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.