RESULTS
Parallel collection of IS, BAL and blood, and chest CT imaging
in 2-year-olds with CF. The ability to collect IS from young children
with CF was shown in prior studies but focused on a limited set of
cytokines [15] and on microbiology [17]. To assess the potential
of IS to yield data on an extended set of inflammatory biomarkers, we
collected it in parallel to BAL from the RML and LIN, blood and chest
CT, all within a single study visit. We attempted and achieved
collection of all samples and data on 11 subjects (demographics inTable 1 ), except for two subjects from whom we were unable to
obtain IS, thus lowering our analyzable set of IS samples to 9
(Table S1 ). Chest CT scoring confirmed that this cohort was at
an early stage of airway disease, with the total disease score (%Dis)
ranging from 0 to 5.9% (Table S2 ).
Similarities and differences in soluble immune mediators in IS
and BAL. Prior studies of BAL have shown that onset of neutrophilic
inflammation is a key determinant of structural lung damage in young
children with CF [18]. Thus, we selected 11 mediators impacting
neutrophil recruitment and activation (Figure 1 ). Five of those
mediators showed similar levels in IS and BAL, namely CXCL1, CXCL8
(IL-8), IL-1α, IL-1RA, and IL-6. Six of those mediators showed
differential levels between IS and BAL: CXCL5, G-CSF, IL-1β, and TNF-α
were higher in IS than in one or both of the BAL fractions while IL-18
was lower in IS than in LIN BAL. Finally, IL-10 was below the limit of
detection in most BAL samples but was measurable in most IS samples. We
also assessed 9 mediators impacting recruitment and activation of
monocytes/macrophages and T cells (Figure 2 ). Four of those
mediators showed similar levels in IS and BAL, namely CCL2, CXCL10,
M-CSF, and VEGF-A. Two of those mediators, CXCL11 and TNFSF10, showed
higher levels in IS than in BAL.Finally, CCL4 and IFN-γ were below
detection levels in most RML BAL samples while GM-CSF was below
detection limit in most IS samples.
Correlations in soluble immune mediator levels among collected
fractions. To explore potential relationships between the three airway
fractions collected, we assessed correlations between IS and RML BAL, IS
and LIN BAL, and RML and LIN BAL in the 20 immune mediators measured
(Figure S2 ). Six neutrophil-associated mediators had
significant positive correlations between IS and one or both of the BAL
fractions, namely, CXCL8, G-CSF , IL1α, IL-1RA, IL-6 and TNF-α. Two
monocyte/macrophage-associated mediators had significant positive
correlations between IS and one or both of the BAL fractions, namely,
M-CSF and VEGF-A. Between RML and LIN BAL fractions, mediators with
significant positive correlations included four neutrophil-associated
mediators (CXCL5, CXCL8, IL-1β, and IL-6) and three monocyte/macrophage
-associated mediators (CXCL11, GM-CSF and VEGF-A).
Next, we assessed cross-correlations of the three airway fluid fractions
with plasma (Figure S3 ). In all three airway samples, we
observed positive correlations between CXCL8 and IL-1β and CXCL11 with
TNFSF10. In IS and RML BAL, TNF-α and TNFSF10 correlated positively and
G-CSF and IL-1RA correlated negatively. In IS and LIN BAL, CCL2 and IL-6
correlated positively. In RML and LIN BAL, CXCL11 correlated positively
with CCL2, CXCL10, IL-1β and TNF-α and IL-1RA correlated positively with
TNF-α. Plasma cross-correlations differed from those in airway samples
except for positive correlations between IL-10 and IL-18 observed in
both plasma and IS; IL-1RA and TNF-α observed in plasma and both BAL
fractions; and CXCL8 and IL-1α as well as IL-1RA with IL-18 observed in
both plasma and RML BAL.
Cellular analysis of IS yields monocyte/macrophage, T cell, and
neutrophil subsets, with phenotypes similar to BAL. By flow cytometry,
we gated live monocytes/macrophages, T cells and neutrophils in BAL as
we showed before [11], and successfully applied the same gating
strategy to IS (Figure S4 ). Relative frequencies of neutrophils
and monocytes/macrophages were similar in IS and BAL fractions, with a
predominance of monocytes/macrophages in both. In contrast, the
frequency of T cells was significantly lower in IS compared to BAL
(Figure 3A ). Neutrophils were activated in IS compared to blood
based on increased surface expression of CD66b (secondary granule
exocytosis marker). However, surface CD63 (primary granule exocytosis
marker) was not significantly increased in IS compared to blood, likely
due to the very early stage of airway disease in this 2-year-old cohort.
CD16 expression was reduced on BAL, but not IS, neutrophils compared to
blood (Figure 3B ). Monocytes/macrophages in IS had
significantly higher surface levels of CD115 (M-CSF receptor), while
surface expression of CD163 (scavenger receptor) trended lower, as
compared to BAL. Monocytes/macrophages in IS also showed decreased
surface expression of CD16, which is sensitive to NE-mediated cleavage
(Figure 3C ).
We further analyzed neutrophil populations by determining the proportion
of neutrophils demonstrating the GRIM phenotype
(CD63high CD16low, as shown inFigure S4 ). The prevalence of these highly exocytic neutrophils
was not significantly different between IS and BAL. Although there was
substantial dynamic range among airway samples, all blood samples had
<1% GRIM neutrophils. Despite the high prevalence of GRIM
neutrophils in some IS samples, soluble NE (following release from the
primary granules of neutrophils) was lower in IS than in BAL
(Figure 4A ). Extracellular NE can be scavenged by neutrophils
and monocytes/macrophages, which we assessed by surface staining with
flow cytometry. In contrast to the difference with soluble NE
measurement, surface NE on neutrophils and monocytes/macrophages did not
differ between IS and BAL, while blood cells had lower surface NE, as
expected (Figure 4B ).