RESULTS
This study sought to understand the interplay among analytes,
relationships to disease severity, and to delineate patterns of
dysregulation through the first five years of life. Serum samples were
collected from a total of 87 pediatric AD patients upon entry, one, and
four years later [9]. The mean age at study entry was 10.4 months
and the population was evenly distributed both by gender and between
Caucasian and African American ethnicities (Figure 1). Incidence of
asthma at Y5 was observed in 65% of patients. AD severity, measured by
SCORAD, was mild to moderate at time of enrollment (range 1-28) and
maintained an average between 10.1-14.7 across the study years (Figure
1). A total of 126 serum proteins, including IgE, were analyzed across
different platforms to identify relevant connections between various
immunological markers and disease progression, using SCORAD, across each
time point of data collections. An important measure of atopy,
circulating IgE, was measured on average to be 29.6 IU/mL at year 1 (Y1;
baseline), 32.7 IU/mL at year 2 (Y2; one year after baseline), and 29.9
IU/mL at year 5 (Y5; four years after baseline). In this patient
population, IgE was only weakly correlated to SCORAD (corr=-0.22) based
on combined analysis across all timepoints of the study (data not
shown). Considering this weak correlation, we then shifted focus to
other key inflammatory serum markers possessing positive correlation
with clinical severity across all 3 visits. The strongest positive
correlation to SCORAD across all visits were CASP-8 (corr=0.48) and
IL-13 (corr=0.47) (Figure 2). Additional proteins positively correlated
with SCORAD in these pediatric AD patients that have been previously
linked to severity in adult AD included TARC (corr=0.37) and MCP-4
(corr=0.3) (Figure 2) [17]. Interestingly, we observed proteins
shown to be elevated in the circulation of adult AD patients to be
negatively correlated with SCORAD across all 3 timepoints taken in the
first 5 years of life from these pediatric AD patients, such as sCD40L
(corr=-0.3), Eotaxin-1 (corr=-0.3), and IL-7 (corr=-0.4) (Figure 2,
Supplemental Table 1) [17].
Understanding the dramatic developmental changes that occur in early
childhood, we utilized volcano plots to visualize differentially
expressed proteins between Y1 and both Y2 and Y5 (Figure 3a-b). Proteins
which had the highest positive correlations with disease severity, such
as IL-13, CASP-8, and TARC (Figure 2a) were observed to have the highest
concentrations early at Y1 relative to Y5 (Figure 3a). We also observed
decreasing serum concentrations of Th2 markers (TARC, IL-13, and MCP-4)
from Y1 to Y5, perhaps indicative of the dramatic elevation at early
stages of life for these patients (Figure 3c, Supplemental Figure 1).
The Th22 marker, IL-22, a highly expressed cytokine in adult AD was not
observed to change over the course of the study, suggesting the absence
of a significant role for IL-22 in the first 5 years of pediatric
disease within this cohort (Supplemental Figure 1) [18]. Similarly,
many Th1/Th17 markers, including CXCL10, IL-17A, and IFNγ shown to be
upregulated in adult AD were unchanged in our study [6]; however,
elevated serum concentration of the related marker, CXCL11, was observed
as early as Y2 (Supplemental Figure 1).
Given the correlation of inflammatory markers with SCORAD, we wanted to
determine how these serum proteins associated with each other. Thus, we
examined correlations among SCORAD-associated analytes across all time
points of the study. We observed strong interconnectivity within two
groups of analytes: 1) CD5, CASP8, IL-12b, TRANCE, and TNFRSF9 and 2)
CXCL12, CCL19, TRAIL, and IL-7 (Figure 4). Surprisingly, IL-13, which
emerged as one of the most significant correlates with SCORAD showed
only moderate associations with other protein concentrations across all
timepoints (Figure 4, Supplemental Table 1). This observation, coupled
with our finding that many inflammatory markers, such as IL-13, decrease
in concentration over the course of the study as patients age and
utilize standard of care therapies initiated further assessment of
pathway nodes connecting these markers to disease severity.
Molecular processes linked to the analytes with higher concentrations
observed at Y1 relative to Y5 were significantly associated with
pathways related to T cell activation and cytokine secretion profiles
including Immune response pathways in T cell differentiation and
cytokine secretion (Figure 3a, Figure 5a). Juxtaposed with the Y1
pathway analysis of T cell driven response mechanisms in early AD
development, Y5 pathway analysis highlights the shift from T cell driven
disease to include innate immunity, links to Langerhans and Dendritic
cells’ presence in allergic dermatitis, and the rise of asthma-related
mast cell mechanisms over time (Figure 5a-b). Proteins increased in
concentration at subsequent visits (Y2 and Y5) relative to Y1 were
sCD40L, ST1A1, 4E-BP1, CXCL12, CXCL11, CCL19 and pathway analysis
connected these to innate mechanisms (Figure 3b, Figure 5b). Most
notably, a 34-fold magnitude increase was observed in the circulating
levels of sCD40L at Y5 as compared to Y1 (Figure 3a). Molecular
processes observed to be upregulated at Y2 from Y1 were also affiliated
with innate immunity (Figure 3b) and tracked with similar innate
cell-influenced mechanisms observed at Y5 from Y1 (Figure 5b). Several
of these markers overlapped between Y2 and Y5 and all markers
significantly higher concentrations in Y5 were also seen in Y2
highlighting the early onset of these mechanisms (Figure 3a-b). These
differences reiterate the idea that blood samples from pediatric AD
patients contain strong Th2-driven signals early, though levels decrease
slightly with age as Th1 and innate-linked inflammatory markers develop.
Utilizing heatmap visualizations, we next assessed the correlations of
serum protein analytes to T cell phenotypes using previously described
flow cytometry analysis of PBMCs from the same individuals demonstrating
sweeping responses for analytes shown to track with changes in T cell
development from Y1 to Y5 (Figure 5c) [11]. We observed distinct
profiles at Y1 between circulating Th2 and Treg cells and
epithelial-derived chemokines, including two proteins most significantly
upregulated at Y1 to Y5; MIP-1a, sCD40L, CCL20, and IL-8 all showed
positive correlations with Th2 populations and were unchanged or
negatively correlated with Treg populations indicating these signaling
mediators promote T-cell mediated immune responses very early in
pediatric development (Figure 5c). sCD40L showed strong correlations at
both Y1 and Y5 with Th2 cells and did not correlate with any other cell
types (Figure 5c). Th2 cytokines, IL-4 and IL-5, strongly correlated
with NKT cell populations at Y1 and Y5, respectively, and IL-9 and
PDFGa2 positively correlated with Treg levels in Y1, but not at Y5
(Figure 5c).
Notwithstanding the complexity of immune responses observed in these
samples, the longitudinal nature of this study suggested that we may be
able to identify predictive biomarkers related to the course of disease.
We utilized the forward selection modeling approach with an Akaike
information criterion (AIC) stopping rule to predict the change of
SCORAD from Y1 to Y5 using the Y1 inflammatory protein concentrations. A
biomarker panel of 18 analytes was selected in the prediction model to
predict progression of severity from infancy to Y5 (Figure 6a,
R2=0.64, p=0.0058). To further illustrate the
robustness of prediction, a cross validation with 100 iterations on the
proposed 18 analytes predicted model resulted in the mean of the R
squares and root of mean square error (RMSE) of 0.77 and 3.5 for the 100
iteration, respectively (Figure 6b). The highlighted serum proteins,
which could be involved in persistence or resolution of AD, include
IL-18, MMP-10, and IL-13 (Figure 6).These analyses highlight the
objective and predictive nature of correlated analytes present in the
serum of infants for subsequent disease severity.