Cell Type-Specific IL-5Rα Distribution and IL-5-Induced CCL11 Expression in Human Nasal Epithelium

Abstract

Allergic airway diseases alter epithelial composition through Th2 cytokines, particularly IL-4, IL-13, and IL-5 [1]. While IL-5 signaling is traditionally associated with eosinophil regulation via its receptor (IL-5R) [2], its role in epithelial cell function remains incompletely understood. IL-5 synergizes with eotaxins in the airways to recruit eosinophils, emphasizing its role in inflammatory cell recruitment [3]. Recent studies have demonstrated IL-5R expression in bronchial epithelial cells [4], but there are conflicting reports about its functionality in nasal epithelial cells [5]. Our study reveals previously unrecognized expression patterns of IL-5R in airway epithelium that may significantly influence therapeutic responses to biologics, particularly in allergic airway diseases.

Using single-cell RNA sequencing of human nasal epithelial cells from healthy controls, we discovered that IL-5Rα expression is predominantly restricted to deuterosomal and multiciliated cells, contrasting with the broader expression of IL-4R and IL-13Rα1 across cell types. Immunofluorescence staining revealed that IL-4R colocalizes with MUC5AC-positive goblet cells in nasal epithelium (Figure S4). IL-13Rα2 showed minimal expression in all populations. This distinct pattern was consistently observed in both ex vivo nasal epithelial cells and in vitro cultures, with IL-4 exposure notably decreasing IL-5Rα expression while modulating other key differentiation markers (Figure 1A–C).

Immunofluorescence analysis confirmed these findings, demonstrating clear IL-5Rα colocalization with acetylated-tubulin (a multiciliated cell marker) in healthy tissues while showing markedly reduced expression in eosinophilic or non-eosinophilic nasal polyp tissues (Figure 1D). Importantly, IL-5Rα showed no colocalization with MUC5AC (a goblet cell marker) under any conditions (Figure 1E,F), establishing its specific localization to the apical surface of multiciliated cells.

Further molecular analysis revealed that IL-4 exposure reduced both IL-5Rα mRNA and protein levels while upregulating MUC5AC expression and modulating other epithelial markers, including KRT5, SOX2, CD44v3, and MUC5B (Figure 2A–G, Figure S5). Consistent with previous studies on airway remodeling in type 2 inflammation [1, 6], our in vitro stimulation with IL-4 resulted in a significant reduction of multiciliated cells with concurrent goblet cell hyperplasia. IL-5R mRNA expression was significantly higher in differentiated (ALI) than in undifferentiated human nasal epithelial cells, with IL-4 suppressing and IL-5 maintaining this expression pattern in differentiated cells only (Figure S1). We further demonstrate that altered expression patterns of key cytokine receptors accompany this phenotypic shift. Since IL-5Rα is present in epithelial cells, we hypothesized that IL-5 could directly stimulate epithelial cells. To test this hypothesis, we investigated whether IL-5 exposure would affect chemokine secretion in epithelial cells as it is associated with eosinophil recruitment. This observation prompted an extended analysis of downstream mediators, including CCL11 and CCL26, suggesting a potentially underrecognized axis of epithelial–eosinophil interaction. Interestingly, IL-5 exposure decreased IL-5Rα protein levels without affecting mRNA expression, suggesting posttranscriptional regulation mechanisms (Figure 2A,H). While recent work has reported a lack of IL-5 signaling in nasal epithelial cells due to absent CSF2RB expression [5], we observed that IL-5 stimulation increased CCL11 mRNA expression, peaking at day 1 (Figure 2I), with an increase in CCL11 protein level detected at day 7 (Figure S2). At the same time, CCL26 remained unchanged (Figure 2J). In contrast, IL-4/IL-13 stimulation induced CCL11 (peaking at day 1) and CCL26 (increasing until day 7), indicating differential regulation of eosinophil-recruiting chemokines and suggesting pathway-specific roles in inflammatory cell recruitment. Taken together, these results indicate that CCL11 is produced by IL-5, IL-4, and IL-13 stimulations primarily in ciliated cells, and CCL26 is produced by IL-4 and 13 stimulations in a wide range of cell types, including both ciliated and goblet cells.

IL-5Rα is predominantly expressed in multiciliated and deuterosomal cells within human nasal epithelial cultures but is progressively downregulated during their differentiation into goblet cells under type 2 inflammatory conditions. Mechanistically, IL-4 likely suppresses FOXJ1-driven multiciliogenesis and promotes MUC5AC expression and goblet cell hyperplasia, partly through STAT6-dependent pathways, as demonstrated for IL-13 [7, 8]. This aligns with our findings of IL-4-induced depletion of deuterosomal and multiciliated cells, as well as a secretory shift in the epithelium [1, 6, 9]. These findings suggest that epithelial phenotypes could predict responses to biologics. The differential expression patterns of IL-5Rα in multiciliated and deuterosomal cells, despite potential signaling limitations [5], may still influence therapeutic responses through direct and indirect mechanisms. Similarly, hyperplasia of basal or suprabasal cells expressing high levels of IL-4Rα and IL-13Rα1 could show better responses to targeted therapies such as dupilumab, indicating the importance of cellular phenotyping in treatment selection. Suppose a high number of ciliated epithelial cells characterizes the epithelial phenotype. In that case, IL-5Rα expression is likely to be high, and the patient is expected to respond well to benralizumab, which targets this receptor. Furthermore, as epithelial cells are a significant source of CCL11 and CCL26, inhibition of IL-5Rα may reduce eosinophil recruitment, thereby contributing to the resolution of inflammation. In our study, although we focused on the epithelial cells, the IL-5Rα is also expressed in mast cells and plasma cells, which are elevated in ECRS tissues. Therefore, ECRS patients with greater ciliated cell populations likely have higher overall IL-5Rα levels and may respond more favorably to benralizumab therapy, which specifically targets IL-5Rα. In this study, we did not analyze differences in IL-5R expression among immune cells like mast cells, eosinophils, and plasma cells. However, when examining online single-cell RNA sequencing data from nasal polyp tissue, we observed that mast cells and plasma cells show higher IL-5R expression (Figure S3). Recent studies have also demonstrated IL-5Rα expression in eosinophils [10], plasma cells [11], and mast cells [10, 12], further supporting our observations. Additional studies will be necessary to investigate this finding more thoroughly.

This newly identified type 2 cytokine receptor expression pattern across epithelial populations provides a framework for understanding variable responses to biological therapies in allergic airway diseases. The broader epithelial distribution of IL-4Rα may underlie the greater clinical efficacy of dupilumab in CRSwNP, whereas the more restricted, phenotype-dependent expression of IL-5Rα could help explain the limited effectiveness of benralizumab in certain epithelial contexts. Even within type 2 inflammation, receptor expression patterns can vary based on disease severity and underlying mechanisms. These receptors' differential expressions and regulation may explain the variable clinical responses observed with different biologics in upper versus lower airway disease. For example, anti-IL-5Rα therapies show robust efficacy in eosinophilic asthma [13] but yield mixed results in chronic rhinosinusitis [14], possibly due to tissue-specific differences in receptor expression and functionality. Future studies correlating epithelial phenotypes with treatment outcomes would increase the usefulness of our findings and enhance the responsiveness of biologics in resistant cases.

Conceptualization: Y.K. and H.J.C. Experiment: Y.K., M.K., J.S. and S.K. Data analysis: Y.K. and H.J.C. Writing reviews and editing: M.S.R., C.H.K., and H.J.C. Funding acquisition: H.J.C. Writing: Y.K. and H.J.C.

The authors declare no conflicts of interest.