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.