Functional immunophenotyping of children with critical status asthmaticus identifies differential gene expression responses in neutrophils exposed to a poly(I:C) stimulus

Abstract

The host immune response to a viral immune stimulus has not been examined in children during a life-threatening asthma attack. We determined whether we could identify clusters of children with critical asthma by functional immunophenotyping using an intracellular viral analog stimulus. We performed a single-center, prospective, observational cohort study of 43 children ages 6-17 years admitted to a pediatric intensive care unit for an asthma attack between July 2019 to February 2021. Neutrophils were isolated from children, stimulated overnight with LyoVec poly(I:C), and mRNA was analyzed using a targeted Nanostring immunology array. Network analysis of the differentially expressed transcripts for the paired LyoVec poly(I:C) samples was performed. We identified two clusters by functional immunophenotyping that differed by the Asthma Control Test score. Cluster 1 (n = 23) had a higher proportion of children with uncontrolled asthma in the four weeks prior to PICU admission compared with cluster 2 (n = 20). Pathways up-regulated in cluster 1 versus cluster 2 included chemokine receptor/chemokines, interleukin-10 (IL-10), IL-4, and IL-13 signaling. Larger validation studies and clinical phenotyping of children with critical asthma are needed to determine the predictive utility of these clusters in a larger clinical setting.

Figure 1

Overall study design sample processing (A). Neutrophils were isolated by negative magnetic bead selection from children ages 6–17 years admitted to the pediatric intensive care unit for an asthma exacerbation. Neutrophils (2 × 10⁶) were stimulated with low molecular weight (LMW) LyoVec poly(I:C) (740 n/ml) or vehicle solution for 20 h at 37 °C in a 5% CO₂ humidified incubator. Following stimulation, neutrophils were isolated, resuspended in RNALater, and stored at – 80 °C until RNA was isolated for batch poly(I:C) gene transcription analysis using the Human Immunology v2 NanoString nCounter Gene Expression CodeSet. Following UMAP dimension reduction (Step 1), k-means clustering was used minimize within-cluster sum of squares and Silhouette analysis was performed to yield two clusters of differentially expressed genes. RNA was also isolated from neutrophils without any treatment (baseline). In Step 2, the differential gene expression of baseline neutrophils was analyzed using the two clusters defined by the poly(I:C) stimulated vs. unstimulated sample cluster analysis shown in Step 1. Data analysis workflow (B). Paired differential gene expression was determined using poly(I:C) stimulated vs. unstimulated samples. Differentially expressed genes were Z-score normalized. Principal component analysis was performed with 20 components, followed by uniform manifold approximation and projection (UMAP) dimensionality reduction. K-means clustering was performed on the two-dimensional UMAP to yield two clusters. The two poly(I:C)-stimulation defined clusters were used to analyze differentially expressed genes from the baseline neutrophils immediately isolated from patients with no stimulation. (A) was created with BioRender.com with a confirmation of publication and licensing rights.

Figure 2

Uniform Manifold Approximation and Projection (UMAP) of component 1 versus component 2 showing two clusters of participants with differentially expressed genes following poly(I:C) stimulation. Cluster 1 (blue circles). Cluster 2 (orange circles). Ellipses indicate the boundaries of one standard deviation (σ) by a solid red line, two σ by a pink dashed line, and 3σ by a blue dotted line.

Figure 3

Bar graph of the top fourteen Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways differentiating cluster 1 from cluster 2 by gene over-representation analysis (gene number) based on the differentially expressed genes following poly(I:C) stimulation.

Figure 4

Box and whisker plots of the top ten differentially expressed genes by cluster from baseline (unstimulated) neutrophils. The middle line is the median, the bottom and top box edges are the 25th and 75th percentiles, respectively, and the whiskers are the minimum interval. (A) C14orf166, (B) Intercellular adhesion molecule 3 (ICAM3), (C) Autophagy-Related Gene 5 (ATG5), (D) B-cell lymphoma 2 (BCL2), (E) C–C chemokine receptor type 5 (CCR5), (F) Colony Stimulating Factor 1 (CSF1), (G) C–X–C Motif Chemokine Receptor 3 (CXCR3), (H) Leukocyte Associated Immunoglobulin Like Receptor 1 (LAIR1), (I) ABL Proto-Oncogene 1, Non-Receptor Tyrosine Kinase (ABL1), (J) CD9.