Transcriptomic Profiles of the Nasal Mucosa Following Birch Pollen Provocation Differ Between Birch Pollen-Allergic and Non-Allergic Individuals

Abstract

Background: Birch pollen (BP) interacts with airway epithelial cells to cause allergic sensitization and allergy in predisposed individuals. However, the basic mechanisms underlying the clinical effects are poorly understood. Changes in gene expression and cytokine secretion in nasal mucosal cells upon BP exposure were determined in BP-allergic and non-allergic individuals.

Methods: BP-allergic (n = 11) and non-allergic individuals (n = 12) participated in nasal provocations with saline and aqueous BP solution. Nasal scrapings and secretions were obtained at baseline and after BP provocation. Bulk RNA sequencing of the nasal scrapings was performed, and cytokines in nasal secretions were quantified.

Results: After BP challenge, we identified 160 differentially expressed genes (DEGs) in the nasal scrapings of allergic individuals and 44 in non-allergic individuals. DEGs encoding S100 proteins, keratins, small proline-rich repeat proteins, and cytokines were predominantly identified, with proinflammatory cytokine transcripts being upregulated only in the allergic cohort. The top canonical pathways in allergic individuals included granulocyte and agranulocyte adhesion and diapedesis, wound healing, IL-8 signaling, and IL-17-related pathways. Enriched pathways in allergic participants were associated with granulocyte chemotaxis, humoral cell responses, and IL-10, IL-4, and IL-13 signaling and were absent in non-allergic individuals. At baseline and after BP challenge, higher amounts of CCL17, CCL20, CCL26, IL-7, IL-16, and IL-33 were detected in nasal secretions of allergic compared to non-allergic individuals.

Conclusion: Our results highlight the activation of important cellular signaling pathways specific to BP-allergic individuals after BP exposure offering new perspectives for studying key players in BP allergy.

Keywords: birch pollen allergy; cytokines; differentially expressed genes; nasal mucosa; nasal provocation.

FIGURE 1

Study overview and differential gene expression analysis. (A) Study design overview: The study was conducted outside the BP season with 11 BP‐allergic and 12 non‐allergic individuals. The participants underwent nasal provocation with saline (baseline) and BP at two separate visits. Nasal secretions and nasal scrapings were collected at 15 min at baseline and at 15, 30, 60, or 120 min post BP challenge. (B) Number of differentially expressed genes (DEGs) with |log2 (fold change)| > 1 (log₂ values) and adjusted p value < 0.05 (−log₁₀ values) at the four different time points in allergic and non‐allergic individuals. A, allergic; NA, non‐allergic. (C) DEGs visualized as volcano plots. Red dots represent upregulated genes, blue dots represent downregulated genes, and gray dots represent genes with no significant difference between baseline and nasal allergen challenge. (D) Overlap of DEGs between allergic and non‐allergic group after nasal BP challenge with enrichment plot of the common DEGs. Circle size represents the adjusted p value, and log2 fold change values are marked in different colors.

FIGURE 2

Pathway analysis. (A) The top 10 significant (−log₁₀ (p value) < 1.3) canonical pathways in the allergic population, obtained by ingenuity pathway analysis using DEGs identified at earlier (15, 30 min) and later (60, 120 min) time points. (B, C) Top 10 significantly enriched pathways from gene set enrichment analysis at the different time points identified using gene ontology biological processes and reactome database in the (B) allergic group and (C) non‐allergic group. The horizontal axis represents normalized enrichment score (NES). Circle size represents the gene to set size ratio in each term. Adjusted p values (p.adjust) are marked in different colors.

FIGURE 3

Cell deconvolution analysis. Box plots depicting cell fractions in the nasal scrapings of study participants analyzed using CIBERSORTx. Pairwise comparisons were performed using Wilcoxon signed‐rank test. The box extends from the 25th to the 75th percentile, and the line in the middle of the box represents the median. The whiskers are plotted from the lowest to the highest value. * indicates p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001.

FIGURE 4

Upstream regulator analysis. Gene network depiction of the top four significant cytokine URs using DEGs in (A) the allergic group at earlier (15, 30 min), (B) at later (60, 120 min) time points, (C) the non‐allergic group at earlier (15, 30 min), and (D) at later (60, 120 min) time points. The cytokine URs determined by IPA are highlighted in bold and represented by square symbols, color‐coded (orange/blue) depending on their activation status (activation z‐score), white squares representing unidentified z‐scores. DEGs from our experimental data are represented by circles and arranged according to their cellular location. Red and green circles represent up and downregulated genes, respectively. The predicted relationship is color coded to indicate activation (orange) or inhibition (blue) and the pointing arrows indicating their relationship (direct/indirect). Relationships that are inconsistent with the prediction (yellow) or that are unpredicted (gray) are also shown.

FIGURE 5

Cytokine profiles of the study cohort. (A) Overview of the 19 detectable cytokines visualized as a heatmap. The values are plotted as log2 cytokine concentration. On the vertical axis, the individual participants are categorized based on the time point of sample acquisition. Values below the detection limit were replaced with the quarters of the lowest detection limit. Cytokines with more than 70% of values beyond the detection limit, measurements with extreme standard deviation and single measurements were excluded from the analysis, which are represented as white boxes. BAFF, B‐cell activating factor; BP, birch pollen; CCL, CC chemokine ligand; G‐CSF, granulocyte‐colony‐stimulating factor; IL, interleukin; S, saline; TNF, tumor necrosis factor; TSLP, thymic stromal lymphopoietin; VEGF, vascular endothelial growth factor. (B) Box plots of cytokines significantly elevated in allergic patients after BP nasal provocation. The box extends from the 25th to the 75th percentile, and the line in the middle of the box represents the median. The whiskers are plotted from the lowest to the highest value. * indicates p < 0.05, **p < 0.0, ***p < 0.001.

FIGURE 6

Top conditions showing similarity in transcriptional profiles to DEGs of allergic and non‐allergic groups. Pie charts visualize the tissue types and/or anatomical parts attributed to the top 50 outcomes from GENEVESTIGATOR for the allergic and non‐allergic groups at earlier and later time points.