IL-5Rα marks nasal polyp IgG4- and IgE-expressing cells in aspirin-exacerbated respiratory disease

Abstract

Background: The cause of severe nasal polyposis in aspirin-exacerbated respiratory disease (AERD) is unknown. Elevated antibody levels have been associated with disease severity in nasal polyps, but upstream drivers of local antibody production in nasal polyps are undetermined.

Objective: We sought to identify upstream drivers and phenotypic properties of local antibody-expressing cells in nasal polyps from subjects with AERD.

Methods: Sinus tissue was obtained from subjects with AERD, chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP), CRS without nasal polyps, and controls without CRS. Tissue antibody levels were quantified via ELISA and immunohistochemistry and were correlated with disease severity. Antibody-expressing cells were profiled with single-cell RNA sequencing, flow cytometry, and immunofluorescence, with IL-5Rα function determined through IL-5 stimulation and subsequent RNA sequencing and quantitative PCR.

Results: Tissue IgE and IgG4 levels were elevated in AERD compared with in controls (P < .01 for IgE and P < .001 for IgG4 vs CRSwNP). Subjects with AERD whose nasal polyps recurred rapidly had higher IgE levels than did subjects with AERD, with slower regrowth (P = .005). Single-cell RNA sequencing revealed increased IL5RA, IGHG4, and IGHE in antibody-expressing cells from patients with AERD compared with antibody-expressing cells from patients with CRSwNP. There were more IL-5Rα⁺ plasma cells in the polyp tissue from those with AERD than in polyp tissue from those with CRSwNP (P = .026). IL-5 stimulation of plasma cells in vitro induced changes in a distinct set of transcripts.

Conclusions: Our study identifies an increase in antibody-expressing cells in AERD defined by transcript enrichment of IL5RA and IGHG4 or IGHE, with confirmed surface expression of IL-5Rα and functional IL-5 signaling. Tissue IgE and IgG4 levels are elevated in AERD, and higher IgE levels are associated with faster nasal polyp regrowth. Our findings suggest a role for IL-5Rα⁺ antibody-expressing cells in facilitating local antibody production and severe nasal polyps in AERD.

Keywords: Aspirin-exacerbated respiratory disease; IL-5Rα; IgE; IgG4; chronic rhinosinusitis; interleukin-5; nasal polyposis; plasma cell.

Figure 1.. Nasal tissue IgE and IgG4 levels are elevated in AERD and relate to nasal polyp recurrence.

Total tissue levels of (A) IgE and (B) IgG4 were measured by ELISA from concha bullosa samples of patients without sinus inflammation (non-CRS controls), sinus mucosa of patients with CRSsNP, and nasal polyp tissue from patients with aspirin-tolerant CRSwNP and AERD. Nasal polyp IgE (C) and IgG4 (D) levels in AERD patients with rapid nasal polyp regrowth (< 6 months) or slower nasal polyp regrown (≥ 6 months). The nasal polyp IgG4, but not IgE levels, from patients with aspirin-tolerant CRSwNP and AERD correlate with lifetime duration of nasal polyposis (E-F). Data in A - D are mean ±SEM, correlation in E and F was calculated by Spearman.

Figure 2.. IgG4⁺ antibody-expressing cells are specifically elevated in nasal polyps from patients with AERD.

(A) Number of IgG4⁺ lymphocytes per HPF from nasal polyp tissue of patients with aspirin-tolerant CRSwNP and AERD, n=5 for each group. Data in A are mean ±SEM. (B - D) Representative samples (CRSwNP, B and AERD, C, magnified in D) of nasal polyp tissue stained with anti-IgG4. Black arrows identify IgG4⁺ cells.

Figure 3.. ScRNA-seq of antibody-expressing cell populations from sinus tissue of subjects with CRSsNP (n=5), CRSwNP (n=3) and AERD (n=3).

(A) UMAP plot of 2,520 antibody-expressing cells from sinonasal tissue of CRSsNP, CRSwNP and AERD patients, indicating 9 clusters identified through a shared nearest neighbor analysis. (B) UMAP plot of sinonasal antibody-expressing cells, colored by disease of origin. Statistical enrichment for AERD disease-of-origin was observed for cluster 2 (P<1×10⁻¹⁵), cluster 3 (P<1×10⁻¹⁵), cluster 4 (P<2×10⁻¹⁵), cluster 6 (P<1×10), and cluster 7 (P<1×10⁻¹⁵) (C) Violin plots of select genes significantly enriched in AERD relative to CRSsNP and CRSwNP within sinonasal antibody-expressing cell populations, including IGHG4 (P<1×10⁻²⁰¹), IGHE (P<2×10⁻³⁴), and IL5RA (P<2×10⁻²¹). Cohen’s d effect size for AERD relative to CRSwNP is 1.57, 0.50, and 0.37, respectively for the 3 transcripts.

Figure 4.. Flow cytometric characterization of plasma cells and immunofluorescence of polyp tissue.

(A) Nasal polyp plasma cell frequency as a percentage of CD45⁺ cells, (B) plasma cell surface expression of IL-5Rα, (C) B cell surface expression of IL-5Rα, and (D) immunofluorescence staining of nasal polyp tissue for plasma cells (white arrows) co-expressing IL-5Rα (green) and CD138 (red).

Figure 5.. Transcriptional consequence of IL-5 stimulation of human nasal polyp plasma cells.

(A) Heatmap representation of differentially expressed transcripts (FDR < 0.1) in plasma cells from two subjects with AERD treated with IL-5 (1 ng/mL, 6 hours) relative to vehicle control treatment. Scale bar indicates Z-score scaled by row. (B) CCND2 fold change with IL-5 stimulation (1 ng/mL, 6 hours) in nasal polyp plasma cells from three subjects with AERD, normalized to glyceraldehyde-3-phosphate dehydrogenase; paired t-test.