Abstract
Rationale While severe coronavirus infections, including Middle East respiratory syndrome coronavirus (MERS-CoV) cause lung injury with high mortality rates, protective treatment strategies are not approved for clinical use.
Objectives We elucidated the molecular mechanisms by which the cyclophilin inhibitors Cyclosporin A (CsA) and Alisporivir (ALV) restrict MERS-CoV to validate their suitability as readily-available therapy in MERS-CoV infection.
Methods Calu-3 cells and primary human alveolar epithelial cells (hAEC) were infected with MERS-CoV and treated with CsA or ALV or inhibitors targeting cyclophilin inhibitor-regulated molecules including Calcineurin, NFAT, or MAP kinases. Novel CsA-induced pathways were identified by RNA sequencing and manipulated by gene knockdown or neutralising antibodies. Viral replication was quantified by qRT-PCR and TCID50. Data were validated in a murine MERS-CoV infection model.
Results CsA and ALV both reduced MERS-CoV titers and viral RNA replication in Calu-3 and hAEC improving epithelial integrity. While neither Calcineurin nor NFAT inhibition reduced MERS-CoV propagation, blockade of c-Jun N-terminal kinase diminished infectious viral particle release but not RNA accumulation. Importantly, CsA induced interferon regulatory factor 1 (IRF1), a pronounced type-III-interferon (IFNλ) response and expression of antiviral genes. Down-regulation of IRF1 or IFNλ increased MERS-CoV propagation in presence of CsA. Importantly, oral application of CsA reduced MERS-CoV replication in vivo, correlating with elevated lung IFNλ levels and improved outcome.
Conclusions We provide evidence that cyclophilin inhibitors efficiently decrease MERS-CoV replication in vitro and in vivo via upregulation of inflammatory, antiviral cell responses, in particular IFNλ. CsA might therefore represent a promising candidate to treat MERS-CoV infection.
Abstract
The cyclophilin inhibitors Cyclosporin A and Alisporivir activate host innate immunity by induction of interferon lambda via activation of IRF1 in human lung epithelial cells and in vivo, resulting in a significant inhibition of MERS-CoV.
Introduction
Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012 in Saudi Arabia [1] and led to recurring human infections with more than 2500 laboratory-confirmed cases and high case fatality rates of about 35% [2]. In ex vivo infection of human lung tissue, MERS-CoV targets bronchial and alveolar epithelial cells (AEC) and leads to a detachment and apoptosis of AEC [3]. Recent reports analysing autopsy material of deceased MERS-CoV-infected patients showed MERS-CoV antigen in AEC and epithelial multinucleated syncytial cell conglomerates in vivo [4, 5]. Accordingly, severe human infection presents as pneumonia with progression to acute respiratory distress syndrome [4, 5].
To date, no vaccine or specific treatment for MERS-CoV - or the recently ongoing pandemic caused by the novel severe acute respiratory syndrome CoV 2 (SARS-CoV-2) - is approved and therapy relies on supportive measures only [2, 6]. While in vitro studies and experiments in non-human primates demonstrated benefits of a combination of type-I-interferon and antiviral compounds including ribavirin against MERS-CoV [7–9], results from retrospective patient cohorts applying similar treatment regimens remain controversial [10–12]. Cyclosporin A (CsA) has been found to inhibit several human-pathogenic CoV in cell lines originating from kidney or liver epithelia [13–16]. However, the molecular mechanisms by which CsA affects CoV, including MERS-CoV, particularly in its primary target cells, the pulmonary epithelium, remain elusive. Moreover, preclinical studies addressing the effect of CsA or related compounds on MERS-CoV replication in vivo have been lacking to date.
CsA is known to block the peptidyl-prolyl cis-trans isomerase (PPI) activity of cyclophilins that is involved in diverse cellular processes (e.g. protein folding, [17]). Additionally, CsA forms together with cyclophilin A (CypA) and calcineurin (CnA) a ternary complex which blocks the CnA-dependent activation of NFAT (nuclear factor of activated T-cells), a process which accounts for the immunosuppressive effect of CsA [18]. In addition, CsA has also been shown to inhibit the MAP kinases JNK (c-Jun N-terminal kinase) and p38 [19, 20].
Here, we aimed to elucidate the distinct signalling pathways by which CsA affects MERS-CoV in clinically relevant models such as primary human AEC and a murine MERS-CoV infection model [21, 22]. We demonstrate that CsA blocks MERS-CoV infectious particle egress, which is dependent on JNK. Moreover, we for the first time provide evidence that CsA triggered the activation of an antiviral defense state in lung epithelial cells. We show that CsA is a potent inducer of Interferon regulatory factor 1 (IRF1), type-III-IFN (IFNλ) and multiple interferon-stimulated genes (ISGs). Additionally, we demonstrate that oral application of CsA induced a robust IFNλ response in vivo and, importantly, significantly reduced MERS-CoV replication and improved disease progression in infected mice.
Materials and Methods
MERS-CoV infection
Experiments with MERS-CoV were performed under biosafety level 4 conditions at the Institute of Virology, Philipps-University of Marburg, Germany. Human alveolar epithelial cells (hAEC) were isolated and cultured as previously described [23]. Human lung tissue was obtained from patients who underwent lobectomy after informed written consent (Departments of Pathology and Surgery, University of Giessen, approved by the University of Giessen Ethics Committee; Az.58/15). Calu-3 or hAEC were infected at a multiplicity of infection (MOI) of 0.1 diluted in DMEM/F12 without FCS at 37°C for 1 h. Cells were washed with DMEM/F12 with 10% FCS and supplemented with stimulatory/inhibitory reagents as indicated. 24 h after infection (pi) cells were processed for quantitative PCR (Maxima-SYBR/ROX qPCR-Mastermix, Thermo Fisher) and supernatant was harvested for virus titration as described previously [24].
In vivo transduction and infection
All animal experiments were performed in accordance with the German animal protection laws and were authorised by the regional authorities (G73/2017). C57BL/6 mice were purchased from Charles River Laboratories and housed under pathogen-free conditions. Mice were intratracheally inoculated with Adenovirus-hDPP4-mCherry (cloned at ViraQuest Inc.) as described [21, 25]. 5 days post transduction, mice were infected intranasally with 1.5×105 TCID50/mL MERS-CoV (EMC/2012). 50 mg·kg−1·day−1 CsA diluted in DMSO or DMSO alone were mixed with a nut/chocolate-creme, and offered to the mice for voluntary uptake. Uptake was controlled daily. CsA feeding started 2 days before MERS-CoV challenge. Mice were sacrificed 4 or 7 days post MERS-CoV infection.
RNA-seq analysis
RNA integrity was assessed on an Experion StdSens RNA Chip (Bio-Rad). RNA-seq libraries were prepared using the TruSeq Stranded mRNA Library Prep kit (Illumina). Libraries were quantified on a Bioanalyzer (Agilent Technologies) and sequenced on an Illumina HiSeq 1500 platform, rapid-run mode, single-read 50 bp (HiSeq SR Rapid Cluster Kit v2, HiSeq Rapid SBS Kit v2, 50 cycles) according to the manufacturer's instructions. Quality control of RNA-seq reads was performed using the FastQC command line tool version 0.11.7. Reads were aligned using STAR version 2.7.0d to an index based on hg38 human genome version. Gene-specific read counts based on hg38 UCSC gene annotations were extracted using FeatureCounts from the Subread package version 1.6.3. Resulting read counts were imported into R. Detection of differentially expressed genes was done using DESeq2 version 1.22.1. Subsequent data analysis and visualisation was done with custom R scripts. GO overrepresentation analysis was performed using the enrichGO function of the clusterProfiler package version 3.10.1. Sequencing data are available at Array Express, accession number E-MTAB-8222.
Statistics
All data are given as mean±sem. Statistical significance was analysed by unpaired two-tailed Student's t-test or by 1-way ANOVA and post-hoc multi-comparison tests as indicated in the respective figures. A p value of less than 0.05 was considered significant. *p<0.05; **p<0.01; ***p<0.005.
Further experimental details can be found in the Online Supplement.
Results
Cyclosporin A (CsA) inhibits MERS-CoV replication and release in lung epithelia
To address the previously proposed antiviral activity of CsA in clinically relevant cells, we infected the human bronchial epithelial cell line Calu-3 and primary human alveolar epithelial cells (hAEC) with MERS-CoV and analysed intracellular viral RNA and infectious particle release in presence of DMSO or CsA (fig. 1). In both Calu-3 and hAEC, CsA treatment led to a >95% decrease of viral RNA (fig. 1a) and a reduction of viral titers in the supernatant by 2.6–2.8 log10, respectively (fig. 1b). Interestingly, and in accordance with reports from autopsy material of MERS-CoV patients [4], MERS-CoV-infected Calu-3 and primary hAEC both showed apoptotic cell loss and formation of multinucleated cell foci (fig. 1c). Addition of CsA reduced cell foci formation and significantly reduced apoptosis induction (fig. 1c and d). In line, both CFTR (cystic fibrosis transmembrane conductance regulator; fig. 1e) and ENaCβ (epithelial sodium channel beta; Supplement Fig.E1) protein expression was improved after addition of CsA to MERS-CoV-infected Calu-3. Moreover, epithelial structural integrity and ability for vectorial water transport were reduced in MERS-CoV-infected control cells and significantly improved to normal levels in MERS-CoV-infected, CsA-treated cells (fig. 1f and g).
Csa treatment affects MERS-CoV infection via CypA- and MAPK-signalling pathways
CsA is known to act via multiple signalling pathways including cyclophilin PPIase activity, the CnA-NFAT axis as well as MAP kinase signalling [17–20]. Using specific inhibitors, we aimed to interfere with CsA-affected pathways to identify relevant molecular signalling events involved in the CsA-mediated reduction of MERS-CoV infection. Inhibition of CnA by its specific inhibitor calcineurin inhibitory peptide (CIP), as well as inhibition of the downstream transcription factor NFAT resulted in minor, statistically non-significant changes in MERS-CoV viral titers in both Calu-3 and hAEC (fig. 2a and b). The non-immunosuppressive derivate of CsA, Alisporivir (ALV), that binds the PPIase but does not induce ternary complex formation of CypA with CnA, reduced viral titers to a similar extent as CsA, suggesting that the CypA-PPIase activity elicits the restrictive effect on MERS-CoV replication rather than ternary complex-mediated signalling events. Moreover, ALV reduced cell foci formation and loss of epithelial integrity to a similar extent as CsA (Supplement Fig. E2). Applying specific MAPK inhibitors against JNK and p38, we revealed that inhibition of the MAP kinase JNK, but not of p38 reduced MERS-CoV titers in both Calu-3 and hAEC (fig. 2a and b). However, neither inhibition of CnA-dependent signalling nor inhibition of JNK or p38 could reproduce the CsA-induced attenuation of MERS-CoV RNA accumulation. In addition, JNK inhibition had no positive effect on cell foci formation or epithelial integrity after MERS-CoV infection (Supplement Fig. E3). These data suggest a role for JNK activity late in MERS-CoV replication, where adverse effects on epithelial integrity are still displayed while viral release is blocked. Of note, application of ALV resulted in a strong reduction of MERS-CoV RNA levels similar to CsA (fig. 2c and d). Together, these results indicate that a CsA-induced, CypA-dependent effect has major impact on early replication steps of MERS-CoV, strongly reducing viral RNA accumulation, even prior to virus release, independently of CnA, NFAT or JNK.
Csa treatment evokes an interferon-driven antiviral state in lung epithelial cells
Our data suggest that, as opposed to its well-known CnA/NFAT-mediated immune-suppressive effects on immune cells, CsA might evoke an antiviral state in human lung epithelial cells. To identify the underlying mechanism, we performed RNA sequencing analysis on CsA- versus DMSO-treated Calu-3 cells. Of note, analysis of enriched gene sets based on GO terms revealed that the most significantly upregulated biological processes after CsA treatment included responses to viruses and, importantly, antiviral interferon responses (fig. 3a). In line with these results, both the type-I-IFN gene IFNB and type-III-IFN genes IFNL1 and IFNL2 were among the top-50, or, in case of IFNL1, top-10 most upregulated genes in CsA-treated cells (fig. 3b and c). Many of the upregulated genes were known interferon-stimulated genes (ISGs), including MX1, MX2, OAS1, OAS2, IFIT1, IFIT2, IFIT3, LAMP3, BST2/tetherin, RSAD2/viperin or CXCL10 (fig. 3b).
To validate our results, we analysed mRNA expression of both type-I and type-III-IFN by qRT-PCR in CsA-stimulated or DMSO-treated Calu-3 cells. We found a moderate upregulation of IFNB (up to 57 fold change over mock) and no significant induction of IFNA (fig. 4a, upper panels). However, we revealed a strong induction of IFNL1 and IFNL2/3 mRNAs (between 150 and 387 fold change over unstimulated mock control, respectively; fig. 4a, lower panels). Quantification of IFNλ1 and IFNλ3 protein release from cell culture supernatants by ELISA demonstrated a robust induction upon CsA addition as early as 12 h after CsA treatment, reaching peak values of 4222±890 ng protein mL−1 at 48 to 56 h after CsA application (fig. 4b). Similarly, treatment with ALV induced robust IFNλ release within reaching a similar maximum release of IFNλ after 72 h (Supplement Fig.E4). We next validated the CsA-induced upregulation of ISGs and confirmed an increased expression of selected ISGs including MxA, PKR, OAS1, IFIT1, IFIT2, IFIT3, Bst2/tetherin, RSAD2/viperin and XAF1 upon 18 h treatment with CsA compared to vehicle-treated control cells (fig. 4c). These data indicate that CsA treatment mounts a distinct interferon-driven antiviral response in lung epithelial cells.
IFNλ-induction is mediated by IRF1 upon CsA treatment in lung epithelial cells
To better understand the transcriptional programs leading to IFNλ induction in CsA-treated cells, we analysed the regulation of interferon regulatory factors (IRFs). Our data reveal significant upregulation of IRF1 mRNA levels upon CsA treatment, but not of IRF3, IRF7 or IRF9 (fig. 5a). IRF1 is known to be a specific activator of IFNL gene expression [26]. Accordingly, we identified a significantly increased number of IRF1-expressing cells in CsA-stimulated Calu-3 cells by immunofluorescence (fig. 5b). In line, IRF1 siRNA knockdown significantly reduced IFNL mRNA levels in CsA treated Calu-3 cells (fig. 5c). Accordingly, IRF1 knockdown inhibited IFNλ release by more than 75% as compared to control (fig. 5d).
Inhibition of the IRF1-IFNλ signalling axis counteracts the MERS-CoV restrictive effect of CsA
To understand the extent to which the inhibition of MERS-CoV propagation in CsA treated cells was mediated by IRF1-mediated production of IFNλ, we performed knockdown of IRF1 or neutralised cell-free IFNλ, respectively. Our data demonstrated that silencing of IRF1 but not treatment by control siRNA lead to a significant increase in MERS-CoV released viral particles in CsA-treated cells (fig. 6a and b). Moreover, neutralising antibodies directed against IFNλ1, IFNλ2 and IFNλ3 or against the less induced IFNβ were applied (fig. 6b). Neutralisation of IFNβ had no significant impact on MERS-CoV replication after CsA treatment, whereas application of anti-IFNλ1/2/3 treatment significantly increased MERS-CoV viral titers by 1.05 log10 level (fig. 6b). These data indicate that the antiviral effects of CsA were at least partially mediated by an IRF1-IFNλ signalling axis, and independent of type-I-IFN.
CsA-treatment upregulates IFNλ and leads to reduced MERS-CoV replication and lung pathology in vivo
To validate the antiviral efficacy of CsA against MERS-CoV in vivo, we used our recently established MERS-CoV infection mouse model that is based on the intratracheal delivery of the human DPP4 receptor to lung epithelial cells via adenoviral transduction, leading to severe MERS-CoV infection that presents as necrotising interstitial pneumonia [22]. We treated mice daily via oral intake of either DMSO or CsA, starting 2 days before mock or MERS-CoV infection. Oral CsA application resulted in CsA serum levels of 202 to 356 ng·mL−1 (mean 270 ±17 ng·mL−1), a concentration that compares to levels reached in patients under CsA treatment (Supplement Figure E5, 34, 35). Accordingly, CsA treatment significantly induced release of IFNλ in the bronchoalveolar lavage fluid (fig. 7a). IFNL induction was significantly elevated in the CsA treatment group compared to DMSO-treated mice at day 7 post MERS-CoV infection (fig. 7b). Oral application of CsA significantly reduced viral titers (3.45 ±0.15 versus 2.1 ±0.36 TCID50/mL in the DMSO versus CsA group) at day 7 post MERS-CoV infection (fig. 7c). CsA treatment did not alter adenoviral transduction efficiency (Supplemental Fig. E6). Of note, expression levels of IFNL inversely correlated with MERS-CoV load in lung homogenates at day 7 pi (fig. 7d). A significant reduction in viral titers and a significant correlation between IFNL induction and MERS-CoV inhibition could also be demonstrated at day 4 pi (Supplement Fig. E7). Expression of the SCNN1B gene (ENaCβ) as a marker of epithelial integrity was improved in lung homogenates of MERS-CoV-infected mice treated with CsA (fig. 7e). While extensive edema formation was present in a substantial portion of MERS-CoV-infected mice, it was absent in the CsA-treated group (Supplement Fig. E8). Importantly, the percentage of lung areas showing histopathological alterations due to MERS-CoV infection was significantly decreased by CsA treatment at day 7 post infection (fig. 7f). Collectively, we demonstrate that oral application of CsA induced IFNλ in the lungs of mice and exerted potent antiviral activity in vivo.
Discussion
With the appearance of SARS-CoV in 2002, MERS-CoV in 2012 and recently SARS-CoV-2, three species of the family Coronaviridae have revealed the ability to be efficiently transmitted from human-to-human and to provoke serious disease with high mortality rates. Both SARS-CoV and MERS-CoV are listed on the WHO blueprint list of priority diseases, and the zoonotic CoV reservoir strains are generally considered and have now been proven to be a source for emerging pandemic viruses.
As no specific treatment is approved for MERS-CoV or SARS-CoV(-2), current treatment strategies are supportive [29, 30]. Treatments including recombinant type-I-IFN and antivirals (e.g. Lopinavir/Ritonavir) have been applied off-label to treat MERS-CoV and yielded only moderate efficacy with controversial results in retrospective studies, and data from prospective studies or randomised controlled trials are lacking [29, 31–33]. Due to its receptor specificity to the human DPP4, only few animal models to study MERS-CoV pathogenesis and MERS-CoV-directed antiviral compounds have been accessible to date. For this study, MERS-CoV infection in the mouse was facilitated via intratracheal delivery of a human DPP4-encoding adenovirus, that might cause low-level inflammation itself and inhomogeneous receptor distribution within the lung, present for a limited time frame. However, even if this model might not fully recapitulate the native cellular distribution or density of the receptor as seen in the human lung, high transduction efficiencies (≥ 95%, data not shown) allow efficient viral spread in the upper and lower respiratory airways with quick progression to severe lung injury [22] and with moderate changes in morbidity [34]. Thus, model-specific neurotropism as seen in some of the transgenic hDPP4 mice [35] or the necessity to adapt virus isolates via multiple passages, which might potentially affect its susceptibility to interventional strategies, are circumvented. While prior exposure to adenovirus evokes moderate histological changes including perivascular and bronchiolar lymphocytic infiltration (data not shown), MERS-CoV infection led to a clearly distinguishable granulocytic, necrotising interstitial pneumonia with alveolar edema formation as described previously [22].
CsA has been implicated as inhibitor of a broad spectrum of virus families, including diverse CoV [14, 36–41]. However, studies on efficacy of CsA against CoV infection relied on results in liver and kidney cell lines [14–16], while results from primary lung epithelial target cells were lacking. Recently, CsA was demonstrated to restrict MERS-CoV ex vivo [13]. Still, insights on mechanistic details and on the question if CsA application would affect MERS-CoV infection in vivo remained elusive.
We now demonstrate that CsA application blocks MERS-CoV both at mRNA level and the amount of infectious viral particles released and significantly improves epithelial barrier integrity after MERS-CoV infection. Using different inhibitors known to block CsA-targeted pathways, we reveal that the CsA-induced blockade of MERS-CoV RNA synthesis can neither be reproduced by inhibition of known CsA-targeted MAP kinases nor by blockade of NFAT activation. Of note, ALV, which blocks CypA PPIase activity efficiently, but affects NFAT-dependent pathways only at very high concentrations [42], diminished MERS-CoV RNA accumulation as efficiently as CsA, suggesting that CypA plays a pivotal role in these processes. In fact, we revealed a previously unknown activation of genes involved in innate immune responses and in limitation of virus replication upon administration of CsA to lung epithelial cells. Moreover, we demonstrate that inhibition of CypA via CsA or ALV, which both potently block the CypA PPIase activity at the used concentrations [42], results in a pronounced upregulation of type-III-IFN on both mRNA and protein level, which was mediated via IRF1 and was accompanied by expression of antiviral ISGs. Among those, especially IFIT1 (Interferon-induced protein with tetratricopeptide repeats 1), has been reported to influence the pathogenesis of MERS-CoV, highlighting the relevance of our findings [43].
Of note, type-III-IFNs have recently emerged as key antiviral players in the innate immune response to viral infections at mucosal and epithelial surfaces [44–47]. They efficiently restrict different respiratory viruses, and act e.g. by limiting spread from the upper to the lower airways [44, 46–48]. As opposed to type-I-IFN, type-III-IFN do not trigger detrimental immune responses that contribute to immunopathology in influenza infection [23, 25, 44, 49]. This might prove to be pivotal in the context of CsA-dependent stimulation of IFNλ during CoV, as severe human CoV infections, like MERS-CoV and– while data are still limited – also SARS-CoV-2, are characterised by an immunopathology with a strong cytokine induction [5, 50, 51].
In addition to defining a novel pro-inflammatory, antiviral expression profile induced by CsA on lung epithelial cells, this study also demonstrated for the first time that oral application of CsA reduces viral load in an in vivo MERS-CoV infection model. CsA is a licensed drug in clinical use since the 1980s. While prolonged treatment (over weeks and months) with CsA can induce side effects (e.g. nephrotoxicity; 48), we here applied a short-interval oral intake of CsA during acute infection. Our results demonstrate that in vivo, oral application over 6 days results in drug serum levels efficiently inhibiting lung viral infection and pneumonia progression, highlighting CsA as a promising drug to be re-purposed for treatment of MERS-CoV.
Notably, our in vitro studies also revealed that neutralisation of type-III-IFNs did not completely reverse the MERS-CoV-restrictive effect of CsA. We therefore suggest that CsA affects MERS-CoV at multiple steps during viral replication. In fact, we show that CsA acts on MERS-CoV propagation via inhibition of JNK, which is another downstream target of CsA [19, 53]. JNK inhibition has no impact on MERS-CoV RNA accumulation but strongly reduces the amount of released infectious virions. While the exact underlying molecular mechanisms remain to be defined, this finding demonstrated that CsA likely exerts additive effects to restrict MERS-CoV replication. While application of recombinant IFNs are approved to treat virus infections and malignancies, severe side effects have been related to systemic IFN application [54]. CsA repurposing for treatment of (MERS-) CoV infection might therefore come with several advantages over IFN treatment, such as additional antiviral effects beyond those mediated by IFNλ alone, a favorable side effect profile upon short-term use, a beneficial effect regarding an overshooting immune response characterising CoV disease [55, 56] and proven oral availability (64, 65). CsA therefore represents a promising therapeutic option to combat human CoV infections, potentially extending over MERS-CoV to the current pandemic SARS-CoV-2 strain and future CoV threats.
Acknowledgements
Work with live MERS-CoV was performed in the BSL-4 facility of the Philipps University, Marburg, Germany. We thank Julia Spengler, Larissa Hamann, Stefanie Jarmer, Dirk Becker and Marc Ringel for excellent technical and experimental assistance. We thank Ralf Bartenschlager for providing Alisporivir.
Footnotes
Sequencing data are available at Array Express, accession number E-MTAB-8222.
This article has supplementary material available from erj.ersjournals.com
Support statement: This work was supported by the German Research Foundation (KFO309 P2/P8; Project ID: 284237345; SFB-TR84 B2, Project ID: 114933180; SFB1021 C05, Project ID: 197785619), by the German Center for Lung Research (DZL), by the German Center for Infection Research (DZIF) and the Cardio-Pulmonary Institute (CPI), EXC 2026, Project ID: 390649896. Cardio-Pulmonary Institute (CPI); Grant: EXC 2026, Project ID: 390649896; Deutsche Forschungsgemeinschaft; DOI: http://dx.doi.org/10.13039/501100001659; Grant: Project ID: 114933180 B2, Project ID: 197785619 C05, Project ID: 284237345 P2/8; Deutsches Zentrum für Infektionsforschung; DOI: http://dx.doi.org/10.13039/100009139; Deutsche Zentrum für Lungenforschung; DOI: http://dx.doi.org/10.13039/501100010564.
Conflict of interest: Dr. Sauerhering has nothing to disclose.
Conflict of interest: Dr. Kupke has nothing to disclose.
Conflict of interest: Dr. Meier has nothing to disclose.
Conflict of interest: Dr. Dietzel has nothing to disclose.
Conflict of interest: Judith Hoppe has nothing to disclose.
Conflict of interest: Dr. Gruber has nothing to disclose.
Conflict of interest: Dr. Gattenloehner has nothing to disclose.
Conflict of interest: Dr. Witte has nothing to disclose.
Conflict of interest: Dr. Fink has nothing to disclose.
Conflict of interest: Nina Hofmann has nothing to disclose.
Conflict of interest: Dr. Zimmermann has nothing to disclose.
Conflict of interest: Dr. Goesmann has nothing to disclose.
Conflict of interest: Dr. Nist has nothing to disclose.
Conflict of interest: Dr. Stiewe has nothing to disclose.
Conflict of interest: Dr. Becker has nothing to disclose.
Conflict of interest: Dr. Herold has nothing to disclose.
Conflict of interest: Dr. Peteranderl has nothing to disclose.
- Received September 16, 2019.
- Accepted June 3, 2020.
- Copyright ©ERS 2020
This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial Licence 4.0.