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American Journal of Respiratory and Critical Care Medicine logoLink to American Journal of Respiratory and Critical Care Medicine
. 2023 Mar 23;207(11):1486–1497. doi: 10.1164/rccm.202211-2096OC

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

Daniel P Cook 1,✉, Christopher M Thomas 1, Ashley Y Wu 1, Mark Rusznak 1, Jian Zhang 1, Weisong Zhou 1, Jacqueline-Yvonne Cephus 1, Katherine N Gibson-Corley 2, Vasiliy V Polosukhin 1, Allison E Norlander 1, Dawn C Newcomb 1,2, David A Stoltz 3,4, R Stokes Peebles Jr 1,2,5
PMCID: PMC10263140  PMID: 36952660

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: cystic fibrosis transmembrane conductance regulator, immune system, innate immunity, adaptive immunity

At a Glance Commentary

Scientific Knowledge on the Subject

In this study, we show that CFTR (cystic fibrosis transmembrane conductance regulator) inhibits airway IL-33 release and negatively regulates epithelial cell DUOX1 (dual oxidase 1) expression. Using Cftr+/+ and Cftr−/− mice with germline mutations in Il-33 or Il1rl1, we found that deletion of the IL-33/ST2 axis significantly reduces allergen-induced inflammation through reduction in type 2 inflammatory mediators and neutrophil recruitment.

What This Study Adds to the Field

Emerging evidence suggests that cystic fibrosis (CF) lung inflammation can occur in the absence of infection. This study highlights an increased production of the potent alarmin IL-33 in both CF human airway epithelial cells and Cftr−/− mice when infection is not present. Moreover, deletion of Il-33 or Il1rl1 in Cftr−/− mice leads to dramatic reduction in allergen-induced type 2 inflammation, as well as neutrophil recruitment to the lung. These studies demonstrate intrinsic immune system defects before infection in CF and suggest the potential of IL-33/ST2–targeting biologic therapy in individuals with CF.

Cystic fibrosis (CF) lung disease is a result of severe and persistent airway inflammation secondary to defects in CFTR (cystic fibrosis transmembrane conductance regulator protein) (1). Recruitment of inflammatory cells in the CF lung leads to tissue damage and release of additional inflammatory mediators (2–4). Recent studies suggest that the CF inflammation resulting from proinflammatory cytokines can occur even in the absence of infection (5–7). Of particular interest is the emerging recognition of type 2 inflammation in the CF lung (5, 8–12). Although historically regarded as a defense mechanism against helminth infections, type 2 inflammation is now recognized to be host protective but also pathogenic (5). Whether type 2 inflammatory responses function in restoration of tissue homeostasis and resolution of inflammation or become chronically activated leading to lung inflammation in CF remains unclear.

In this context, the IL-1 cytokine family member, IL-33, has recently emerged as an important initiator of type 2 immune responses (13, 14). Constitutively produced by airway epithelia and stored in the nucleus (15), IL-33 is passively released during cellular damage and/or secreted during stress conditions (16–18). Once secreted, IL-33 activates CD4+ T-helper 2 (Th2) cells and other immune cells through binding to the cytokine receptor IL-1 receptor-like 1 (ST2), leading to increased production of classical type 2 inflammatory cytokines, including IL-5 and IL-13 (19, 20). Interestingly, IL-33 is elevated in lung biopsy specimens (21) and BAL fluid (BALF) (22) from individuals with CF compared with non-CF control subjects. Moreover, exposure of human CF airway epithelial cells (AECs) to Pseudomonas aeruginosa bacteria increased IL-33 expression compared with wild-type counterparts (23). Despite these observations, IL-33 biology in CF disease remains poorly understood.

To investigate the biological role for CFTR regulation of IL-33, we took advantage of both human and murine models of CF. Here, we show that IL-33 is increased in CF human AECs and Cftr−/− murine airway tissues compared with non-CF AECs or lungs from Cftr+/+ mice, respectively. We used an extract of Alternaria alternata (AE), a ubiquitous fungal aeroallergen, as the airway antigen challenge. Using this model, we are the first to report that CFTR deletion in mice results in increased IL-33 secretion that is dependent on the NADPH oxidase, DUOX1 (dual oxidase 1). In addition, CFTR potentiation and correction using CFTR modulators in AE-challenged CF AECs reduces IL-33 release. Moreover, we demonstrate that germline deletion of IL-33 or ST2 dramatically decreases classical type 2 inflammation and neutrophil recruitment in allergen-challenged Cftr−/− mice compared with Cftr+/+ mice, demonstrating a potential avenue for the use of biologic therapies targeting type 2 endotypes in CF lung disease.

Methods

The materials and methods used, including human AEC models, allergen-challenged mouse models, sample collection and assessment of BAL and cytokines, histopathology, CD4+ T cell studies, and gene and protein expression are reported in the online supplement.

Mice

Cftrtm1 UncTg(FABPCFTR)1Jaw/J mice were obtained from The Jackson Laboratory (stock no: 002364). These mice are knockouts for the murine Cftr gene (Cftr−/−) but express human CFTR in the gut under control of the FABP1 (fatty acid binding protein1) promoter, which prevents acute intestinal obstruction. Mice heterozygous for the Cftr mutation (Cftr+/−) were crossed with IL-1 receptor like 1 (Il1rl1−/−, encoding the ST2 receptor) or IL-33 (Il33−/−) knockout mice (both the kind gifts of Andrew McKenzie). All strains were on a C57BL/6 background. These strains were crossbred to generate Il33−/−Cftr+/− and Il1rl1−/−Cftr+/− mice homozygous for the FABP-hCFTR transgene that served as breeders. The resulting various genetic combinations Il33−/−Cftr+/+, Il33−/−Cftr−/−, Il1rl1−/−Cftr+/+, Il1rl1−/−Cftr−/−, Cftr+/+, and Cftr−/− (all strains homozygous for the FABP-hCFTR transgene) were used for studies. Genotyping of experimental mice was performed by Transnetyx. All animal use procedures were approved by the Institutional Animal Care and Use Committee of Vanderbilt University Medical Center.

Statistics

All data were analyzed with GraphPad Prism 9 (GraphPad Software). Data are expressed as individual data points ± SEM. For analyses that compared two groups, we used an unpaired Student’s t test. Statistical significance for more than two genotypes and more than two conditions (AE vs. phosphate-buffered saline [PBS] or PBS vs. ML171) was assessed by two-way ANOVA with Bonferroni multiple pairs comparisons test. Values of P < 0.05 were considered significant between two groups.

Results

CFTR Deficiency Increased Total and Released IL-33 in Human AECs and Mice

To investigate whether human CF AECs express IL-33, we performed immunohistochemistry staining for IL-33 using non-CF (NuLi, CFTRWT/WT) and CF (CuFi, CFTRΔF508/ΔF508) human AECs grown at the air–liquid interface (ALI). Both non-CF and CF polarized AECs expressed IL-33 localized to the nucleus (Figure 1A). Consistent with greater CF expression of IL-33, both IL33 mRNA (Figure 1B) and IL-33 protein (Figures 1C and 1D) were increased in CF AECs compared with non-CF AECs. To determine whether the increased IL-33 expression in CF AECs increased IL-33 release, AECs were treated apically with 30 μg of AE for 1 hour. Similar to the quantitative PCR and Western blot data, IL-33 expression in cellular lysate by ELISA was increased in either PBS or AE-treated CF AECs compared with similarly treated non-CF AECs (Figure 1E). AE treatment increased IL-33 in the apical and basal compartments of AE-challenged CF AECs compared with AE-challenged non-CF AECs (Figures 1F and 1G), consistent with AE-mediated IL-33 release. Given our finding of increased IL-33 in human AECs at baseline and in cellular supernatant after AE challenge, we hypothesized that IL-33 expression is increased in the lungs of Cftr−/− mice. To assess whether Cftr−/− mice have similar increases in IL-33 expression, cells positive for the EpCAM (epithelial cell adhesion molecule) were isolated after lung digestion from Cftr+/+ or Cftr−/− mice. Similar to human AECs, EpCAM-positive cells from Cftr−/− mice displayed increased Il33 mRNA expression compared with Cftr+/+ mice (Figure 1H). In addition, Cftr−/− mice had significantly increased amounts of IL-33 protein in whole lung homogenate at baseline compared with Cftr+/+ mice (Figure 1I).

Figure 1.

Figure 1.

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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 [CFTRWT/WT (cystic fibrosis transmembrane conductance regulatorWT/WT)]) and CF (CuFi [CFTRΔF508/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.

To determine if IL-33 release is increased in Cftr−/− mice, we used an in vivo model similar to our human AEC experiments. Mice were challenged intranasally with either AE (7.5 μg) or PBS for 1 hour, at which time BAL was performed and lungs were harvested for IL-33 protein measurement (Figure 1J). Similar to baseline, IL-33 protein was significantly increased in Cftr−/− total lung homogenates compared with Cftr+/+ controls (Figure 1K). Although release of IL-33 into BALF from both genotypes was significantly increased in the AE-challenged groups compared with PBS-treated control mice, the AE-challenged Cftr−/− mice had significantly more IL-33 release compared with Cftr+/+ AE-challenged mice (Figure 1L). IL-33 protein from whole lung homogenate was not significantly different between challenged and unchallenged genotypes (Figure 1K), suggesting release of preformed IL-33, and not de novo synthesis of IL-33, led to the increased IL-33 in Cftr−/− BALF. Taken together, these human and murine data indicate that CFTR deficiency leads to increased total cellular IL-33 and augmented IL-33 release in the setting of allergen challenge.

AE-induced IL-33 Release Was Dependent on CFTR Channel Function in AECs

To assess whether IL-33 release was dependent on CFTR function, non-CF and CF (CFTRΔF508/ΔF508) AECs were incubated in the presence of the CFTR potentiator ivacaftor (1 μM), and the CFTR correctors elexacaftor (3 μM) and tezacaftor (3 μM, elexacaftor-tezacaftor-ivacaftor [ETI]) for either the entirety of culture after seeding or 24 hours before challenge (Figure 2A). DMSO was used as a control group. Cells were then challenged with 30 μg of AE, and 1-hour IL-33 concentrations in the cell lysate, apical, and basal compartments were measured. DMSO-treated CF AECs had increased amounts of intracellular IL-33 compared with DMSO-treated non-CF AECS (Figure 2B). ETI treatment significantly reduced AE-mediated IL-33 release in CF AECS compared with DMSO-treated CF AECs, with no significant ETI effect seen in non-CF AECS. IL-33 concentrations in apical and basal compartments after AE challenge showed significant reductions with ETI incubation when provided during polarization of AECs compared with DMSO-treated CF AECs (Figures 2C and 2D). No significant effect of ETI was seen with AE challenge in non-CF AECs in either the apical or basal compartments. These data demonstrate that IL-33 release is dependent on CFTR, and increased CFTR function by ETI in CF AECs reduces IL-33 release.

Figure 2.

Figure 2.

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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.

DUOX1 Was Increased in CF Models, and DUOX1 Was Necessary for IL-33 Release in Cftr−/− Mice

Because AEC IL-33 release in response to AE is DUOX1 dependent (16), we tested for DUOX1 expression in non-CF and CF-unchallenged AECs. Immunohistochemistry staining of DUOX1 in unchallenged AECs revealed a cytosolic staining pattern in both CF and non-CF AECs (Figure 3A). DUOX1 mRNA was significantly upregulated in CF AECs compared with non-CF AECs at baseline (Figure 3B). Similarly, Western blot analysis showed increased amounts of DUOX1 protein in CF AECs compared with non-CF controls (Figures 3C and 3D). Similar to human AECs, Cftr−/− mice displayed increased Duox1 transcript expression (Figure 3E) and protein (Figures 3F and 3G) in whole lung homogenate at baseline compared with Cftr+/+ mice. To test whether the increased IL-33 release in Cftr−/− mice was DUOX1 dependent, Cftr+/+ and Cftr−/− mice were treated with intranasal instillation of either the DUOX1 inhibitor ML171 (24) or PBS for 3 days before AE challenge and subsequent 1-hour harvest (Figure 3H). DUOX1 inhibition significantly reduced IL-33 release in BALF in both genotypes, returning IL-33 BALF concentrations in Cftr−/− mice to Cftr+/+ concentrations, an effect not seen in PBS-treated control mice (Figure 3I). These data suggest that increased DUOX1 expression in CFTR deficiency is necessary for IL-33 release.

Figure 3.

Figure 3.

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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 [CFTRWT/WT] and n = 6 CuFi [CFTRΔF508/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.

IL-33 Stimulation of Cftr−/− CD4+ T Cells Increased Th2 Effector Function Compared with Cftr+/+ CD4+ T Cells

Because IL-33 is known to stimulate type 2 inflammation, in part through direct differentiation of CD4+ T cells toward a Th2 phenotype (25), we next investigated whether the IL-33 receptor, ST2, was present on Cftr−/− and Cftr+/+ murine CD4+ cells. CD4+ CD62LhiCD44lo (naive) cells from Cftr+/+ and Cftr−/− mice were isolated from murine spleens and cultured in the presence of IL-33 (20 ng/ml), plate-bound anti-CD3e, and anti-CD28 for 3 days (Figure 4A). Il1rl1 mRNA (the transcript encoding for ST2) was significantly increased in cultured Cftr−/− CD4+ T cells compared with Cftr+/+ controls (Figure 4B). Single-cell analysis of cultured cells by flow cytometry revealed a larger percentage of ST2+ CD4+ T cells (Figure 4C) and a higher median fluorescence intensity of ST2 in Cftr−/− CD4+ T cells compared with Cftr+/+ CD4+ T cells (Figure 4D). Cytokine measurements from the supernatant of IL-33 polarized CD4+ T cells revealed increased expression of IL-5 (Figure 4E) and IL-13 (Figure 4F) by Cftr−/− CD4+ T cells compared with Cftr+/+ CD4+ T cells. Similar flow cytometry analysis of cultured CD4+ T cells revealed a larger percentage of IL-13+ CD4+ T cells (Figure 4C) and a higher median fluorescence intensity of IL-13 in Cftr−/− CD4+ T cells compared with Cftr+/+ CD4+ T cells (Figure 4D). No differences were seen in median fluorescence intensity values for IFN-γ, IL-17, or PD-1 (programmed cell death protein 1) in IL-33 or IL-4/anti–IFN-γ culturing conditions between Cftr−/− and Cftr+/+ CD4+ T cells (see Figure E3 in the online supplement). These data demonstrate that IL-33 enhanced Th2 polarization and increased Th2 effector function in Cftr−/− murine CD4+ T cells compared with Cftr+/+ control CD4+ T cells.

Figure 4.

Figure 4.

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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+ CD62LhiCD44lo (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.

Deletion of the IL-33/ST2 Axis Reduced AE-induced Inflammation in CF Mice

Given increased expression of ST2 and increased effector cytokine expression in murine Cftr−/− Th2 cells, we hypothesized that loss of IL-33 or ST2 reduces AE-induced inflammation in CF mice. To test this hypothesis, Cftr+/+ and Cftr−/− mice were sensitized and challenged with either AE (7.5 μg) or PBS control using an adaptive model of allergic airway inflammation (Figure 5A). The effects of IL-33 and ST2 deletion on CF pulmonary pathology, including airway inflammation, goblet cell metaplasia, vascular remodeling, and eosinophil recruitment, were examined using a composite score of these histopathologic markers. Cftr−/− mice lungs had significantly more pulmonary pathology in response to AE challenge compared with Cftr+/+ AE-challenged controls (Figures 5B and 5C). Cftr−/− mice lacking either IL-33 (Il33−/− Cftr−/−) or the receptor ST2 (Il1rl1−/−Cftr−/−) demonstrated significantly less histologic pathology in response to AE challenge compared with similarly challenged Cftr−/− mice (Figures 5B and 5C).

Figure 5.

Figure 5.

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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.

To determine the immune cells contributing to the observed pulmonary pathology, AE-induced cellular inflammation was assessed in the BALF (Figures 6A–6D). BALF cell differential counts revealed marked increases in inflammation in AE-challenged Cftr+/+ and Cftr−/− mice compared with PBS challenge, with dramatic increases in eosinophils and lymphocytes. Genetic deficiency of IL-33 or ST2 significantly decreased overall type 2 inflammatory response, including reduction in eosinophils (Figure 6B) and lymphocytes (Figure 6C). Neutrophil recruitment was also significantly increased in AE-challenged Cftr+/+ mice and further statistically increased in Cftr−/− mice (Figure 6D). Surprisingly, the recruitment of neutrophils was significantly decreased in AE-challenged IL-33 or ST2-deficient Cftr−/− mice compared with Cftr+/+ mice. Taken together, these data suggest that IL-33 elevations in Cftr−/− mice lead to enhanced pulmonary inflammation characterized by eosinophil, lymphocyte, and neutrophil recruitment in response to allergen sensitization and challenge. In addition, deletion of IL-33 or ST2 significantly reduced the amount of inflammation in AE-challenged Cftr−/− mice.

Figure 6.

Figure 6.

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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.

AE Challenge Increased Type 2 Cytokine and Neutrophil Chemokine Expression in Cftr−/− Mice

IL-33 release induced type 2 cytokines in response to adaptive AE challenge in an adaptive model of allergic inflammation (25). In agreement with these findings, BALF from AE-challenged Cftr+/+ and Cftr−/− mice showed increased IL-5 (Figure 7A) and IL-13 concentrations (Figure 7B). Loss of IL-33 or ST2 significantly reduced type 2 cytokine expression in Cftr−/− mice. To assess whether these cytokines were dependent on adaptive immune responses, serum IgE amounts were measured. IgE was significantly increased in AE-challenged Cftr+/+ mice compared with PBS-challenged mice, and AE challenge further significantly increased IgE in Cftr−/− mice compared with Cftr+/+ mice (Figure 7C). Loss of IL-33 or ST2 significantly reduced the amount of serum IgE produced in Cftr−/− mice (Figure 7C). Analysis of the BALF from AE-challenged mice revealed marked and IL-33–dependent increases of the mouse functional IL-8 homolog CXCL1, suggesting a role for IL-33 in neutrophilic inflammation in this allergen-challenge model (Figure 7D). Although IL-17a was increased with AE challenge in both Cftr+/+ and Cftr−/− lung homogenate, no significant differences were detected when IL-33 or ST2 was deleted (Figure E4). To test whether DUOX1 inhibition similarly decreases type 2 inflammation and neutrophil recruitment, we performed AE challenge using our adaptive model in Cftr−/− mice treated with 1) PBS; 2) ML171 (DUOX1 pharmacologic inhibitor) before challenge; or 3) ML171 before sensitization and challenge (Figure E5A). In Cftr−/− mice, DUOX1 inhibition before sensitization and challenge significantly reduced inflammatory cell recruitment, including eosinophils and lymphocytes (Figures E5B–E5D), as well as IL-5 and IL-13 cytokine concentrations (Figures E5F and E5G). DUOX1 inhibition in Cftr−/− mice also reduced recruited BALF neutrophils and lung CXCL1 (Figures 5E and 5H). These data show that endogenous IL-33 contributes to allergen-induced inflammation in Cftr−/− mice and that DUOX1 and IL-33/ST2 signaling are necessary for the increased type 2 and neutrophilic response to AE in Cftr−/− mice.

Figure 7.

Figure 7.

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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.

Discussion

Prior studies have shown a type 2 immune response in the human CF lung (10, 12, 26). Relatedly, diseases of type 2 inflammatory disorders, such as allergic bronchopulmonary aspergillosis, asthma, and nasal polyposis, are increased in individuals with CF and carriers of CFTR mutations, suggesting a direct link of CFTR and type 2 immunity in humans (27, 28). However, the mechanisms leading to type 2 and allergic-like inflammatory abnormalities in the CF lung are not well characterized. The largest confounding factor to assessing the role of IL-33 in human CF tissues is the presence of chronic airway inflammation and infection. Here, we used human airway epithelial cells from non-CF and CF donors as well as Cftr−/− mice, which lack any spontaneous airway infection, to provide a unique opportunity to study the role of the type 2 inflammatory cytokine, IL-33, in the setting of CFTR deficiency. Our results are the first to show that human CF AECs and Cftr−/− mice have increased allergen-induced IL-33 release compared with control mice. These results are further supported by our novel finding that the epithelial NADPH oxidase DUOX1, a known regulator of IL-33 release (16, 24), is increased in Cftr−/− mice compared with Cftr+/+ mice, and DUOX1 inhibition leads to significantly decreased IL-33 release in Cftr−/− mice. Importantly, we report that IL-33 increased Th2 effector function in Cftr−/− CD4+ T cells. Finally, we demonstrated that germline deletion of IL-33 or its functional receptor, ST2, in Cftr−/− mice significantly reduces not only conventional type 2 inflammation markers, including IL-5, IL-13, and IgE expression but also CXCL1 expression and resultant neutrophilia (Figure 7E).

Previous studies have shown IL-33 to be elevated in CF lung biopsy specimens (21), CF BALF (22), and Pseudomonas-treated CF human AECs (23). Our current data confirm these prior human findings and, importantly, extend our observations regarding IL-33 to a murine model of Cftr−/− lacking spontaneous infection, thereby removing the confounding pathogen-associated inflammation inherent with the disease. We found that both human CF AECs and Cftr−/− murine lungs had increased IL-33 at baseline compared with either healthy or Cftr+/+ controls. Given that IL-33 functions as a potent alarmin released under stress conditions and necrotic death (8, 13, 19, 29), we hypothesized that challenge with acute AE would result in greater release of IL-33 from CF samples compared with non-CF controls. Indeed, in both CF human AECs and Cftr−/− mice there was greater IL-33 release after acute allergen challenge compared with non-CF human AECs and Cftr+/+ mice, respectively. Furthermore, deletion of the potent alarmin IL-33 or ST2 in Cftr−/− mice resulted in significant reduction in both type 2 and neutrophilic inflammation after allergen challenge.

There is increasing evidence that loss of CFTR function causes inflammation even in the absence of pathological microbial infection (6, 30, 31). Finding increased IL-33 in CF human and Cftr−/− murine lung cells before allergen challenge suggests that the CF environment is skewed toward IL-33 synthesis at baseline and poised for excessive IL-33 release in the setting of tissue injury. Using ETI in CF AECs, we demonstrate a dependency of IL-33 release on CFTR function. We show that DUOX1 is a necessary regulator of IL-33 release in CF, and CFTR deficiency results in upregulation of the NADPH oxidase. Dual oxidase enzymes are dependent on CFTR thiocyanate secretion (32, 33), and DUOX1-mediated recruitment of immune cells to the airway epithelial surface has been hypothesized to contribute to oxidative and proteolytic stress seen in CF disease (34–36). Others have shown that IL-33 release requires DUOX1 generation of H2O2 to activate the tyrosine kinases Src and EGFR to promote processing and nonclassical secretion of IL-33 (16). Here we show that CFTR expression regulates DUOX1 expression and the ability of the NADPH oxidase to affect IL-33 release.

We do not fully understand how CFTR loss leads to increased DUOX1 expression. Typical DUOX1 activation in respiratory epithelial cells is dependent on the activation of calcium signaling, initiated by activation of cell surface protease–activated receptors and transient receptor potential channels (37). CF airway epithelial cells display abnormal calcium regulation (38), and increased intracellular calcium concentrations enhance the inflammatory profile of CF airway cells (39). Therefore, increased calcium due to CFTR deficiency may account for the increased DUOX1 expression seen in CF human AECs and Cftr−/− murine lungs. A recent study suggested that calmodulin, a major regulator of calcium homeostasis, directly interacts with CFTR, and the presence of the CFTR-F508del mutation increases concentrations of intracellular calcium in epithelial cells (40). Another study demonstrated that CFTR potentiator treatment of CF neutrophils was able to decrease intracellular calcium concentration, with improvement in cell intrinsic antimicrobial functions (41). Future studies will be required to mechanistically determine how loss of CFTR activity leads to increased DUOX1 expression.

We were initially surprised to find that deletion of IL-33 or ST2 and DUOX1 inhibition in Cftr−/− mice led to decreased neutrophil recruitment after AE challenge. IL-33 knockout in another CF-like lung disease murine model, transgenic mice overexpressing Scnn1b (sodium channel non-voltage gated 1 β subunit), decreased age-related recruitment of eosinophils and Th2 inflammation (42). However, this model failed to show any differences in neutrophil chemokines or recruitment and yet revealed worse overall pulmonary inflammation in the IL-33KO Scnn1b–overexpressing mice compared with transgenic mice with IL-33 expression. These differences may reflect 1) our use of AE as a stimulant for IL-33 release; 2) whether IL-33 was released via necrotic or nonclassical pathways; 3) IL-33 or ST2 expression on other cell types that express CFTR; or 4) contributions of other disease mechanisms, including airway surface liquid changes or mucus pathology.

Although IL-33 has been implicated in neutrophil recruitment in response to bacterial infections (43–45), we showed that IL-33 deletion significantly reduced neutrophil recruitment in Cftr−/− mice challenged with AE. Neutrophil recruitment is associated with increased chemokine CXCL1 (KC) concentrations in Cftr−/− mice. Consistent with these findings, we did not see any significant changes in total lung KC between PBS-treated Cftr+/+ and Cftr−/− mice. However, in the setting of an allergen challenge, KC became significantly elevated in Cftr+/+ and more so in Cftr−/− mice, an effect that was significantly reduced with IL-33 or ST2 deletion. These findings in Cftr−/− mice challenged with a common environmental allergen and devoid of infection indicate a potential etiology for priming of the immune system before infection and a role of IL-33 in early CF disease.

Our studies have both advantages and limitations. Advantages include: 1) The use of comparative methodologies highlighting a conserved effect of CFTR deficiency on IL-33 release in both humans and mice. 2) The use of germline deletion of both IL-33 and ST2 in mice provided a powerful approach to assess respective gene function in CF, a method that would be more challenging in CF models in larger animals. 3) We took advantage of the lack of spontaneous lung disease in mice to determine the primary effect of allergen challenge on the IL-33/ST2 axis in CF. and 4) To the best of our knowledge, we were the first to demonstrate in an in vivo CF model the inflammatory response to Alternaria, a common environmental allergen causing allergic sensitization in humans with CF. Limitations include: 1) Differences were observed in released IL-33 amounts between in vitro human AECs and in vivo murine studies. Although AE challenge was able to induce IL-33 release in both Cftr+/+ and Cftr−/− mice, AE challenge was only able to induce measurable IL-33 release in CF AECs. These differences may reflect contributions of other cell types in vivo to maximize IL-33 release or species differences to AE challenge. Despite these model differences, in both human CF AECs and Cftr−/− mice IL-33 release was augmented compared with non-CF or Cftr+/+ control mice. 2) We cannot exclude the possibility of IL-33 or ST2 effects on other cell types of the innate and adaptive immune system. As a cytokine, IL-33 binds to the specific receptor ST2 to induce type 2 inflammation through activation of Th2 cells, ILC2s, mast cells, eosinophils, macrophages, and dendritic cells (46). Previous studies demonstrated a regulatory T cell (Treg) functional deficiency in both CF human and Cftr−/− murine models (47), emphasizing the importance of nonepithelial contributions to type 2 inflammation. Further studies will be required to understand how alterations in IL-33 directly affect these other cell populations. 3) Although we primarily explored the role of type 2 inflammation, other aspects of inflammation may be impacted by IL-33 alterations, including the Th17 pathway, which has also been shown to be prevalent in CF disease as well as the suppressive effects of Tregs (48, 49). Both of these forms of inflammation, in addition to the alterations in Th2 inflammation reported, serve as attractive areas of further study to better understand the excessive inflammatory response seen in CF.

In summary, our findings demonstrate that CF human AECs and Cftr−/− mice produce and release elevated amounts of the potent alarmin IL-33 in response to allergen through DUOX1-mediated release. Moreover, IL-33–induced increase in Th2 cytokines from Cftr−/− CD4+ T cells compared with Cftr+/+ T cells supports a role for the IL-33/ST2 axis in allergen-induced inflammation in Cftr−/− mice. The dysregulation of IL-33 reflects priming of the immune system, before any infectious insult, to provide excessive type 2 inflammation characteristic of the disease. Several IL-33 and ST2 targeting biologics are in clinical development (phase 1 or 2) for asthma and other allergic disease. These biologics may provide pharmacological approaches to managing persistent CF pulmonary inflammation in the setting of highly effective CFTR modulator therapies. Equally exciting is that this work highlights current animal models of CF as archetypes to better understand the mechanistic interactions pertaining to IL-33 synthesis and release as well as the complex relationship between innate and adaptive immunity.

Acknowledgments

Acknowledgment

The authors thank Dr. Joseph Zabner and Phil Karp (University of Iowa) for providing NuLi and CuFi cell lines and a standard culturing protocol. They also thank Lan Wu for providing IL-33 knockout mice.

Footnotes

Supported by NIH grants R01 AI 124456 (R.S.P.), R01 AI 145265 (R.S.P.), U19 AI 095227 (R.S.P.), and R21 AI 145397; Division of Intramural Research, National Institute of Allergy and Infectious Diseases grant R01 AI 111820 (R.S.P.); U.S. Department of Veterans Affairs Biomedical Laboratory Research and Development Service grant 101BX004299 (R.S.P.); and Cystic Fibrosis Foundation grant COOK20L0. The Translational Pathology Shared Resource is supported by Division of Cancer Prevention, National Cancer Institute/NIH Cancer Center Support grant 5P30 CA68485-19.

Author Contributions: D.P.C., C.M.T., A.E.N., D.C.N., D.A.S., and R.S.P. contributed to conception and design of the research. D.P.C., C.M.T., A.Y.W., M.R., J.Z., W.Z., and J.-Y.C. performed experiments and data acquisition. D.P.C., C.M.T., A.Y.W., K.N.G.-C., V.V.P., A.E.N., D.C.N., D.A.S., and R.S.P. analyzed the data. D.P.C., C.M.T., A.Y.W., K.N.G.-C., V.V.P., A.E.N., D.C.N., D.A.S., and R.S.P. interpreted results of experiments. D.P.C., C.M.T., and R.S.P. prepared the figures. D.P.C., C.M.T., D.A.S., and R.S.P. drafted the manuscript. D.P.C., C.M.T., A.Y.W., M.R., J.Z., W.Z., J.-Y.C., K.N.G.-C., V.V.P., A.E.N., D.C.N., D.A.S., and R.S.P. edited, reviewed, and approved the final version of the manuscript.

This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org.

Originally Published in Press as DOI: 10.1164/rccm.202211-2096OC on March 23, 2023

Author disclosures are available with the text of this article at www.atsjournals.org.

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