An ALARMINg Type 2 Response in Cystic Fibrosis—The Key to Understanding ABPA?

Cystic fibrosis (CF) airway disease is marked by significant regional heterogeneity and severe neutrophil-predominant inflammation with high levels of proinflammatory cytokines and ineffective innate and adaptive mucosal immunity contributing to microbial persistence (1, 2). Despite the preponderance of neutrophilic inflammation, people with CF (pwCF) have a high rate of allergic bronchopulmonary aspergillosis (ABPA), characterized by increased type 2 inflammation (3). CF transmembrane conductance regulator (CFTR) mutations have also been linked to ABPA in non-CF cases, suggesting a direct relationship between CFTR activity and ABPA, but the molecular basis for this association remains unclear (4). In this issue of the Journal, Cook and colleagues (pp. 1486–1497) shed light on the potential mechanisms underlying this observed link (5).

The mucosal epithelium is a key upstream regulator of type 2 inflammation as stressed or injured cells express several cytokines and alarmins that promote type 2 immunity, including thymic stromal lymphopoietin (TSLP), IL-25, and, notably, IL-33 (6). In CF, IL-33 localizes to the nuclei of basal airway epithelial cells (AECs) on biopsied tissues from explanted lungs obtained from people with end-stage disease and has also been shown to be elevated in BAL fluid (7, 8). Here, Cook and colleagues confirm the presence of elevated IL-33 levels in the CF airway. The authors revealed increased IL-33 production in human CF AECs as compared with non-CF AECs when treated with allergen (Alternaria alternata) extract in vitro. Similarly, epithelial cells derived from Cftr^−/− mice also showed increased Il33 transcripts. Additionally, on allergen challenge, Cftr^−/− mice had significantly increased IL-33 protein in the lung as compared with Cftr^+/+ mice.

SNPs within the IL-33 promoter and the IL-33 receptor ST2 (Il1rl1) have been associated with increased genetic risk of asthma development (9). Consistent with prior work in asthma as well as pulmonary fibrosis showing that IL-33 activates and modulates the Th2 inflammatory response in an ST2 (Il1rl1)-dependent manner (6, 10), the authors also studied whether naive CD4⁺ T cells from wild-type or Cftr^−/− mice have differential Th2 responses to IL-33. They found enhanced Th2 differentiation of naive CD4⁺ T cells in the presence of IL-33 in Cftr^−/− cells compared with that in wild-type mice. This enhanced differentiation was also associated with higher levels of ST2 (Il1rl1). In line with this known role of Il-33 in the regulation of Th2 differentiation in vitro, the authors showed that deletion of Il33 or Il1rl1 attenuated allergen-induced type 2 inflammation in vivo in the Cftr^−/− background.

Cook and colleagues have mechanistically linked lung epithelial cell IL-33 release to intrinsic CFTR function and suggest that this may occur by means of the NADPH oxidase DUOX1. DUOX1-mediated H₂O₂ production and the generation of other reactive oxygen species by AECs have not been extensively explored in CF. It is plausible that DUOX1 initiates a pathway for IL-33 release by means of hydroxyl radical–mediated membrane lipid peroxidation. IL-33 release could also be regulated by DUOX1, and the authors also found that DUOX1 was upregulated at both the transcript and protein levels in CF cells. Oxidation of IL-33, through the formation of disulfide bonds, inactivates it, suggesting that DUOX1 activity may control local IL-33 activity or duration. Further supporting the critical role of CFTR in direct IL-33 regulation, the authors showed that treatment of F508del epithelial cells with CFTR modulator therapies, including elexacaftor-tezacaftor-ivacaftor (ETI), reduced IL-33 release in CF AECs.

In CF patients, the resting membrane potential of the epithelium is likely altered as a function of altered CFTR activity and could lead to IL-33 production. In support, knockout of the colonic chloride transporter, SLC26A3, implicated in congenital diarrheal diseases, also results in enhanced IL-33 production and enhanced epithelial-immune crosstalk (11). Indeed, bacterial enterotoxins from enterotoxigenic Escherichia coli that cause secretory diarrhea promote CFTR-mediated chloride efflux from intestinal epithelial cells and cause IL-33 production localized to small intestinal tissue (12). Although it is unknown whether the enterotoxins modulate CFTR activity or whether DUOX1 is implicated in its release, it will be of interest to determine whether CFTR activity is altered after enterotoxin exposure. Comparative studies between the lung and gastrointestinal mucosa should help sort out nuances of CF, as they are both derived from the embryonic foregut. In other models of ion transport, IL-33 has been shown to modulate Th2 inflammatory response and MUC5aC protein production in the sodium channel non–voltage-gated 1 β subunit (Scnn1b)-Tg+ mouse model that overexpresses the sodium channel in AECs, thereby displaying a CF-like phenotype with airway surface liquid dehydration and significant mucus obstruction (13).

ABPA is mediated by fungal-specific Th2 responses that express both IL-5 and IL-13 (3, 14), and it will be interesting to see whether ETI administration modifies ABPA prevalence in CF. To this end, a recent single-center study observed reductions in fungal colonization as well as reduced fungal-specific IgE after ETI administration (15). The data here further support the investigation of anti–IL-33 or anti-ST2 in CF patients with recalcitrant APBA or ABPA in ETI-ineligible patients. This work also calls to question the potential role of vitamin D in modulating IL-33 expression. Most pwCF have pancreatic insufficiency and are thus frequently deficient in the fat-soluble vitamins. Still, the immune modulatory role of vitamin D in CF remains an area that has been relatively underexplored. In pwCF and ABPA, an exaggerated Th2 response has been correlated with lower mean serum vitamin D levels (3). The role of vitamin D in IL-33 expression has recently been studied in psoriasis and may be an intriguing avenue of further research in the CF population (16).

In summary, in the CF airway where ongoing epithelial damage is known to occur, elevated IL-33 release may explain the increased type 2 immunity in CF. As Cook and colleagues highlight here, our understanding of type 2 inflammation in CF continues to evolve, as does our understanding of the intrinsic and complex role that CFTR function plays in mucosal immunity. Modulation of the IL-33/ST2 axis represents an interesting and potential therapeutic target in CF as well as in ABPA.