Clinical & Translational Immunology 2022; e1376. doi: 10.1002/cti2.1376 www.wileyonlinelibrary.com/journal/cti ORIGINAL ARTICLE Epigenetic regulation of inflammation by microRNAs in post-infectious bronchiolitis obliterans Ruth P Duecker1 , Ines De Mir Messa2, Silvija-Pera Jerkic1, Annalena Kochems1, Gabriele Gottwald1, Antonio Moreno-Galdó2,3, Martin Rosewich1, Lucia Gronau1, Stefan Zielen1, Andreas Geburtig-Chiocchetti5, Hermann Kreyenberg4 & Ralf Schubert1 1 Division for Allergy, Pneumology and Cystic Fibrosis, Department for Children and Adolescence, Goethe University, Frankfurt, Germany 2 Allergy, Pulmonology and Cystic Fibrosis Section, Department of Pediatrics, Vall d’Hebron Hospital Universitari, Universitat Autònoma de Barcelona, Barcelona, Spain 3 CIBER of Rare Diseases (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain 4 Division for Stem Cell Transplantation and Immunology, Department for Children and Adolescence, Goethe University, Frankfurt, Germany 5 Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, Goethe University, Frankfurt, Germany Correspondence Abstract RP Duecker, Division for Allergy, Pneumology and Cystic Fibrosis, Department for Children Objectives. Post-infectious bronchiolitis obliterans (PiBO) is a rare, and Adolescence, Goethe University, chronic disease initiated by severe infection and followed by Theodor-Stern-Kai 7, 60590 Frankfurt am perpetuating inflammation and obliteration of the small airways. Main, Germany. MicroRNAs (miRNAs) have been proposed to play a central role as E-mail: ruthpia.duecker@kgu.de epigenetic regulators, which control resolution and prevent the uncontrolled progress of inflammation. The aim of this study was Received 27 March 2021; to define biomarkers on the level of post-transcriptional gene Revised 14 November 2021 and regulation in order to characterise PiBO. Methods. A total of 39 3 February 2022; patients with well-defined PiBO and 31 controls from two centres, Accepted 4 February 2022 Barcelona, Spain, and Frankfurt, Germany, were analysed by next- generation sequencing (NGS). The evaluation of the biological doi: 10.1002/cti2.1376 targets of the miRNAs was performed by pathway enrichment Clinical & Translational Immunology analysis and protein–protein interaction network analysis 2022; 11: e1376 respectively. Results. Patients with PiBO had significantly lower lung function values and increased airway inflammation in induced sputum as indicated by total cell counts, neutrophils, IL- 1β, IL-6, IL-8 and TGF-β compared to controls. Next-generation sequencing analysis revealed a total of 22 dysregulated miRNAs, which passed significance threshold for Padj ≤ 0.001 with 17 being upregulated and 5 being downregulated. Of these dysregulated miRNAs, miR-335-5p, miR-186-5p, miR-30b-5p and miR-30c-5p were further validated using qRT-PCR. Interestingly, these miRNAs are functionally implicated in cytokine–cytokine receptor interaction, TGF-β signalling and FoxO signalling pathway and significantly correlated with lung function values (FEV1). Conclusion. Our results demonstrate an aberrant miRNA expression profile in PiBO, which impacts pathways responsible for the regulation of inflammation and fibrosis. The defined miRNAs are useful ª 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of 2022 | Vol. 11 | e1376 Australian and New Zealand Society for Immunology, Inc. Page 1 miRNA in post-infectious bronchiolitis obliterans RP Duecker et al. biomarkers and should be assessed as potential target in the field of miRNA therapeutics. Keywords: fibrosis, inflammation, microRNA, post-infectious bronchiolitis obliterans of miRNA expression. A study cohort of Spanish INTRODUCTION and German patients with PiBO was investigated Post-infectious bronchiolitis obliterans (PiBO) is a to overcome biases in regional and lifestyle rare, chronic pulmonary disease characterised by factors. Clinical, laboratory and computational inflammation and fibrous wall thickening of the analyses were performed to identify relevant bronchioles, narrowing their lumens and restricted miRNAs, which have the potential as biomarkers air circulation, which emerges after severe in PiBO and which should be investigated as pulmonary infection.1,2 Although the pathogenesis targets for new therapies. of PiBO is still largely unclear, the lack of resolution of the ongoing inflammation seems to be an RESULTS important part of the disease process. A pathomechanism with a predominant neutrophilic In total, 39 patients with PiBO and 31 controls inflammation in the bronchioles together with from the Children’s Hospitals in Barcelona and increased concentrations of inflammatory cytokines Frankfurt were enrolled into the study (Table 1). has been proposed.3,4 This inflammation stimulates No differences were found in age or gender the migration of fibroblasts and myofibroblasts between the sites. Two patients from the into the luminal exudate, resulting in intraluminal Frankfurt cohort had to be excluded from the NGS granular tissue formation. Thus, the new tissue analysis. No blood for NGS was available from one formation can lead to a complete occlusion of the patient. One patient sample had to be removed small airways.5 Recent observations in induced from the data analysis because of too-low reads. sputum of PiBO patients demonstrated ongoing neutrophilic inflammation without termination.6 Pulmonary function This unresolved inflammation in PiBO and the identification of endogenous control mechanisms As expected, measurement of lung function in the inflammatory response is of critical parameters showed a significant reduction of FVC importance. [patients (F) 76.3 (range 54.5–94.2%; P < 0.001); Studies in other immune-mediated lung diseases patients (B) 80.6 (range 49.3–98.4%; P < 0.01); such as asthma, cystic fibrosis, idiopathic controls 90.8 (range 73.9–102.7)], FEV1 [patients interstitial pneumonia or COPD suggest an (F) 59.8 (range 40.2–96.9%; P < 0.001); patients (B) epigenetic dysregulation of the immune response 62.0 (range 29.03–99.0%; P < 0.001); controls 97.4 to external and internal stimuli.7 The post- (range 73.5–116.4%)], FEV1/FVC [patients (F) 0.7 transcriptional regulation by microRNAs (miRNAs) (range 0.4–1.0%; P < 0.001); patients (B) 0.7 is a central epigenetic mechanism.8 MiRNAs are a (range 0.4–1.0%; P < 0.001); controls 0.9 (range highly conserved group of small (22–25 0.7–1.0%)] and FEF75 [patients (F) 25.1 (range 9.2– nucleotides), relatively stable, non-coding RNA 195.7%; P < 0.01); patients (B) 52.1 (range 7.1– molecules, which control gene expression 97.9%; P < 0.001); controls 107.4 (range 41.3– primarily via mRNA silencing.9 Dysregulation of 184.7%)] among patients with PiBO compared to miRNA expression leads to aberrant gene controls (Table 1, Supplementary tables 3–5). This expression and might be responsible for was true for patients from the Frankfurt cohort as pathological conditions.10 In this regard, miRNA well as for patients from Barcelona. expression profiles might have the potential as biomarkers in inflammatory lung diseases. Bronchial inflammation However, so far, only limited data about miRNA expression in PiBO exist. Therefore, the aim of the To assess airway inflammation, induced sputum present study was to investigate the regulation of was analysed for inflammatory cells and mediators inflammation and fibrotic processes in the context in the Frankfurt patient and control group. In the 2022 | Vol. 11 | e1376 ª 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 2 Australian and New Zealand Society for Immunology, Inc. RP Duecker et al. miRNA in post-infectious bronchiolitis obliterans Table 1. Lung function of patients with post-infectious bronchiolitis obliterans from Frankfurt (F) and Barcelona (B) compared to controls Controls Patients (F) Patients (B) N 31 19 20 Age (years) 14 (7 to 28) 13 (6 to 28) 13 (6 to 20) Sex (m/f) 19/12 13/6 11/9 FVC (%) 90.8 (73.9 to 02.7) 76.3*** (54.5 to 94.2) 80.6** (49.3 to 98.4) FVC (z score) −0.77 (−2.23 to 0.24) −2.04***(−3.84 to −0.46) −1.68** (−4.11 to −1.30) FEV1 (%) 97.4 (73.5 to 116.4) 59.8*** (40.2 to 96.9) 62.0*** (29.3 to 99.0) FEV1 (z score) −0.54 (−2.20 to −1.41) −3.42*** (−4.86 to −0.25) −3.08*** (−5.64 to −0.08) FEV1/FVC 0.9 (0.7 to 1.0) 0.7*** (0.4 to 1.0) 0.7*** (0.4 to 1.0) FEV1/FVC (z score) 0.33 (−2.26 to 2.49) −2.19*** (−4.45 to 2.19) −3.58*** (−4.50 to 1.42) FEF75 (%) 107.4 (41.3 to 184.7) 25.1** (9.2 to 195.7) 52.1*** (7.1 to 97.9) FEF75 (z score) 0.21 (−2.30 to 2.01) −3.20*** (−5.03 to 2.07) −1.70*** (−5.30 to 0.1) Values are expressed as percentage and z score (median and range). FEF, forced expiratory flow; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity. *P < 0.05, **P < 0.01, ***P < 0.001, significant to controls. patient group, neutrophils and inflammatory Barcelona and Frankfurt were successfully cytokines such as IL-1β, IL-6 and IL-8 were assessed. Analysis revealed ~158 Mio reads with an significantly increased compared to controls average read count per sample of 2 323 748 reads (Figure 1a–e). In addition, TGF-β, which is an per miRNA. Overall, 877 miRNAs were detected in essential cytokine for the induction of the fibrotic at least one peripheral blood sample, of which response, was significantly increased in induced 288 had 5 or more reads in each sample and 538 sputum of PiBO patients (Figure 1f). miRNAs were detected with 5 or more reads in at least 50% of the samples. The groups showed no differences (ANOVA P-values > 0.1) with respect miRNA expression to age, sex, site, RNA quality and read depth. Differences in the global miRNA expression Statistical analysis revealed a total of 22 between 37 PiBO patients and 31 controls from dysregulated miRNAs passing the significance (a) (b) (c) *** *** ** 10000 100 100 Macr ophages (% ) Neutrophils (% ) IL-1β (pg mL–1) 80 80 1000 60 60 40 40 100 20 20 0 0 10 Controls Patients Controls Patients Controls Patients (d) (e) (f) 100000 ** 1000000 *** 20000 * TGF-β (pg mL–1) 10000 IL-8 (pg mL–1) IL-6 (pg mL–1) 100000 15000 1000 10000 10000 100 1000 5000 10 1 100 0 Controls Patients Controls Patients Controls Patients Figure 1. Bronchial inflammation. (a) Neutrophils and (b) macrophages as well as the inflammatory cytokines (c) IL-1β, (d) IL-6, (e) IL-8 and (f) TGF-β were analysed in induced sputum from patients with PiBO (n = 14) and healthy controls (n = 13) by cell differentiation and cytometric bead array, respectively. *P < 0.05, **P < 0.01, ***P < 0.001. ª 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of 2022 | Vol. 11 | e1376 Australian and New Zealand Society for Immunology, Inc. Page 3 miRNA in post-infectious bronchiolitis obliterans RP Duecker et al. threshold of Padj ≤ 0.001 (195 miRNAs for TGF-β signalling pathway and FoxO signalling P < 0.05), of which 17 were upregulated and 5 pathway (Figure 4). Network analysis showed a downregulated (Figure 2a, Supplementary table 2). strong interaction of the target proteins between Of these dysregulated miRNAs, miR-1287-5p, miR- the pathways (PPI enrichment P-value < 1.0−16). 335-5p, miR-5695, miR-30b-5p, miR-186-5p, miR- Three targets, TGFB2, TGFBR1 and TGFBR2, were 190b, let-7f-1-3p, miR-30c-5p, miR-26a-2-3p, miR- located in all three pathways; IL-6 and EGFR were 4677-3p and miR-199a-5p share dysregulated located in cytokine–cytokine receptor interaction; expression after separate analyses of the two and 11 targets, ACVR1, ACVR2B, AMHF, BMP2, centres (Figure 2b; Supplementary figure 1a and BMPR1A, BMPR1B, BMPR2, GDF5, INHBA, INHBB b). Of these, four miRNAs were successfully and INHBE, were located in cytokine–cytokine confirmed by TaqMan qPCR namely miR-335-5p, receptor interaction and in the FoxO signalling miR-30b-5p, miR-186-5p and miR-30c-5p pathway. Most important, we identified the (Figure 2c–f). These four were considered in the inflammatory cytokines IL-1A, IL-6 and CXCL8 (IL- further computational analysis. The expression 8) as well as TGFB2. levels for miR-1287-5p, let-7f-1-3p and miR-199a-5p only showed a tendency between patients and Knockdown of miR-335-5p, miR-186-5p, miR- controls, and no differences were found for miR- 30b-5p and miR-30c-5p enhances IL-6 protein 5695, miR-26a-2-3p and miR-4677-3p levels in A549 cells (Supplementary figure 1c–h). Expression of miR- 190b could not be detected using TaqMan in most To demonstrate the functional output in altering of the samples. protein expression and in order to validate the role of miR-335-5p, miR-186-5p, miR-30b-5p and miR-30c-5p as regulators of IL-6 expression, we KEGG pathway analysis measured the secretion of IL-6 in a lung epithelial To identify the contribution of the miRNAs to cell inflammation system. As shown in Figure 5a, biological processes, KEGG pathway enrichment an increase of IL-6 secretion of A549 cells was analysis has been performed, and targets of the evident after the separate inhibition of miR-335- individual miRNAs were assessed. In total, four 5p, miR-186-5p, miR-30b-5p and miR-30c-5p and miRNAs of interest were predicted to be involved after the inhibition of all four miRNAs at every in 34 pathways including cancer, cell signalling, level of CM stimulation. Combination of these cell proliferation and apoptosis, cell–cell experiments comparing fold change of IL-6 interaction; cytokine–cytokine receptor interaction secretion of the inhibited miRNAs with the (P < 0.05); and TGF-β signalling pathway (P < 0.05) respective CM stimulation showed that IL-6 and target 3775 genes (Figure 3). Shared targets expression after inhibition of the four miRNAs for miR-186-5p and miR-30b/c-5p were ST3GAL5, separately as well as in the knockdown of all four LRRC3C, PLXNA1 and LPCAT1. miRNAs simultaneously was significantly increased Single analysis of the individual miRNAs (miR-335-5p, 1.23  0.04; miR-186-5p, 1.23  0.04; concentrating on inflammatory processes revealed miR-30b-5p, 1.34  0.08; miR-30c-5p, 1.22  0.05; that 61 target genes of miR-335-5p are strongly all miRNAs inhibited together, 2.46  0.43; involved in cytokine–cytokine receptor interaction P < 0.05; Figure 5b). (P < 0.0009), 20 target genes of miR-186-5p are strongly involved in the TGF-β signalling pathway miRNA expression of miR-335-5p, miR-186- (P < 0.0002), and 18 target genes of miR-30b-5p 5p, miR-30b-5p and miR-30c-5p in induced and miR-30c-5p are strongly involved in the FoxO sputum signalling pathway (P < 0.0095) respectively (Table 2; Supplementary figure 2). miRNA expression in induced sputum samples of PiBO patients was compared with miRNA profile of age-matched healthy individuals (Figure 6a). Protein–protein interaction in inflammation miR-335-5p and miR-186-5p were significantly Based on the KEGG analysis, we used the STRING upregulated in sputum of PiBO patients compared web server to visualise the network of the to controls (miR-335-5p controls 1.74  0.1, PiBO protein–protein interaction of the miRNA targets 2.36  0.22, miR-186-5p controls 637.5  120.5, involved in cytokine–cytokine receptor interaction, PiBO 1849  503.8; P < 0.05). No significant 2022 | Vol. 11 | e1376 ª 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 4 Australian and New Zealand Society for Immunology, Inc. RP Duecker et al. miRNA in post-infectious bronchiolitis obliterans (a) (c) miR-335-5p 10 *** 8 6 2-ddCT miR-335-5p 4 -log10 (nom P-val) miR-30b-5p 2 miR-186-5p 0 Controls PiBO miR-30c-5p (d) padj <0.001 miR-30b-5p 4 *** 3 2-ddCT 2 p <0.05 1 0 Controls PiBO log2(FC) (e) miR-186-5p 10 *** (b) 8 6 2-ddCT 4 miR-1287-5p miR-192-5p 2 miR-335-5p 0 miR-30d-5p Controls PiBO miR-5695 miR-30b-5p miR-140-3p 11 111 (f) 29miR-183-5p miR-186-5p miR-190b let-7f-1-3p miR-30c-5p miR-1307-3p miR-7-1-3p miR-3140-3p 5 miR-30c-5p *** miR-26a-2-3p miR-215-5p 4 miR-4677-3p miR-340-3p 3 2-ddCT miR-199a-5p Frankfurt Barcelona 2 1 0 Controls PiBO Figure 2. Global miRNA expression of PiBO patients (n = 36) and controls (n = 30). (a) Volcano plot showing 877 detected miRNAs, of which 195 were significant dysregulated at nominal level P < 0.05 and 22 were significant with a Padj < 0.001. (b) Intra-centre evaluation of the dysregulated miRNAs. Intersections shows miRNAs with Padj < 0.05 in the individual analysis of the respective centre. Only these miRNAs were used for further analysis. (c–f) Expression levels of miR-335-5p, miR-30b-5p, miR-186-5p and miR-30c-5p confirmed by TaqMan qPCR analysis. ***P < 0.001. ª 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of 2022 | Vol. 11 | e1376 Australian and New Zealand Society for Immunology, Inc. Page 5 miRNA in post-infectious bronchiolitis obliterans RP Duecker et al. Figure 3. Interaction of the dysregulated miRNAs. (a) Circle chart of Kyoto encyclopaedia of genes and genomes (KEGG) pathway analysis based on microRNA signature was performed with miRNet (miRTarBase v8.0) for all the identified miRNAs together. (b) Dissected miRNA–target interactions and functional associations through network-based visual analysis (miRNet). miR-335-5p, miR-30b-5p, miR-186-5p and miR-30c-5p regulate 3775 genes, four of which are shared targets (red dots). Targets involved in cytokine–cytokine receptor interaction (blue dots) and in TGF-β signalling (green dots) are shown. 2022 | Vol. 11 | e1376 ª 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 6 Australian and New Zealand Society for Immunology, Inc. RP Duecker et al. miRNA in post-infectious bronchiolitis obliterans Table 2. miRNA targets in inflammation miRNA Targets database KEGG pathway P-value Genes miR-335-5p TarBase v7.0 Cytokine–cytokine receptor interaction 0.0009 61 miR-186-5p microT-CDS (5.0) TGF-beta signalling pathway 0.0002 20 miR-30b-5p microT-CDS (5.0) FoxO signalling pathway 0.0095 18 miR-30c-5p microT-CDS (5.0) FoxO signalling pathway 0.0095 18 differences could be detected for miR-30b-5p and analysis identified four aberrant miRNAs, miR-335- miR-30c-5p. 5p, miR186-5p, miR-30b-5p and miR-30c-5p, which are involved in inflammatory and fibrotic processes such as cytokine–cytokine receptor Expression of miR-335-5p, miR-186-5p, miR- interaction, TGF-β and FoxO signalling. 30b-5p and miR-30c-5p correlates with the The role of these miRNAs in the regulation in lung function parameter FEV1 inflammatory processes was confirmed by our Next, we performed correlation analyses to in vitro experiments, showing that knockdown of investigate the relationship between circulating miR-335-5p, miR-186-5p, miR-30b-5p and miR-30c- miRNA expression in the blood and lung function 5p enhances IL-6 protein levels in stimulated A549 parameter such as forced expiratory volume in 1 s cells. In their function as post-transcriptional gene (FEV1). Expression of miR-335-5p, miR-186-5p, miR- regulators, miRNAs inhibit the translation or 30b-5p and miR-30c-5p significantly correlated with transcription of inflammatory target mRNAs. The FEV1 (miR-335-5p P < 0.001, r = −0.4345, miR-186-5 downregulation of these miRNAs leads to an P < 0.01, r = −0.3582, miR-30b-5p P < 0.001, increase in protein synthesis. In their study, Long r = −0.4322, miR-30c-5p P < 0.01, r = −0.3990, et al.17 impressively demonstrated that Figure 6b) of PiBO patients and healthy controls. upregulation of miR-335 has protective effects against sepsis-induced myocardial injury and that downregulation reverses the protective effect. DISCUSSION Several studies have connected miR-335-5p to the A potent role of miRNAs in the regulation of activation of pathways, which are directly related resolution circuits of inflammation has been to inflammation and the TGF-β pathway.18–20 This proposed.11 MicroRNAs are regulatory factors is highly supported by our laboratory and involved in most biological processes and have to be computational findings, which show the considered to play a key role in the development and involvement of miR-335-5p in the cytokine and manifestation of inflammatory lung diseases, such as chemokine network, which interacts closely with CF, IPF, COPD, PiBO and asthma.12 Unresolved the TGF-ß pathway and reflects the bronchial neutrophilic inflammation also contributes to the inflammatory and fibrotic processes in PiBO. Our chronicity of lung disease in PiBO.4,6,13 This prompted analysis demonstrated that miR-335-5p induces us to use global NGS to get deeper insights into post- the secretion of various pro-inflammatory transcriptional regulation of gene expression in chemokines/cytokines including IL-1α, IL-6 and IL-8 patients with PiBO. As described, PiBO patients as well as targeting members of the TGF-β exhibited impaired pulmonary function parameters signalling such as TGFβ2, TGFβR1 and TGFβR2. In as it has been described previously.13–15 Airway this regard, upregulation of miR-335-5p reduced inflammation in induced sputum was confirmed by inflammation in a chronic rhinosinusitis mouse increased neutrophils and pro-inflammatory model.19 In addition, confirmation of modelling cytokines such as IL-1ß, IL-6 and IL-8 in German pathway analyses was proved by Du et al.,20 who samples.4 In addition, we detected increased TGF-β showed that miR-335-5p inhibits TGF-β1-induced levels in the sputum of the patients pointing to epithelial–mesenchymal transition in non-small- fibrotic processes in the bronchial tissue.16 cell lung cancer via ROCK1. Beside the tight In accordance with increased levels of connection of miR-335-5p and the TGF-β pathway, neutrophils and pro-inflammatory cytokines such we also found ROCK1 as part of the protein– as IL-1ß, IL-6 and IL-8 in induced sputum, our protein interaction. ª 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of 2022 | Vol. 11 | e1376 Australian and New Zealand Society for Immunology, Inc. Page 7 miRNA in post-infectious bronchiolitis obliterans RP Duecker et al. Expression of miR-335-5p has been found to be increased in obliterative airway remodelling in decreased in parenchymal lung fibroblasts from transplanted and non-transplanted lungs.31 Thus, current smokers and to be reduced in the serum our findings support the hypothesis that BMPs are from patients with Parkinson’s disease, important players in the pathomechanism of investigating miRNAs targeting cell death and/or airway remodelling. inflammation pathways.18,21 In contrast, in our Further impact on inflammation and on TGF-β present study miR-335-5p expression was elevated signalling, controlling the expression of SMAD2 and in blood samples from PiBO patients compared to TGFβR1, has been described for the miR-30 controls. This fact might be because of the nature, family.32,33 Increased miR-30b expression inhibits quality and duration of the inflammation in PiBO, endothelial cell capillary morphogenesis through which drives the expression of miR-335-5p upregulated TGF-β2 expression.34 In addition, miR- without significant effect on the resolution of 30c attenuates atrial fibrosis via inhibition of inflammation. However, the high expression of proliferation, differentiation, migration and collagen miR-335-5p qualifies this specific miRNA as a production by targeting TGFβR2.35 Our KEGG potential marker for inflammation in PiBO. pathway analysis revealed that upregulated miR-30b Beside miR-335-5p, NGS analysis also showed a and miR-30c were highly associated with the FoxO highly significant differential expression of miR- signalling pathway. FoxO family members have been 186-5p and of the miR-30 family members, miR- increasingly recognised to be involved in chronic 30b-5p and miR-30c-5p. miR-186-5p has been inflammation and immune homeostasis.36 FOXO1 shown to impact regulation of proliferation and transcription factors affect a number of cell types apoptosis strongly and has been proposed to be that are important in the host immune response involved in the pathogenesis of COPD.22,23 To our including the mucosal dermis, macrophages and knowledge, this is the first study, which connects neutrophils.37 Overexpression of FOXO1 enhances directly increased miR-186-5p expression in neutrophil-mediated inflammation by increasing association with fibrotic processes and TGF-β inflammatory expression of TNF-α and IL-1β. Recent signalling. TGF-β controls proliferation, studies have been shown that FOXO1 is involved in differentiation and other functions including cystic fibrosis-related diabetes.38 Accordingly, the inflammation and fibrosis.24 Dysregulation of the network analysis of miR-30 targets shows TGF-β2, TGF-β pathway results in activation of TGFβR1 and TGFβR2, which connects cytokine– myofibroblasts, excessive production of cytokine receptor interaction and the TGF-β pathway. extracellular matrix (ECM) and inhibition of ECM The data are underlined by the fact that the shared degradation.25 Early extracellular matrix changes targets for miR-186-5p and miR-30b/c-5p, LPCAT1, and changes in VEGF/VEGFR2 axis have been LRRC3C, PLXNA1 and ST3GAL5 are also involved in demonstrated to be associated with the inflammatory and fibrotic processes respectively.39–42 development of bronchiolitis obliterans syndrome Dysregulated miRNAs have been identified as (BOS) after lung transplantation or hematopoietic biomarkers for various inflammatory lung stem cell transplantation.26,27 Like in BOS, patients diseases.12 Although several miRNAs, miR-21, miR- with PiBO undergo fibrotic processes after lower 29a, miR-103 and miR-191, have been suggested as respiratory tract infection, which is in line with our biomarkers for BOS, the significance of circulating findings of TGF-β concentrations in induced sputum miRNAs for the diagnosis PiBO is still unclear.43,44 in our patient cohort.28 Moreover, VEGF A was Our study confirms some of the dysregulated found to be a target of miR-335-5p. Interestingly, miRNAs in BOS, such as miR-29a, miR-146a, miR-151, we identified a group of bone morphogenetic miR-181b and others, but clearly highlighted miR- proteins (BMPs) and their receptors, ACVR1, 335-5p, miR186-5p, miR-30b-5p and miR-30c-5p as ACVR2B, BMP2, BMPR1A, BMPR1B and BMPR2, possible new biomarkers for PiBO. In addition, the located in cytokine–cytokine receptor interaction strong correlations between the expression of these and the FoxO signalling pathway, which are also miRNAs and FEV1 underline their role as new members of the transforming growth factor-β (TGF- biomarkers in the identification of PiBO patients. β) family.29 Alteration in BMP-signalling has been In conclusion, our study identifies four miRNAs, linked to immune regulation, autoimmunity and namely miR-335-5p, miR-186-5p, miR-30b-5p and infection diseases.30 Moreover, BMPs such as TGF- miR-30c-5p, which impact pathways responsible β1 and FGF-β2 as well as BMP-4 and BMP-7 are for the regulation of inflammation and fibrosis in 2022 | Vol. 11 | e1376 ª 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 8 Australian and New Zealand Society for Immunology, Inc. RP Duecker et al. miRNA in post-infectious bronchiolitis obliterans IL-6 TGFB2 CXCL8 IL-1A Cytokine-cytokine receptor interacon hsa04060 TGF-beta signaling pathway hsa04350 FoxO signaling pathway hsa04068 Figure 4. Interaction of miRNA targets. STRING diagram of target protein-protein interaction of the miR-335-5p target proteins in cytokine- cytokine receptor interaction (red bullets), miR-186-5p protein targets in the TGF-β signalling pathway (green bullets) and miR-30b/c-5p target proteins in the FoxO signalling pathway (blue bullets). Lines indicate interaction evidence. ª 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of 2022 | Vol. 11 | e1376 Australian and New Zealand Society for Immunology, Inc. Page 9 miRNA in post-infectious bronchiolitis obliterans RP Duecker et al. Figure 5. Inhibition of hsa-miR-335-5p, hsa-miR-186-5p, hsa-miR-30b-5p and hsa-miR-30c-5p via transfection with corresponding anti-miRs in A549 cells. (a) IL-6 expression was assessed in A549 cells after 24-h stimulation with three different cytokine mixtures (CM1, CM2 and CM3) and inhibition of the four miRNAs using cytometric bead array against positive control (CM; n = 3). (b) IL-6 expression is illustrated as Fold Change combining all three CM stimulations together after inhibition of the four miRNAs separately and taken all four miRNAs together (n = 3). Figure 6. (a) NGS in samples of induced sputum of PiBO patients compared to control samples (n = 3) showing the reads for hsa-miR-335-5p, hsa-miR-186-5p, hsa-miR-30b-5p and hsa-miR-30c-5p. (b) Correlations between expression (reads) of verified miRNAs and the lung function parameter FEV1 analysed in peripheral blood of PiBO patients compared to healthy controls. *P < 0.05. 2022 | Vol. 11 | e1376 ª 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 10 Australian and New Zealand Society for Immunology, Inc. RP Duecker et al. miRNA in post-infectious bronchiolitis obliterans Figure 7. Overview of the study design. Clinical, laboratory and computational analysis, which were performed in the present study. PiBO. The identified miRNAs should be considered Prior to the start of the study, the subjects or their legal as novel biomarkers, which can support disease guardians had to sign the informed consent form. The study was approved by the Ethics Committee of the Goethe diagnosis and amplify treatment options. University Frankfurt (166/16) and is registered by Moreover, we consider these newly identified ClinicalTrials.gov Identifier: NCT02627833. miRNAs as subjects for further investigations, Blood samples using the PAXgene Blood RNA System for with promising potential in the field of miRNA miRNA were collected and all patients performed lung function therapeutics. tests. Induced sputum was collected and processed from the Frankfurt patient cohort and stored until analysis (Figure 7). METHODS Pulmonary function tests Study design Baseline lung function tests were assessed at both centres using the same body plethysmograph (VIASYS Healthcare Figure 7 gives an overview of the study design, which GmbH, Hoechberg, Germany). The following parameters consists of clinical and laboratory as well as computational have been recorded: FVC, FEV1, FEV1/FVC and FEF. analyses. Clinical and laboratory analysis included lung Reversibility was defined as positive with a post- function tests, cell differentiation and cytokine analysis in bronchodilator change of ⩾ 12% and 200 mL. induced sputum, and miRNA analysis in the peripheral blood by next-generation sequencing (NGS). A systematic bias in miRNA regulation because of regional and lifestyle Sputum cell differentiation and factors was excluded by using two study cohorts from Germany and Spain, respectively, and data were validated inflammatory mediators by TaqMan PCR. Combined KEGG pathway enrichment Sputum cell differentiation46 and analysis of inflammatory analysis of all the identified miRNAs was performed in total cytokines/chemokines by cytometric bead array were (combined analysis) to detect common targets and in performed as described previously.47 Samples were individual miRNAs (single analysis) to detect miRNA specific measured with a BD FACS ArrayTM cytometer (BD targets. Finally, identified targets were visualised in a Biosciences-Pharmingen, USA). FCAP Array Software (BD protein–protein interaction (PPI) network. Biosciences-Pharmingen) was used to analyse the data. Patients miRNA sequencing and analysis Patients with PiBO and controls were recruited from the The PAXgene Blood miRNA Kit (Qiagen, Hilden, Germany) Vall d’Hebron Hospital Universitari, Barcelona, Spain, and was used to extract total RNA, including miRNA, from from the Department for Children and Adolescents, Goethe peripheral blood and sputum samples according to the University Frankfurt, Germany, from October 2015 to April manufacturer’s recommendations. Nanodrop Lite 2019. The diagnosis of PiBO was made according to spectroscopy (Thermo Scientific, Dreieich, Germany) was National Institutes of Health Clinical Centre (NIH-CC), and used to determine RNA concentration, and the Agilent RNA new onset of changes on HRCT including mosaic pattern, 6000 Nano kit and Agilent 2100 Bioanalyzer was used to hyperinflation and bronchial wall thickening.45 determine RNA integrity (RIN) (Agilent Technologies, Santa ª 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of 2022 | Vol. 11 | e1376 Australian and New Zealand Society for Immunology, Inc. Page 11 miRNA in post-infectious bronchiolitis obliterans RP Duecker et al. Carla, CA, USA). The QIAseq miRNA Library Kit was used to the candidate miRNAs in relation to inflammation revealed create miRNA libraries (Qiagen). Finally, cDNA cytokine–cytokine interaction, TGF-β signalling pathway and concentrations were determined using the Qubit dsDNA FoxO signalling pathway. Targets were grouped together, Assay Kit, Qubit Assay Tubes and a Qubit 3.0 Fluorometer and analysis of networks was conducted using the software (Thermo Fisher Scientific, Dreieich, Germany). DNA quality tool String v.10 (https://string-db.org/) with standard was verified using Agilent High Sensitivity DNA Reagents parameters for visualising target interaction networks. with High Sensitivity DNA Chips and an Agilent 2100 Bioanalyzer (Agilent Technologies). Libraries were frozen at –20°C until miRNA sequencing. Knockdown of hsa-miR-335-5p, hsa-miR-186- Next-generation sequencing was performed using MiSeq 5p, hsa-miR-30b-5p and hsa-miR-30c-5p in Reagent Kit v3, PhiX Sequencing Control v3 and MiSeqTM A549 cells Desktop Sequencer (Illumina Inc., San Diego, USA). Data were preprocessed using the Qiagen Web Portal service, Human lung epithelial cell line A549 was cultured with 10% which converts coverage files into raw count matrices foetal calf serum (FCS) (Sigma-Aldrich, Taufkirchen, (https://geneglobe.qiagen.com/de/analyze). Differential Germany) and 1% penicillin–streptomycin (Life expression analysis was performed in RStudio 1.2.1335 Technologies, Darmstadt, Germany). Cells were seeded at (https://cran.r-project.org/) as previously described.48 Fdr 2 × 106 cells at 37°C in a humidified atmosphere containing correction was applied to each miRNA that passed the 5% CO2. For in vitro experiments, A549 cells were detached DESeq quality threshold, and miRNAs were considered to from tissue culture flasks by accutaseTM cell isolation be < 0.05 with Paje. miRNAs with Padj < 0.001 were used solution (Sigma-Aldrich Chemie). 25 000 cells per 96 wells for further analysis. were seeded in medium without penicillin/streptomycin. Cells were stimulated with a pro-inflammatory cytokine mixture (CM) containing 4 U mL–1 IFN-γ, 0.5 U mL–1 IL-1β and miRNA validation by TaqMan qPCR 0.2 ng mL–1 TNF-α (CM1); 40 U mL–1 IFN-γ, 5 U mL–1 IL-1β and 2 mL–1 TNF-α (CM2); and 400 mL–1 IFN-γ, 50 U mL–1 IL-1β and Next-generation sequencing-based expression levels of the 20 ng mL–1 mL–1 TNF-α (CM3) (Peprotech, Hamburg, following miRNAs were validated by TaqMan qPCR Germany). For miRNA knockdown, cells were reversely (TaqMan® Advanced miRNA Assays; Thermo Fisher): hsa- transfected with either miRVana anti-hsa-miR-335-5p, anti- miR-1287-5p, hsa-miR-335-5p, hsa-miR-5695, hsa-miR-30b- hsa-miR-186-5p, anti-hsa-miR-30b-5p and anti-hsa-miR-30c- 5p, hsa-miR-186-5p, hsa-miR-190b, hsa-7f-1-3p, hsa-miR-30c- 5p (Thermo Fisher) at 20 nM, using Lipofectamine RNAiMAX 5p, hsa-miR-26a-2-3p, hsa-miR-4677-3p and hsa-miR-199a-5p (1:1 v/v; Invitrogen, Karlsruhe, Germany) in Opti-MEM® as described previously.48 miR-24-3p was used as media (Thermo Fisher). mirVana miRNA Inhibitor Negative endogenous control based on low variance and high Control (Thermo) was used as a negative control (in abundance over control samples (Supplementary table 2). Figure not shown) and the sole cytokine mixture stimulation cDNA preparation was performed by extending 5 ng t-RNA (CM) without miRNAs served as positive control. The effect of at the 30 end of mature transcripts through poly (A) transfection of miRNA inhibitors on IL-6 protein was assessed addition. The 50 ends were extended by adapter ligation, at 48 h after transfection via cytometric bead array. followed by universal reverse transcription and amplification using the TaqMan® Advanced miRNA cDNA Synthesis Kit and a thermal cycler (GeneAmp Cycler PCR Statistics Systems 9700 v3.12., Thermo Fisher). The thermocycler programme for RT cycles was programmed according to the Expression levels of miRNAs, determined by qPCR, are manufacturer’s instructions. For qPCR, 5 µL of a 1:10 (v:v, in TE presented as relative quantification (RQ) of gene buffer) diluted cDNA was introduced to the oligonucleotide. expression, whereas the rest of the data are presented as StepOnePlus Real-Time PCR Systems and StepOnePlusTM median (10–90 percentile). All experiments have been software were used to perform qPCR (Thermo Fisher carried out at least three times. Differences between groups Scientific). Evaluation was performed by the 2ΔΔC t method in were determined by one-way ANOVA (multiple groups) Expression Suite software v1.1 (Fisher Scientific). with post hoc Bonferroni analysis. The comparison of the two groups was performed by Mann–Whitney U test. For the correlation analysis, we used the Spearman test. Pathway enrichment analysis of target Calculations were performed with RStudio 1.2.1335 (https:// genes and protein–protein interaction cran.r-project.org/) or GraphPad Prism 7 software (GraphPad network software, La Jolla, California) and Excel (Microsoft Office, München, Germany). Statistical significance was defined as Kyoto encyclopaedia of genes and genomes (KEGG) P < 0.05. pathway analysis based on microRNA signature was performed with miRNet (miRTarBase v8.0) for all the identified miRNAs together and with DIANA-mirPath v.3.0 Sample size estimation to detect targets of individual miRNAs.49 For DIANA- mirPath v.3.0, three different algorithms, namely, TarBase Sample size calculations were based on previous results v.7.0, microT-CDS and TargetScan 7.0, were manually published by our group.6 In this study, 20 children and screened for pathways involved in inflammation and targets adolescents with PiBO were compared with 20 age-matched were selected according to the highest P-value. Targets of controls. As described, we found significant differences in 2022 | Vol. 11 | e1376 ª 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 12 Australian and New Zealand Society for Immunology, Inc. RP Duecker et al. miRNA in post-infectious bronchiolitis obliterans lung function parameters as well as neutrophils and Methodology; Writing – review & editing. Gabriele Gottwald: cytokines in induced sputum.6 Since no reports on miRNAs in Data curation; Formal analysis; Writing – review & editing. relation to inflammation in PiBO are available, we assumed Antonio Moreno: Data curation; Investigation; Writing – that two cohorts with 20 patients for the measurement of review & editing. Martin Rosewich: Data curation; miRNAs are sufficient for an explorative investigation. Investigation; Methodology; Writing – review & editing. Lucia Gronau: Data curation; Formal analysis; Methodology; Visualization; Writing – review & editing. Stefan Zielen: ACKNOWLEDGMENTS Conceptualization; Funding acquisition; Investigation; Project administration; Resources; Supervision; Writing – review & The authors thank Katrin Krug und Petra Schoen for their editing. Andreas Geburtig-Chioccetti: Data curation; Formal excellent technical assistance. This project was supported by analysis; Visualization; Writing – review & editing. Hermann the Starke Lunge Foundation. Starke Lunge is a non-profitable Kreyenberg: Data curation; Formal analysis; Methodology; foundation registered in Germany (Regierungspräsidium Visualization; Writing – review & editing. Ralf Schubert: Darmstadt: AZ: I 1325d 04/11(6)-78), which supports research Conceptualization; Data curation; Formal analysis; Funding projects in paediatric rare lung diseases, specifically in BO. acquisition; Investigation; Methodology; Project Open Access funding enabled and organized by Projekt DEAL. administration; Software; Supervision; Visualization; Writing – original draft; Writing – review & editing. CONFLICT OF INTEREST Dr Duecker reports grants from Starke Lunge Foundation, REFERENCES during the conduct of the study. Dr de Mir Messa reports grants from Starke Lunge Foundation, during the conduct of 1. Kurland G, Michelson P. Bronchiolitis obliterans in the study; personal fees from GSK, other from Novartis and children. Pediatr Pulmonol 2005; 39: 193–208. other from Aldo Union, outside the submitted work. Dr 2. Colom AJ, Teper AM. Post-infectious bronchiolitis Moreno-Galdó reports grants from Starke Lunge Foundation, obliterans. Pediatr Pulmonol 2019; 54: 212–219. during the conduct of the study; personal fees from AbbVie, 3. Cazzato S, Poletti V, Bernardi F et al. Airway personal fees from AbbVie, personal fees from Sanofi, other inflammation and lung function decline in childhood from Novartis, other from AbbVie and other from Actelion post-infectious bronchiolitis obliterans. Pediatr outside the submitted work. Dr Rosewich reports grants from Pulmonol 2008; 43: 381–390. Starke Lunge Foundation, during the conduct of the study; 4. Rosewich M, Zissler UM, Kheiri T et al. Airway other from Engelhard Arzneimittel, other from GSK inflammation in children and adolescents with Arzneimittel GmbH and other from Bencard GmbH, outside bronchiolitis obliterans. Cytokine 2015; 73: 156–162. the submitted work. Dr Zielen reports grants and personal 5. Smith KJ, Fan LL. Insights into post-infectious fees from bene-Arzneimittel GmbH, grants and personal fees bronchiolitis obliterans in children. Thorax 2006; 61: from Biotest GmbH, grants from Vifor Pharma Deutschland 462–463. GmbH, grants from ALK Arzneimittel, personal fees from 6. Eckrich J, Herrmann E, Voss S et al. Short-term variation Novartis GmbH, grants and personal fees from Böhringer of lung function and airway inflammation in children Ingelheim, personal fees from Lofarma GmbH, personal fees and adolescents with bronchiolitis obliterans. Lung from IMS HEALTH GmbH & Co. OHG, personal fees from GSK, 2016; 194: 571–579. personal fees from Stallergen, personal fees from Procter and 7. Liu Y, Li H, Xiao T et al. Epigenetics in immune- Gamble, personal fees from Allergopharma GmbH, personal mediated pulmonary diseases. Clin Rev Allergy Immunol fees from Engelhard Arzneimittel GmbH, personal fees from 2013; 45: 314–330. AstraZeneca, personal fees from Sanofi-Aventis GmbH, 8. Guo H, Ingolia NT, Weissman JS et al. Mammalian personal fees from Allergy Therapeutics and personal fees microRNAs predominantly act to decrease target mRNA from Aimmune Therapeutics, outside the submitted work. Dr levels. Nature 2010; 466: 835–840. Schubert reports grants from Starke Lunge Foundation, 9. Cai Y, Yu X, Hu S et al. A brief review on the during the conduct of the study. All other authors declare no mechanisms of miRNA regulation. Genomics Proteomics conflict of interest. The funders had no role in the design of Bioinformatics 2009; 7: 147–154. the study; in the collection, analyses or interpretation of data; 10. Alipoor SD, Adcock IM, Garssen J et al. The roles of in the writing of the manuscript; or in the decision to publish miRNAs as potential biomarkers in lung diseases. Eur J the results. Pharmacol 2016; 791: 395–404. 11. Recchiuti A, Serhan CN. Pro-resolving lipid mediators (SPMs) and their actions in regulating miRNA in novel AUTHOR CONTRIBUTION resolution circuits in inflammation. Front Immunol 2012; 3: 298. Ruth Pia Duecker: Data curation; Formal analysis; Investigation; 12. Stolzenburg LR, Harris A. The role of microRNAs in Methodology; Software; Visualization; Writing – original draft. chronic respiratory disease: recent insights. Biol Chem Ines De Mir Messa: Data curation; Formal analysis; 2018; 399: 219–234. Investigation; Resources; Writing – review & editing. Silvija 13. Jerkic SP, Michel F, Donath H et al. Calprotectin as a Jerkic: Data curation; Formal analysis; Investigation; new sensitive marker of neutrophilic inflammation in Methodology; Supervision; Visualization; Writing – review & patients with bronchiolitis obliterans. Mediators editing. Annalena Kochems: Data curation; Investigation; Inflamm 2020; 2020: 4641585. ª 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of 2022 | Vol. 11 | e1376 Australian and New Zealand Society for Immunology, Inc. Page 13 miRNA in post-infectious bronchiolitis obliterans RP Duecker et al. 14. Colom AJ, Maffey A, Garcia Bournissen F et al. 29. Gomez-Puerto MC, Iyengar PV, Garcı́a de Vinuesa A et al. Pulmonary function of a paediatric cohort of patients Bone morphogenetic protein receptor signal transduction with postinfectious bronchiolitis obliterans. A long term in human disease. J Pathol 2019; 247: 9–20. follow-up. Thorax 2015; 70: 169–174. 30. Sanjabi S, Oh SA, Li MO. Regulation of the immune 15. Jerkic S-P, Brinkmann F, Calder A et al. Postinfectious response by TGF-β: from conception to autoimmunity and bronchiolitis obliterans in children: diagnostic workup infection. Cold Spring Harb Perspect Biol 2017; 9: a022236. and therapeutic options: a workshop report. Can Respir 31. Jonigk D, Theophile K, Hussein K et al. Obliterative J 2020; 2020: 5852827. airway remodelling in transplanted and non- 16. Elssner A, Jaumann F, Dobmann S et al. Elevated levels transplanted lungs. Virchows Arch 2010; 457: 369–380. of interleukin-8 and transforming growth factor-beta in 32. Zhou T, Chen Y-L. The functional mechanisms of miR- bronchoalveolar lavage fluid from patients with 30b-5p in acute lung injury in children. Med Sci Monit bronchiolitis obliterans syndrome: proinflammatory role 2019; 25: 40–51. of bronchial epithelial cells. Munich Lung Transplant 33. Braun J, Hoang-Vu C, Dralle H et al. Downregulation of Group. Transplantation 2000; 70: 362–367. microRNAs directs the EMT and invasive potential of 17. Long X, Huang Y, He J et al. Upregulation of miR-335 anaplastic thyroid carcinomas. Oncogene 2010; 29: exerts protective effects against sepsis-induced 4237–4244. myocardial injury. Mol Med Rep 2021; 24: 806. 34. Howe GA, Kazda K, Addison CL. MicroRNA-30b controls 18. Ong J, van den Berg A, Faiz A et al. Current smoking is endothelial cell capillary morphogenesis through associated with decreased expression of miR-335-5p in regulation of transforming growth factor-β2. PLoS One parenchymal lung fibroblasts. Int J Mol Sci 2019; 20: 2017; 12: e0185619. 5176. 35. Xu J, Wu H, Chen S et al. MicroRNA-30c suppresses the 19. Gu X, Yao X, Liu D. Up-regulation of microRNA-335-5p pro-fibrogenic effects of cardiac fibroblasts induced by reduces inflammation via negative regulation of the TGF-β1 and prevents atrial fibrosis by targeting TGFβRII. TPX2-mediated AKT/GSK3β signaling pathway in a J Cell Mol Med 2018; 22: 3045–3057. chronic rhinosinusitis mouse model. Cell Signal 2020; 36. Peng SL. Forkhead transcription factors in chronic 70: 109596. inflammation. Int J Biochem Cell Biol 2010; 42: 482–485. 20. Du W, Tang H, Lei Z et al. miR-335-5p inhibits TGF-β1- 37. Graves DT, Milovanova TN. Mucosal immunity and the induced epithelial-mesenchymal transition in non-small FOXO1 transcription factors. Front Immunol 2019; 10: cell lung cancer via ROCK1. Respir Res 2019; 20: 225. 2530. 21. Oliveira SR, Dionı́sio PA, Correia Guedes L et al. 38. Smerieri A, Montanini L, Maiuri L et al. FOXO1 content Circulating inflammatory miRNAs associated with is reduced in cystic fibrosis and increases with IGF-I Parkinson’s disease pathophysiology. Biomolecules treatment. Int J Mol Sci 2014; 15: 18000–18022. 2020; 10: 945. 39. Dukhinova M, Kuznetsova I, Kopeikina E et al. Platelets 22. Feng H, Zhang Z, Qing X et al. miR-186-5p promotes mediate protective neuroinflammation and promote cell growth, migration and invasion of lung neuronal plasticity at the site of neuronal injury. Brain adenocarcinoma by targeting PTEN. Exp Mol Pathol Behav Immun 2018; 74: 7–27. 2019; 108: 105–113. 40. Söderman J, Berglind L, Almer S. Gene expression- 23. Li R, Xu F, Wu X et al. CUL1-mediated organelle fission genotype analysis implicates GSDMA, GSDMB, and pathway inhibits the development of chronic LRRC3C as contributors to inflammatory bowel disease obstructive pulmonary disease. Comput Math Methods susceptibility. Biomed Res Int 2015; 2015: 834805. Med 2020; 2020: 5390107. 41. Tomati V, Pesce E, Caci E et al. High-throughput 24. Stewart AG, Thomas B, Koff J. TGF-β: master regulator screening identifies FAU protein as a regulator of of inflammation and fibrosis. Respirology 2018; 23: mutant cystic fibrosis transmembrane conductance 1096–1097. regulator channel. J Biol Chem 2018; 293: 1203–1217. 25. Kramer EL, Clancy JP. TGF-β as a therapeutic target in 42. Cheng S, Chen H, Wang A et al. Lentiviral vector- cystic fibrosis. Expert Opin Ther Targets 2018; 22: 177– mediated delivery of lysophosphatidylcholine 189. acyltransferase 1 attenuates airway inflammation in 26. Müller C, Andersson-Sjöland A, Schultz HH et al. Early ovalbumin-induced allergic asthmatic mice. Asian Pac J extracellular matrix changes are associated with later Allergy Immunol 2015; 33: 320–329. development of bronchiolitis obliterans syndrome after 43. Budding K, Rossato M, van de Graaf EA et al. Serum lung transplantation. BMJ Open Respir Res 2017; 4: miRNAs as potential biomarkers for the bronchiolitis e000177. obliterans syndrome after lung transplantation. Transpl 27. Larsson-Callerfelt A-K, Müller C, Andersson-Sjöland A Immunol 2017; 42: 1–4. et al. VEGF synthesis and VEGF receptor 2 expression 44. Xu Z, Yang W, Steward N et al. Role of circulating in patients with bronchiolitis obliterans syndrome microRNAs in the immunopathogenesis of rejection after lung transplantation. Respir Med 2020; 166: after pediatric lung transplantation. Transplantation 105944. 2017; 101: 2461–2468. 28. Yu J. Postinfectious bronchiolitis obliterans in children: 45. Walther S, Rettinger E, Maurer HM et al. Long-term lessons from bronchiolitis obliterans after lung pulmonary function testing in pediatric bronchiolitis transplantation and hematopoietic stem cell obliterans syndrome after hematopoietic stem cell transplantation. Korean J Pediatr 2015; 58: 459–465. transplantation. Pediatr Pulmonol 2020; 55: 1725–1735. 2022 | Vol. 11 | e1376 ª 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Page 14 Australian and New Zealand Society for Immunology, Inc. RP Duecker et al. miRNA in post-infectious bronchiolitis obliterans 46. Koc-Günel S, Schubert R, Zielen S et al. Cell distribution with experimental support. Nucleic Acids Res 2015; 43: and cytokine levels in induced sputum from healthy W460–W466. subjects and patients with asthma after using different nebulizer techniques. BMC Pulm Med 2018; 18: 115. Supporting Information 47. Eickmeier O, Huebner M, Herrmann E et al. Sputum biomarker profiles in cystic fibrosis (CF) and chronic Additional supporting information may be found obstructive pulmonary disease (COPD) and association online in the Supporting Information section at between pulmonary function. Cytokine 2010; 50: 152– 157. the end of the article. 48. Fussbroich D, Kohnle C, Schwenger T et al. A combination of LCPUFAs regulates the expression of This is an open access article under the terms of miRNA-146a-5p in a murine asthma model and human the Creative Commons Attribution- NonCommercial-NoDerivs License, which permits alveolar cells. Prostaglandins Other Lipid Mediat 2020; use and distribution in any medium, provided 147: 106378. the original work is properly cited, the use is 49. Vlachos IS, Zagganas K, Paraskevopoulou MD et al. non-commercial and no modifications or DIANA-miRPath v3.0: deciphering microRNA function adaptations are made. ª 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of 2022 | Vol. 11 | e1376 Australian and New Zealand Society for Immunology, Inc. Page 15