IL-33-responsive innate lymphoid cells are an important source of IL-13 in chronic rhinosinusitis with nasal polyps

Abstract

Rationale: Chronic rhinosinusitis (CRS) without nasal polyps (CRSsNP) and CRS with nasal polyps (CRSwNP) are associated with Th1 and Th2 cytokine polarization, respectively; however, the pathophysiology of CRS remains unclear. The importance of innate lymphoid cells in Th2-mediated inflammatory disease has not been clearly defined.

Objectives: The objective of this study was to investigate the role of the epithelial cell-derived cytokine IL-33 and IL-33-responsive innate lymphoid cells in the pathophysiology of CRS.

Methods: Relative gene expression was evaluated using quantitative real-time polymerase chain reaction. Innate lymphoid cells in inflamed ethmoid sinus mucosa from patients with CRSsNP and CRSwNP were characterized using flow cytometry. Cytokine production from lymphoid cells isolated from inflamed mucosa of patients with CRS was examined using ELISA and intracellular cytokine staining.

Measurements and main results: Elevated expression of ST2, the ligand-binding chain of the IL-33 receptor, was observed in inflamed sinonasal mucosa from CRSwNP compared with CRSsNP and healthy control subjects. An increased percentage of innate lymphoid cells was observed in inflamed sinonasal mucosa from CRSwNP compared with CRSsNP. ST2(+) innate lymphoid cells are a consistent source of IL-13 in response to IL-33 stimulation. Significant induction of IL-33 was observed in epithelial cells derived from patients with CRSwNP compared with patients with CRSsNP in response to stimulation with Aspergillus fumigatus extract.

Conclusions: These data suggest a role for sinonasal epithelial cell-derived IL-33 and an IL-33-responsive innate lymphoid cell population in the pathophysiology of CRSwNP demonstrating the functional importance of innate lymphoid cells in Th2-mediated inflammatory disease.

Figure 1.

Elevated expression of ST2 in inflamed sinonasal mucosa from chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) as compared with CRS without nasal polyps (CRSsNP) and healthy control subjects (HC). Expression of (A) ST2, (B) IL1RAP, and (C) IL-33 relative to β-actin was determined in inflamed sinonasal mucosa from CRSsNP (n = 20), CRSwNP (n = 36), and HC (n = 8) by quantitative real-time polymerase chain reaction. Each point represents the average of duplicate results for an individual patient sample, and the horizontal line represents the mean. Results were analyzed by one-way analysis of variance with Tukey posttest, *P < 0.05 and ***P < 0.001.

Figure 2.

Elevated percentage of innate lymphoid cells in chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP). (A) Identification of innate lymphoid cells in inflamed sinonasal mucosa. Representative data from one patient with CRS without nasal polyps (CRSsNP) and one patient with CRSwNP are shown. (B) Percentage of innate lymphoid cells (ILC) in inflamed sinonasal mucosa from CRSsNP (n = 4) and CRSwNP (n = 6). (C) The surface phenotype of ILC from patients with CRSwNP was analyzed by flow cytometry. Data shown are representative of four independent patients. Shaded histogram represents isotype control, and open histogram represents staining with antigen-specific antibody. (D) Percentage of cells lacking lineage markers (lineage^neg), CD117^neg CD127⁺ ILC in inflamed sinonasal mucosa from CRSsNP (n = 4) and CRSwNP (n = 6). (E) Percentage of lineage^neg CD127⁺ CRTH2⁺ ILC in inflamed sinonasal mucosa from CRSsNP (n = 3) and CRSwNP (n = 4). Each point (B, D, E) represents an individual patient sample, and the horizontal bar represents the mean. Results were analyzed by Mann-Whitney test. P < 0.05 was considered significant.

Figure 3.

Increased IL-13 production from cells lacking lineage markers (lineage^neg) CD127⁺ innate lymphoid cells in chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) in response to IL-33 stimulation. IL-13 production from CD45⁺ cells isolated from inflamed sinonasal mucosa of (A) patients with CRSwNP (n = 10), or (B) patients with CRS without nasal polyps (CRSsNP) (n = 3) stimulated with recombinant IL-2, IL-33, or both was determined by ELISA. (C) IL-13 production from CD45⁺ cells from CRSwNP (n = 10) and CRSsNP (n = 3) stimulated with recombinant IL-2 and IL-33 was determined by ELISA. Each point represents the results for an individual patient sample, and the horizontal line represents the mean. Results were analyzed by one-way analysis of variance with Tukey posttest (A, B) or Mann-Whitney test (C). P < 0.05 was considered significant. **P < 0.01 and ***P < 0.001.

Figure 4.

Innate lymphoid cells (ILC) from chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) produce IL-13 in response to IL-33. CD45⁺ cells from inflamed mucosa of patients with CRSwNP (n = 6) were stimulated with recombinant IL-2 alone or together with recombinant IL-33. The percentage of IL-13⁺ ILC was determined by intracellular staining. Data from one representative patient (A) and cumulative data from six independent patients (B) are shown. (C) IL-13 production from lineage^neg CD127⁺ CRTH2⁺ ILC sorted from inflamed ethmoid sinus mucosa from CRSwNP and stimulated with recombinant IL-2 alone or together with recombinant IL-33 was determined by ELISA. (D) The total percentage of IL-13⁺ cells within the lineage⁺ and lineage^neg population was determined. (E) IL-13 production from CD45⁺ cells from inflamed mucosa of patients with CRSwNP stimulated with recombinant IL-2 and IL-33 or plate-bound anti-CD3 and soluble anti-CD28 alone or together with recombinant IL-2, IL-33, or both was determined by ELISA. (F) IL-13 production from total or lineage-depleted CD45⁺ cells sorted from inflamed ethmoid sinus mucosa of CRSwNP, plated at a density of 10⁶/ml (total) or 9.3 × 10⁴/ml (lineage-depleted) to represent the frequency of lineage⁺ cells within the total CD45⁺ population, and stimulated for 4 days with IL-2 alone or together with IL-33 was determined by ELISA. Each point represents the results for an individual patient sample, and the horizontal line represents the mean. Data from one experiment representative of two (C, F) or three (E) independent experiments each with one patient are shown. Results were analyzed by one-way analysis of variance with Tukey posttest (A) or Wilcoxon signed rank test (B, D). P < 0.05 was considered significant.

Figure 5.

Induction of IL-33 expression in sinonasal epithelial cells from patients with chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) in response to stimulation with Aspergillus fumigatus extract. Relative expression of IL-33 in sinonasal epithelial cells from patients with CRS without nasal polyps (CRSsNP) (n = 7) (A) or patients with CRSwNP (n = 12) (B) cultured for 24 hours in medium alone or with an extract of A. fumigatus (AF), Alternaria alternata (AA), or Cladosporium herbarum (CH). (C) Fold increase in IL-33 expression in sinonasal epithelial cells from CRSsNP (n = 7) and CRSwNP (n = 12) in response to stimulation with fungal extracts. Each point represents the average of duplicate results from an individual patient and the horizontal line represents the mean. Results were analyzed by paired t test (A, B) or unpaired t test (C). P < 0.05 was considered significant.