers appear to dwindle more precociously in IS than in BAL at the onset of CF airway disease, a finding that warrants validation in a larger, longitudinal cohort of patients.
There are limitations to this pilot study. First, IS collection from young children requires additional maneuvers (use of high frequency chest wall oscillation and pharyngeal suctioning) and additional infection control precautions for the caregiver (see details in Supplemental Methods). Another limitation lies in the limited volume and cell yield inherent to IS collection in young children. This highlights the importance of applying sample-sparing/multiplexed and highly sensitive assays like the ones used in this study to the analysis of IS. Of note, in order to conduct side-by-side comparison of IS to BAL, we chose to normalize the calculated concentrations of soluble mediators and NE to that of total proteins. This method is likely a better approach than normalizing to volume, considering the possible differences in absorption of saline into the airway tissue for BAL, the variable output per lavage attempt, and the expected differences in density of IS samples. However, we previously noted total protein in BAL increases with PRAGMA-%Dis score [11, 33], likely as leukocytes secrete proteins into the lumen. As a result, protein normalization may also normalize in part to inflammatory burden. Finally, an important limitation of IS collection is the possibility for contamination of the sample by other fluids, including saliva and nasal passage secretions. In this study, we took particular precautions during the collection procedure to minimize such contamination (see Supplemental Methods for details).
In sum, the concomitant recruitment of neutrophils and acquisition of the GRIM phenotype, disappearance of T cells, and cytokine profile showing both pro- and anti-inflammatory mediators signifies that these patients who have minimal signs of lung damage by chest CT scan may still presen with measurable immunological signatures of progressing towards chronic CF lung disease. In this pilot study, we demonstrated that IS can be employed successfully in young children with CF to collect valuable data for multiparameter analysis of early CF lung disease. These findings warrant further testing of IS into disease monitoring efforts and interventional trials in early CF.