Allergen-induced activation of natural killer cells represents an early-life immune response in the development of allergic asthma

Abstract

Background: Childhood asthma in inner-city populations is a major public health burden, and understanding early-life immune mechanisms that promote asthma onset is key to disease prevention. Children with asthma demonstrate a high prevalence of aeroallergen sensitization and T_H2-type inflammation; however, the early-life immune events that lead to T_H2 skewing and disease development are unknown.

Objective: We sought to use RNA sequencing of PBMCs collected at age 2 years to determine networks of immune responses that occur in children with allergy and asthma.

Methods: In an inner-city birth cohort with high asthma risk, we compared gene expression using RNA sequencing in PBMCs collected at age 2 years between children with 2 or more aeroallergen sensitizations, including dust mite, cockroach, or both, by age 3 years and asthma by age 7 years (cases) and matched control subjects who did not have any aeroallergen sensitization or asthma by age 7 years.

Results: PBMCs from the cases showed higher levels of expression of natural killer (NK) cell-related genes. After cockroach or dust mite allergen but not tetanus antigen stimulation, PBMCs from the cases compared with the control subjects showed differential expression of 244 genes. This gene set included upregulation of a densely interconnected NK cell-like gene network reflecting a pattern of cell activation and induction of inflammatory signaling molecules, including the key T_H2-type cytokines IL9, IL13, and CCL17, as well as a dendritic cell-like gene network, including upregulation of CD1 lipid antigen presentation molecules. The NK cell-like response was reproducible in an independent group of children with later-onset allergic sensitization and asthma and was found to be specific to only those children with both aeroallergen sensitization and asthma.

Conclusion: These findings provide important mechanistic insight into an early-life immune pathway involved in T_H2 polarization, leading to the development of allergic asthma.

Keywords: Asthma; allergens; dendritic cells; natural killer cells; transcriptomics.

Fig. 1. Differential expression of NK cell genes is associated with allergic asthma

(A) Site, not case-control assignment, accounted for the greatest source of gene expression variability in unstimulated PBMCs by PCA. (B) The set of differentially expressed genes between Early and Neither in unstimulated PBMC samples (NS linear model with fixed effects for site, gender, blood draw year; n_Early = 19, n_Neither = 30). 13 genes were significant at FDR<0.05 and FC>1, indicated by a red dot. Labelled genes (arrowheads) are NK cell marker genes in the IRIS database that have an FDR<0.05. Genes with a semi-transparent red dot have a FC>1 and FDR<0.25. Genes with a semi-transparent blue dot have a FC<1 and FDR<0.25. (C) Genes that showed higher expression in Early compared to Neither were highly enriched for IRIS NK cell marker genes and no other cell type by Fisher’s exact test of overlap between significant genes and IRIS marker genes. (D) There were no differentially expressed genes with FDR<0.05 between Early and Neither after tetanus toxoid stimulation (tetanus stimulation linear model with fixed effects for site, gender, blood draw year; n_Early = 19, n_Neither = 30).

Fig. 2. Allergen stimulation of PBMCs leads to robust gene expression changes

(A) Stimulus accounts for the greatest source of gene expression variability in PBMCs by PCA. Ellipses represent 95% confidence intervals. (B) CR-Early individuals demonstrated significant differences in expression of 206 genes in response to CR stimulation compared to Neither (FDR<0.05). (C) DM-Early individuals demonstrated significant differences in expression of 91 genes in response to DM stimulation compared to Neither (FDR<0.05) (Stimulation linear modeling with fixed effects for site, gender, blood draw year, home allergen levels, and stimulation and random effect for individual; n_CR-Early = 16, n_DM-Early = 13, n_Neither = 30). Differentially expressed genes in both comparisons are enriched for similar GO BP pathways. (D) Significantly upregulated genes in the Early group in NS, CR, and DM stimulated samples show a high degree of overlap as depicted in a bipartite network (P=3.6E-75; Fisher’s exact test, CR and DM upregulated genes).

Fig. 3. Allergen stimulation of PBMCs cause NK cell activation and Th2-like inflammation

(A) The 244 genes could each be assigned to 1 of the 6 cell subsets using marker gene cell deconvolution. Sensitivity analysis showed that the gene assignments were relatively stable over 100 bootstrap simulations (SPEC sensitivity analysis). (B) The NK cell gene set shows significantly higher baseline expression in Early compared to Neither and increases significantly with allergen stimulation in both the CR-Early and DM-Early groups; bounds represent 95% confidence intervals (Stimulation linear models with fixed effects for site, gender, blood draw year, home allergen levels, and stimulation; random effect for individual; p-value and FC represent group term controlling for stimulation). (C) NK cell genes represent a densely connected network of known gene-gene interactions. (D) Boxplot of normalized expression of IL9, IL13, and CCL17 showing group difference with allergen stimulation (Pink=CR-Early, Blue=DM-Early, Grey=Neither).

Fig. 4. NK cell gene activation is specific to allergic asthma

(A) The Late group demonstrated significant differences in expression of similar genes in response to CR stimulation compared to controls (31 genes at FDR<0.05, 326 genes at FDR<0.25). (Stimulation linear model with fixed effects for site, gender, blood draw year, home allergen levels, and stimulation, and random effect for individual; n_Late = 7, n_Neither = 14). (B) The NK cell gene set showed significantly higher baseline expression in Late compared to Neither, and expression increased significantly with CR stimulation in Late (p-value and FC represent group term controlling for stimulation; number of individuals in each comparison is represented in parentheses; bounds represent 95% confidence intervals) (C) The NK cell gene set shows significantly higher baseline expression in all cases (Red; “Both” = Early and Late groups, n=20) compared to Neither (Black, n=102), asthma only (Blue; “Asthma”, n=40), and CR allergy only (Green; “Allergy”, n=35) groups and increases with CR stimulation only in the Both group. (D) Boxplot of normalized expression of IL9, IL13, and CCL17 showing group difference with allergen stimulation (Pink=CR-Early, Blue=DM-Early, Grey=Neither). (E) The average expression value of the NK cell gene set showed correlation with Bla g 1 levels measured in the living room (p<0.05) and bedroom (p=0.08) in the Both group.