Farm dust reduces mucin production in rhinovirus‐infected primary bronchial epithelial cells

Growing up on traditional farms is associated with protection against the development of wheezing, respiratory allergies, and asthma.1 Rhinovirus (RV) infections in early life are a substantial risk factor for asthma development and are linked to mucus hypersecretion in the lungs.2, 3 This results in impaired mucociliary clearance, which contributes to obstruction of (small) airways, processes implicated in asthma pathogenesis.4 Interestingly, the protective farm-effect against virus-induced early-life wheezing has been linked to exposure to cowsheds.5 This may be explained in part by the observation that farm dust extract (FD) inhibits epithelial inflammatory responses, increases airway epithelial barrier function, and decreases RV infection in house dust mite (HDM)-induced allergic airway inflammation in mice and in an human airway epithelial culture model.6, 7 We hypothesize that FD also restricts RV-induced mucus production, thus providing further explanation for this protective farm-effect. Here, we aimed to investigate whether treatment with FD modulates baseline and RV-induced expression and release of the mucin 5AC (MUC5AC) and mucin 5B (MUC5B) as well as goblet cell numbers in air–liquid interface (ALI) cultures of differentiated PBEC (ALI-PBEC). In this model, we have previously shown that RV infection increases the expression of MUC5AC and MUC5B.8 We also assessed gene expression of the goblet cell marker CLCA1 and of genes (FOXA2, FOXA3, and SPDEF) known to be involved in the regulation of MUC5AC production. ALI-PBEC were pretreated with FD for 24 h and subsequently infected with RV-A16 or stimulated with IFN-β (as a control) in the presence or absence of FD for 48 h (Figure 1A). Rhinovirus infection and IFN-β both increased MUC5AC and MUC5B mRNA expression, whereas apical MUC5AC release was only elevated after RV infection. We found that FD reduced mRNA expression of MUC5AC and MUC5B in all conditions, whereas FD-mediated inhibition of MUC5AC release was only observed in RV-infected cells (Figure 1B–D). In addition, the expression of CLCA1 was increased after RV infection, but unaffected by FD (Figure 1E). We further investigated the effects of FD on RV-A16-replication by assessing RV-A16 viral (v)RNA levels 24-, 48-, and 72-h postinfection. We found that at the multiplicity of infection (MOI) that we used for this study, RV-A16 vRNA was not significantly affected by FD. This indicates that the FD-mediated interference with RV-induced mucin expression and release was independent of its effects on viral replication (Figure 1F). Next, we investigated the effects of FD on the number of goblet cells and on protein levels of MUC5AC using confocal immunofluorescence staining. We found that FD reduced the MUC5AC protein content of cells without altering the number of goblet cells (Figure 1G–J), which is in line with the absence of an effect on CLCA1 expression. The expression of MUC5AC is stimulated by both SAM-pointed domain-containing ETS transcription factor (SPDEF) and Forkhead box protein A3 (FOXA3) and repressed by Forkhead box protein A2 (FOXA2).4 We therefore assessed the expression of these genes to explore the mechanism underlying FD-mediated inhibition of MUC5AC. We found that FD inhibited the expression of FOXA3 in all conditions (including control-exposed cells), whereas the effects of FD on SPDEF nor FOXA2 reach statistical significance (Figure 2A–C). These results suggest that FD-induced repression of MUC5AC is mediated in part through the inhibition of FOXA3. We further investigated whether FD affects the expression of genes that are either activated (the epithelial alarmin TSLP) or repressed [interferon regulatory factor-1 (IRF1) and Toll-like receptor 3 (TLR3)] by FOXA3.9 The expression of TSLP, TLR3, and IRF1 was induced after RV-infection without being altered by FD (Figure 2D–F). We furthermore did not observe any inhibitory effect of FD on RV-induced Indoleamine 2,3-dioxygenase 1 (IDO1) expression (Figure 2G). This is relevant, since IDO1, an enzyme involved in tryptophan metabolism that may generate an endogenous ligand for the aryl hydrocarbon receptor (AhR), was found to be involved in the ability of IFN-β and IFN-γ to increase the expression of mucins including MUC5AC and MUC5AB through the AhR.10 Moreover, FD did inhibit the expression of TLR3, IRF1, and IDO1 upon IFN-β-exposure (Figure 2E–G), and this suggest that in RV-infected cells, FD-mediated inhibition of MUC5AC and MUC5B might be partly explained by its ability to affect IFN-β-signaling. Further studies using additional analyses, such as transcriptomics, kinomics, or metabolomics, may help to explain the mechanisms underlying the inhibitory effects of FD on mucin expression. This study demonstrates that exposure to FD reduces the expression of MUC5AC and MUC5B and its transcription factor FOXA3, not only after RV infection, but also at baseline. Mucus production is essential for mucociliary clearance of inhaled particles from the airways and serves as an important host defense mechanism. Therefore, it is important to clarify whether inhibitory effects of FD on mucin expression may have disadvantageous effects on, for example, respiratory host defense under homeostatic conditions. Whether FD reduces mucus hypersecretion upon RV infection and thus limits childhood wheeze and asthma development remains to be investigated. Sajuthi et al.11 demonstrated that MUC5AC and FOXA3 genetic variants are linked to airway mucus pathobiology in asthma, and our results here suggest that FD could alleviate MUC5AC-driven effects in asthma. So far, we have not identified the inhibitory component of FD or for instance used dust extracts from urban homes as controls to further specify the underlying mechanism of FD on mucin expression. Interestingly, a recent publication showed that a fraction, isolated from farm dust and containing animal and plant transport proteins loaded with microbial and/or plant metabolites, was sufficient to provide protection against allergic airways inflammation in mice.12 Searching for active components in FD that mediate these effects may serve as a lead for the development of novel asthma prevention strategies. Jasmijn A. Schrumpf: Conceptualization; writing – original draft; investigation; formal analysis; writing – review and editing; visualization. Dennis K. Ninaber: Investigation. Christoph Müller: Resources. Bettina Rankl: Resources. Erika von Mutius: Resources; conceptualization; funding acquisition. Hermelijn H. Smits: Conceptualization; writing – review and editing; funding acquisition; supervision. Pieter S. Hiemstra: Conceptualization; writing – review and editing; funding acquisition; supervision. This study is supported by a grant from the Lung Foundation Netherlands: AWWA grant #12.0.17.001. JAS and DKN declare no conflicts of interest related to this work. CM is inventor of the following patents: PCT application number EP21189353, entitled “Proteins identified from barn dust extract for the prevention and treatment of diseases” and PCT application, serial number PCT/EP2019/085016, entitled “Barn Dust Extract for the Prevention and Treatment of Diseases.” BR is inventor in PCT application number EP21189353, entitled “Proteins identified from barn dust extract for the prevention and treatment of diseases.” EM report a grant form Gottfried Wilhelm Leibniz Award 2013 of the German Research Foundation. EM is inventor of the following patents: EP2361632 (“Specific environmental bacteria for the protection from and/or the treatment of allergic, chronic inflammatory and/or autoimmune disorders”), EP1411977 (“Composition containing bacterial antigens used for the prophylaxis and the treatment of allergic diseases”), EP1637147 (“Stable dust extract for allergy Protection”), PCT/US2021/016918, entitled “Therapeutic Fractions and Proteins from Asthma-Protective Farm Dust,” PCT application number EP21189353, entitled “Proteins identified from barn dust extract for the prevention and treatment of diseases,” in PCT application, serial number PCT/EP2019/085016, entitled “Barn Dust Extract for the Prevention and Treatment of Diseases.” EM received honoraria as expert from Chinese University of Hongkong, European Commission, HiPP GmbH & Co KG, AstraZeneca, Imperial College London, OM Pharma, ALK-Abello Arzneimittel GmbH, and Boehringer Ingelheim International GmbH. EM received payment from Massachusetts Medical Society, Springer-Verlag GmbH, Elsevier Ltd., Boehringer Ingelheim International GmbH, European Respiratory Society (ERS), Universiteit Utrecht, Faculteit Diergeneeskunde, Universität Salzburg, Springer Medizin Verlag GmbH, Japanese Society of Pediatric Allergy and Clinical Immunology (JSPACI), Klinikum Rechts der Isar, University of Colorado, Paul-Martini-Stiftung, Astra Zeneca, Imperial College London, Children's Hospital Research Institute of Manitoba, Kompetenzzentrum für Ernährung (Kern), OM Pharma S.A., Swedish Pediatric Society for Allergy and Lung Medicine, Chinese College of Allergy and Asthma (CCAA), ALK-Abello Arzneimittel GmbH, Abbott Laboratories, Deutscher Apotheker Verlag GmbH & Co. KG, Japanese Society of Allergology. HHS and PSH report grants from Lung Foundation Netherlands. Cells were isolated from macroscopically normal lung tissue obtained from patients undergoing resection surgery for lung cancer at the Leiden University Medical Center, the Netherlands. Patients from which this lung tissue was derived were enrolled in the biobank via a no-objection system for coded anonymous further use of such tissue (www.coreon.org). However, since 01-09-2022, patients are enrolled in the biobank using active informed consent in accordance with local regulations from the LUMC biobank with approval by the institutional medical ethics committee (B20.042/Ab/ab and B20.042/Kb/kb). The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer-review/10.1111/pai.14008. Data S1: Table S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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