Single cell transcriptomics reveal type-2 inflammatory gene regulation in airway epithelium driven by allergen-specific immunotherapy

Background: Airway epithelium in patients with allergic airway disease actively releases and is subjected to the influence of type-2 cytokines with observable changes in basal epithelial cells. Symptoms that grass pollen-allergic patients experience due to this priming can be controlled by allergen-specific immunotherapy (AIT). However, the impact of AIT on type-2 related mediators of airway epithelial cells is unknown. Methods: Protein levels of type-2 cytokines in nasal secretions of grass pollen-allergic patients were analyzed during 3-year AIT-regimen using multiplex-ELISA. Source and IL-4-dependence of type-2 cytokines were examined in IL-4-primed primary ALI cultures using single-cell transcriptomics. Results: Type-2 cytokines CCL-26 and POSTN oscillated seasonally, derived from basal epithelial cells and were induced by IL-4-priming. In contrast, TSLP and IL-33 derived from basal cells, albeit independently of IL-4. POSTN and IL-24 nasal levels changed corresponding to 3-year AIT progression. Four characteristic epithelial type-2 cytokines stood out: IL-33 changed independent of season, IL-4-exposure, or AIT; CCL-26 was triggered upon IL-4-priming and was induced during pollen season, but not influenced by AIT; IL-24 decreased following three years of AIT; and POSTN, which was increased in IL-4-primed basal epithelial cells and oscillated during pollen season, also responded to long-term AIT. Conclusions: Atopic reprogramming of type-2 epithelial cytokines seems to persist despite long-term AIT, which may be one mechanism explaining why AIT only restores allergen tolerance for seven to ten years. Type-2 related epithelial cytokines are differentially expressed which may relate to the distinct expression of basal as opposed to differentiated secretory or ciliated epithelial cells. Overall design: The single-cell suspension was subjected to library preparation for single-cell 5' gene expression analysis using the Chromium Next GEM Single Cell 5' Library Kit v1.1 (10x Genomics, Leiden, The Netherlands), the Chromium Next GEM Single Cell Gel Bead Kit v1.1 (10x Genomics), and the single-cell partitioning instrument Chromium Controller (10x Genomics) according to the manufacturer's instructions. The Bioanalyzer High Sensitivity DNA Analysis (Agilent, Waldbronn, Germany) and KAPA Library Quantification Kit (Hoffmann-La Roche, Basel, Switzerland) were used to control the quality and quantity of the libraries prior to submission for sequencing.