Cystic Fibrosis Reprograms Airway Epithelial IL-33 Release and Licenses IL-33-Dependent Inflammation

Abstract

Rationale: Type 2 inflammation has been described in people with cystic fibrosis (CF). Whether loss of CFTR (cystic fibrosis transmembrane conductance regulator) function contributes directly to a type 2 inflammatory response has not been fully defined. Objectives: The potent alarmin IL-33 has emerged as a critical regulator of type 2 inflammation. We tested the hypothesis that CFTR deficiency increases IL-33 expression and/or release and deletion of IL-33 reduces allergen-induced inflammation in the CF lung. Methods: Human airway epithelial cells (AECs) grown from non-CF and CF cell lines and Cftr^+/+ and Cftr^-/- mice were used in this study. Pulmonary inflammation in Cftr^+/+ and Cftr^-/- mice with and without IL-33 or ST2 (IL-1 receptor-like 1) germline deletion was determined by histological analysis, BAL, and cytokine analysis. Measurements and Main Results: After allergen challenge, both CF human AECs and Cftr^-/- mice had increased IL-33 expression compared with control AECs and Cftr^+/+ mice, respectively. DUOX1 (dual oxidase 1) expression was increased in CF human AECs and Cftr^-/- mouse lungs compared with control AECs and lungs from Cftr^+/+ mice and was necessary for the increased IL-33 release in Cftr^-/- mice compared with Cftr^+/+ mice. IL-33 stimulation of Cftr^-/- CD4⁺ T cells resulted in increased type 2 cytokine production compared with Cftr^+/+ CD4⁺ T cells. Deletion of IL-33 or ST2 decreased both type 2 inflammation and neutrophil recruitment in Cftr^-/- mice compared with Cftr^+/+ mice. Conclusions: Absence of CFTR reprograms airway epithelial IL-33 release and licenses IL-33-dependent inflammation. Modulation of the IL-33/ST2 axis represents a novel therapeutic target in CF type 2-high and neutrophilic inflammation.

Keywords: adaptive immunity; cystic fibrosis transmembrane conductance regulator; immune system; innate immunity.

Figure 1.

IL-33 release is increased in Alternaria extract (AE)-treated human and murine cystic fibrosis (CF) models. (A) Immunohistochemistry staining for IL-33 in differentiated human non-CF (NuLi [CFTR^WT/WT (cystic fibrosis transmembrane conductance regulator^WT/WT)]) and CF (CuFi [CFTR^{ΔF508/ΔF508}]) cell cultures grown at the air–liquid interface. Scale bars, 10 μm. (B) Quantitative PCR (qPCR) analysis of IL-33 expression in non-CF and CF airway epithelial cells (AECs) (n = 4 Transwell membranes per genotype). (C) Detection of IL-33 by Western blot analysis in non-CF and CF AECs (n = 4, 3 Transwell membranes combined per n per genotype). (D) Densitometric analysis of IL-33 expression in non-CF and CF AECs normalized to β-actin. (E–G) IL-33 by ELISA in cellular (E) lysate, (F) apical, and (G) basal compartments of non-CF (n = 3 [phosphate-buffered saline (PBS)] and 4 [AE] Transwell membranes) and CF (n = 3 [PBS] and 8 [AE] Transwell membranes) AECs. (H) qPCR analysis of IL-33 expression in Cftr^+/+ and Cftr^−/− EpCAM (epithelial cell adhesion molecule)-positive epithelial cells (n = 4 mice per genotype, normalized to Gapdh). (I) IL-33 by ELISA in whole lung homogenate from Cftr^+/+ and Cftr^−/− mice (n = 6 mice per genotype). (J) Schematic diagram showing AE exposure with subsequent 1-hour harvest. (K and L) IL-33 by ELISA in (K) whole lung homogenate and (L) BAL from Cftr^+/+ and Cftr^−/− mice (n = 3 mice per genotype for PBS-treated conditions and n = 6 mice per genotype for AE-challenged conditions). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. H&E = hematoxylin and eosin; IN admin = intranasal administration; ns = not significant.

Figure 2.

IL-33 release in response to Alternaria extract (AE) challenge is dependent on CFTR (cystic fibrosis transmembrane conductance regulator) channel function in airway epithelial cells (AECs). (A) Schematic diagram showing AE challenge in three groups (DMSO, DMSO with addition of elexacaftor-tezacaftor-ivacaftor [ETI] 24 h before AE challenge [DMSO/ETI], and ETI throughout seeding, polarization, and AE challenge in non–cystic fibrosis (non-CF) and CF AECs. (B–D) IL-33 by ELISA in cellular (B) lysate, (C) apical, and (D) basal compartments of DMSO- and ETI-treated non-CF and CF AECs (n = 5 Transwell membranes per group). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. C = at time of challenge; ns = not significant; P = at time of seeding/polarization.

Figure 3.

Alternaria extract (AE)-induced IL-33 release is dependent on DUOX1 (dual oxidase 1) in Cftr^−/− (cystic fibrosis transmembrane conductance regulator^−/−) mice. (A) Immunohistochemistry staining for DUOX1 in differentiated non–cystic fibrosis (CF) and CF cell cultures grown at the air–liquid interface. Scale bars, 15 μm. (B) Quantitative PCR (qPCR) analysis of Duox1 expression in non-CF and CF airway epithelial cells (n = 3 NuLi [CFTR^WT/WT] and n = 6 CuFi [CFTR^{ΔF508/ΔF508}] Transwell membranes). (C) Detection and (D) quantification of DUOX1 by Western blot analysis in non-CF and CF cells (n = 4, 3 Transwell membranes combined per n per genotype). (E) qPCR analysis of Duox1 expression in murine Cftr^+/+ and Cftr^−/− whole lung homogenate (n = 6 Cftr^+/+ and n = 3 Cftr^−/− mice, normalized to Gapdh). (F) Detection and (G) quantification of DUOX1 by Western blot analysis in Cftr^+/+ and Cftr^−/− cells (n = 4 per genotype). (H) Schematic diagram showing DUOX1 pharmacologic inhibitor (ML171) administration before AE challenge with subsequent 1-hour harvest. (I) IL-33 by ELISA in BALF from AE-challenged Cftr^+/+ and Cftr^−/− mice treated with phosphate-buffered saline (PBS) or ML171 (n = 4 mice per genotype for PBS-treated conditions and n = 5 mice per genotype for ML171-treated conditions). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns = not significant.

Figure 4.

ST2 expression is increased in murine Cftr^−/− (cystic fibrosis transmembrane conductance regulator^−/−) airway epithelial cells, and IL-33 increases T-helper cell type 2 (Th2) effector function. (A) Schematic diagram showing isolation and culture conditions of CD4⁺ CD62L^hiCD44^lo (naive) lymphocytes isolated from Cftr^+/+ and Cftr^−/− murine spleens. (B) Quantitative PCR (qPCR) analysis of Il1rl1 expression in Cftr^+/+ and Cftr^−/− cultured CD4⁺ T cells (n = 4 mice per genotype, normalized to Gapdh). (C) Representative gating strategy for ST2 expression in cultured Cftr^+/+ and Cftr^−/− CD4⁺ T cell populations gated on live lymphoid cells. (D) ST2 median fluorescence intensity (MFI) of cultured Cftr^+/+ and Cftr^−/− CD4⁺ T cells (n = 4 mice per genotype). (E) IL-5 and (F) IL-13 by ELISA in cellular supernatant from Cftr^+/+ and Cftr^−/− CD4⁺ T cells grown in culture stimulated with murine IL-33 (n = 7 mice per genotype). (G) Representative gating strategy for IL-13 expression in cultured Cftr^+/+ and Cftr^−/− CD4⁺ T cell populations gated on live lymphoid cells. (H) IL-13 MFI of cultured Cftr^+/+ and Cftr^−/− CD4⁺ T cells (n = 4 mice per genotype). **P < 0.01 and ****P < 0.0001.

Figure 5.

Deletion of IL-33 reduces Alternaria extract (AE)-induced inflammation in Cftr^−/− (cystic fibrosis transmembrane conductance regulator^−/−) mice. (A) Schematic diagram showing adaptive model of AE-induced inflammation. (B) Representative photomicrographs of lung sections stained with hematoxylin and eosin for phosphate-buffered saline (PBS) and AE-challenged mice. Scale bars, 1 mm; inlet scale bars, 150 μm. (C) Histological scoring of AE-treated mice (n = 3–15 per genotype). Gray circles indicate PBS-challenged mice, and blue circles indicate AE-challenged mice. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 6.

Inflammatory cell count of the BAL fluid (BALF) decreases with IL-33 or ST2 deletion in Alternaria extract (AE)-sensitized and challenged Cftr^−/− (cystic fibrosis transmembrane conductance regulator^−/−) mice. The number of (A) macrophages, (B) eosinophils, (C) lymphocytes, and (D) neutrophils in the BALF of phosphate-buffered saline (PBS) or AE-challenged mice (n = 4–10 per genotype). Gray circles indicate PBS-challenged mice, and blue circles indicate AE-challenged mice. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns = not significant; PMNs = polymorphonuclear neutrophils.

Figure 7.

IL-33 or ST2 deletion reduces Alternaria extract (AE)-induced T-helper cell type 2 (Th2) cytokines, serum IgE, and airway neutrophils in Cftr^−/− (cystic fibrosis transmembrane conductance regulator^−/−) mice. (A and B) BAL fluid concentrations of (A) IL-5 and (B) IL-13 by ELISA in treated mice. (C) IgE concentrations by ELISA in serum from treated mice (n = 3–9 depending on genotype). (D) Whole lung homogenate concentrations of CXCL1 (KC, n = 4–8 depending on genotype). Gray circles indicate phosphate-buffered saline (PBS)-challenged mice and blue circles indicate AE-challenged mice. (E) Schematic representation of the induction of the IL-33/ST2 axis in the cystic fibrosis (CF) adaptive immune response. CFTR deficiency leads to increased DUOX1 (dual oxidase 1) expression and, in the setting of allergen-induced epithelial injury, increased amounts of released IL-33. The active form of IL-33 activates ST2-high CF Th2 cells and, in addition to CXCL1, contributes to neutrophil migration. The activation of Th2 cells leads to IL-5 and IL-13 synthesis and subsequent eosinophilia and IgE expression by matured B cells. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.