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Eicosanoids have been historically involved in the pathogenesis of various inflammatory diseases. Lipoxins (LXs) and epi-LXs show physiological effects relevant to inflammation regulation. In this study, we focused on LX precursors based on the hypothesis that their entrance and metabolism into the cell may facilitate their targeting at the inflammation site. Because compound chirality is of considerable importance in the efficacy of therapeutic agents, our aim was to study the anti-inflammatory effects of various epimers of LXA4 precursors compared to LXA4. Blood polymorphonuclear cells (PMNs) were incubated with 15(S)- or 15(R)-hydroxyeicosatetraenoic acid (HETE), 14(R)-,15(S)-, or 14(S),15(S)-diHETE, and LXA4 and then stimulated with the calcium ionophore A23187. We found that 15(R)-HETE rather than 15(S)-HETE was preferentially metabolized and that 15-epi-LXs were produced in larger amounts than LXs. In contrast, when PMNs were incubated with the diastereoisomers of 14,15(S)-diHETE, 14-epi-LXB4 was produced in lower amounts than LXB4. Enantiomers of 15-HETE and diastereoisomers of 14,15-diHETE and LXA4 were able to significantly decrease LTB4 release by PMNs. These results suggest a potential resolution of the inflammatory process through endogenous anti-inflammatory mediators released by the way of trans-cellular metabolism. [Copyright &y& Elsevier]

COPD affects millions of people and is now ranked as the third leading cause of death worldwide. This largely untreatable chronic airway disease results in irreversible destruction of lung architecture. The small lung hypothesis is now supported by epidemiological, physiological and clinical studies. Accordingly, the early and severe COPD phenotype carries the most dreadful prognosis and finds its roots during lung growth. Pathophysiological mechanisms remain poorly understood and implicate individual susceptibility (genetics), a large part of environmental factors (viral infections, tobacco consumption, air pollution) and the combined effects of those triggers on gene expression. Genetic susceptibility is most likely involved as the disease is severe and starts early in life. The latter observation led to the identification of Mendelian inheritance via disease-causing variants of SERPINA1 – known as the basis for alpha-1 anti-trypsin deficiency , and TERT. In the last two decades multiple genome wide association studies (GWAS) identified many single nucleotide polymorphisms (SNPs) associated with COPD. High significance SNPs are located in 4q31 near HHIP which encodes an evolutionarily highly conserved physiological inhibitor of the Hedgehog signaling pathway (HH). HHIP is critical to several in utero developmental lung processes. It is also implicated in homeostasis, injury response, epithelial-mesenchymal transition and tumor resistance to apoptosis. A few studies have reported decreased HHIP RNA and protein levels in human adult COPD lungs. HHIP+/- murine models led to emphysema. HH pathway inhibitors, such as vismodegib and sonidegib, are already validated in oncology, whereas other drugs have evidenced in vitro effects. Targeting the Hedgehog pathway could lead to a new therapeutic avenue in COPD. In this review, we focused on the early and severe COPD phenotype and the small lung hypothesis by exploring genetic susceptibility traits that are potentially treatable, thus summarizing promising therapeutics for the future. [ABSTRACT FROM AUTHOR]

Human pluripotent stem cells (hiPSC) represent a unique opportunity to model lung development and chronic bronchial diseases. We generated a hiPSC line from a highly characterized healthy heavy smoker male donor free from emphysema or tobacco related disease. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using integration-free Sendai virus. The cell line had normal karyotype, expressed pluripotency hallmarks, and differentiated into the three primary germ layers. The reported UHOMi007-A iPSC line may be used as a control to model lung development, study human chronic bronchial diseases and drug testing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 University Hospital Centre of Montpellier. Published by Elsevier B.V. All rights reserved.)

Airway-liquid interface cultures of primary epithelial cells and of induced pluripotent stem-cell-derived airway epithelial cells (ALI and iALI, respectively) are physiologically relevant models for respiratory virus infection studies because they can mimic the in vivo human bronchial epithelium. Here, we investigated gene expression profiles in human airway cultures (ALI and iALI models), infected or not with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), using our own and publicly available bulk and single-cell transcriptome datasets. SARS-CoV-2 infection significantly increased the expression of interferon-stimulated genes ( IFI44 , IFIT1 , IFIT3 , IFI35 , IRF9 , MX1 , OAS1 , OAS3 and ISG15 ) and inflammatory genes ( NFKBIA , CSF1 , FOSL1 , IL32 and CXCL10 ) by day 4 post-infection, indicating activation of the interferon and immune responses to the virus. Extracellular matrix genes ( ITGB6 , ITGB1 and GJA1 ) were also altered in infected cells. Single-cell RNA sequencing data revealed that SARS-CoV-2 infection damaged the respiratory epithelium, particularly mature ciliated cells. The expression of genes encoding intercellular communication and adhesion proteins was also deregulated, suggesting a mechanism to promote shedding of infected epithelial cells. These data demonstrate that ALI/iALI models help to explain the airway epithelium response to SARS-CoV-2 infection and are a key tool for developing COVID-19 treatments.

Epithelial cytokines are involved in the orchestration of T1/T2 inflammatory patterns. We question the persistence of this trait in air-liquid interface (ALI) epithelial cultures and whether this local orientation can be related to systemic patterns (e.g., blood eosinophil counts [BECs]). We investigated alarmin release related to high versus low T2 phenotypes associated with chronic airway diseases. ALIs were reconstituted from 32 control, 40 chronic obstructive pulmonary disease, and 20 asthmatic patients. Interleukin-8 (IL-8; a T1-cytokine), IL-25, IL-33, and thymic stromal lymphopoietin (T2-alarmins) concentrations were assessed in subnatants at steady state and used to explain blood neutrophil and eosinophil counts. IL-25 and IL-8 levels were highest in asthma ALI-subnatants, whereas IL-33 was sparsely detected. Thymic stromal lymphopoietin levels were similar among groups. All asthma cell cultures were T1-high/T2-high, while chronic obstructive pulmonary disease and controls tended to be mixed. BECs were independently explained by both disease and in-culture T2-alarmin levels, irrespective of the T2-alarmin considered. The epithelial ALI-T2 signature was more frequently high in patients with a BEC > 300/mm 3 . Despite removal from an in vivo environment for ≥2 months, ALIs release disease-specific cytokine "cocktails" into their subnatants, suggesting continued persistence of alarmin orientation in differentiated cell line environments., (© 2023 Wiley-VCH GmbH.)

(1) Background: We have previously shown that sputum rheology can discriminate between patients with COPD and other muco-obstructive lung diseases, and that it is correlated with mucin content and sputum eosinophilia. We now hypothesize that it could be a more-accurate guide than clinical evaluation for the prescription of azithromycin to prevent exacerbations of COPD and to reduce exposure to antibiotics; (2) Methods: "COPD CaRhe" is a multicentric, randomized, controlled trial comparing outcomes in two parallel arms (36 vs. 36 patients). Patients will be recruited in the university hospitals of Montpellier, Bordeaux, and Toulouse, in France, and they should have a diagnosis of COPD with frequent exacerbations (≥3/year). Enrollment will occur during a routine visit to a respiratory department, and follow-up visits will occur every 3 months for a period of 1 year. At each visit, a 3-month prescription of azithromycin will be provided to those patients who obtain a score of <70 on the Cough and Sputum Assessment Questionnaire (CASA-Q) or a critical stress score of σc > 39 on a rheological assessment of sputum, depending upon their randomization group. The primary outcome will be the number of exacerbations of COPD; (3) Discussion: By using sputum rheology, the COPD CaRhe study may provide clinicians with an objective biomarker to guide the prescription of azithromycin while reducing the cumulative exposure to macrolides.

Background: Obesity is known to diminish lung volumes and worsen asthma. However, mechanistic understanding is lacking, especially as concerns small-airway responsiveness. The objective of this study was therefore to compare small-airway responsiveness, as represented by the change in expiratory:inspiratory mean lung density ratios (MLD e/i , as determined by computed tomography [CT]) throughout methacholine testing in obese versus non-obese women with asthma., Methods: Thoracic CT was performed during methacholine bronchoconstriction challenges to produce standardized response curves (SRC: response parameter versus ln[1 + % PD20], where PD20 is the cumulative methacholine dose) for 31 asthma patients (n = 18 non-obese and n = 13 obese patients). Mixed models evaluated obesity effects and interactions on SRCs while adjusting for age and bronchial morphology. Small airway responsiveness as represented by SRC slope was calculated for each third of the MLD e/i response and compared between groups., Results: Obesity-associated effects observed during experimental bronchoconstriction included: (i) a significant baseline effect for forced expiratory volume in 1 second with lower values for the obese (73.11 ± 13.44) versus non-obese (82.19 ± 8.78; p = 0.002) groups prior to methacholine testing and (ii) significantly higher responsiveness in small airways as estimated via differences in MLD e/i slopes (group×ln(1 + % PD20 interaction; p = 0.023). The latter were pinpointed to higher slopes in the obese group at the beginning 2/3 of SRCs (p = 0.004 and p = 0.021). Significant obesity effects (p = 0.035 and p = 0.008) indicating lower forced vital capacity and greater % change in MLD e/I (respectively) throughout methacholine testing, were also observed., Conclusion: In addition to baseline differences, small-airway responsiveness (as represented by the change in MLD e/i ) during methacholine challenge is greater in obese women with asthma as compared to the non-obese., (© 2022 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Introduction: Therapeutic education for patients with asthma has been shown to reduce asthma morbidity. The high availability of smart phones provides the opportunity to furnish patient training via specifically designed chatbot applications. The goal of this protocol is to perform a first pilot comparison of traditional face to face versus chatbot-guided patient therapeutic education programmes for patients with asthma., Methods and Analysis: Eighty adult patients with a physician-confirmed diagnosis of asthma will be enrolled in a two-parallel-arm, randomised (1:1) controlled pilot trial. A single-Zelen consent procedure is deployed to first enrol all participants in the comparator arm, that is, the standard patient therapeutic education programme at the University Hospitals of Montpellier, France. This means of patient therapeutic education is based on reoccurring interviews and discussion with qualified nursing staff as per usual care. Following baseline data acquisition, randomisation will be performed. Those patients randomised to the comparator arm will not be informed of the second arm. Those patients randomised to the experimental arm will be proposed access to a specifically designed chatbot (Vik-Asthme) as the second tested means of patient training (refusals continue with standard training, though analysed as intention to treat). The primary outcome is change in the total Asthma Quality of Life Questionnaire score at the end of follow-up (6 months). Secondary outcomes cover asthma control, spirometry, general health status, programme adherence and burden for medical staff, exacerbations and medical resource use (medications, consults, emergency visits, hospitalisation and intensive care)., Ethics and Dissemination: This study ('AsthmaTrain' protocol version 4-20220330) has been approved by the Committee for the Protection of Persons Ile-de-France VII on 28 March 2022 (reference number 21.03617.000059). Enrolment began on 24 May 2022. Results will be published in international peer-reviewed journals., Trial Registration Number: NCT05248126., Competing Interests: Competing interests: IV, DG, FC and FV report no competing interests. CMS reports grants and personal fees from Astra Zeneca, grants from GSK, outside the submitted work. NM reports personal fees from Astra Zeneca, grants from GSK, outside the submitted work. AB reports grants, personal fees, non-financial support and other from GSK, grants, personal fees, non-financial support and other from Astra Zeneca, grants, personal fees, non-financial support and other from Boeringher Ingelheim, personal fees, non-financial support and other from Novartis, personal fees, non-financial support and other from Chiesi Farma, non-financial support and other from Teva, personal fees, non-financial support and other from Sanofi Regeneron, grants, personal fees, non-financial support and other from Actelion/Jansen, other from United Therapeutics, other from Pulsar, personal fees, non-financial support and other from Roche, outside the submitted work., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Mucus is a viscoelastic aqueous fluid that participates in the protective barrier of many mammals' epithelia. In the airways, together with cilia beating, mucus rheological properties are crucial for lung mucociliary function, and, when impaired, potentially participate in the onset and progression of chronic obstructive pulmonary disease (COPD). Samples of human mucus collected in vivo are inherently contaminated and are thus poorly characterized. Human bronchial epithelium (HBE) cultures, differentiated from primary cells at an air-liquid interface, are highly reliable models to assess non-contaminated mucus. In this paper, the viscoelastic properties of HBE mucus derived from healthy subjects, patients with COPD and from smokers are measured. Hallmarks of shear-thinning and elasticity are obtained at the macroscale, whereas at the microscale mucus appears as a heterogeneous medium showing an almost Newtonian behaviour in some extended regions and an elastic behaviour close to boundaries. In addition, we developed an original method to probe mucus adhesion at the microscopic scale using optical tweezers. The measured adhesion forces and the comparison with mucus-simulants rheology as well as mucus imaging collectively support a structure composed of a network of elastic adhesive filaments with a large mesh size, embedded in a very soft gel., (© 2022 The Author(s).)

Background: Mucus is known to play a pathogenic role in muco-obstructive lung diseases, but little is known about the determinants of mucus rheology. The purpose of this study is to determine which sputum components influence sputum rheology in patients with muco-obstructive lung diseases., Methods: We performed a cross sectional prospective cohort study. Spontaneous sputum was collected from consecutive patients with muco-obstructive lung diseases. Sputum rheology was assessed using the Rheomuco® rheometer (Rheonova, Grenoble); the elastic modulus G', viscous modulus G″, and the critical stress threshold σc were recorded. Key quantitative and qualitative biological sputum components were determined by cytology, nucleic acid amplification tests and mass spectrometry., Results: 48 patients were included from January to August 2019. Among them, 10 had asthma, 14 COPD and 24 non-CF bronchiectasis (NCFB). The critical stress threshold σc predicted a sputum eosinophilia superior to 1.25% with 89.19% accuracy (AUC = 0.8762). G' and G″ are positively correlated with MUC5AC protein concentration ((rho = 0.361; P = .013) and (rho = 0.335; P = .021), respectively). σc was positively correlated with sputum eosinophilia (rho = 0.394; P = .012), MUC5B (rho = 0.552; P < .001) and total protein (rho = 0.490; P < .001) concentrations. G' and G″ were significantly higher in asthma patients (G' = 14.49[7.18-25.26]Pa, G'' = 3.0[2.16-5.38]Pa) compared to COPD (G' = 5.01[2.94-6.48]Pa, P = .010; G'' = 1.45[1.16-1.94]Pa, P = .006) and to NCFB (G' = 4.99[1.49-10.49]Pa, P = .003; G'' = 1.46[0.71-2.47]Pa, P = .002)., Conclusion: In muco-obstructive lung diseases, rheology predicts sputum eosinophilia and is correlated with mucin concentrations, regardless of the underlying disease., Clinical Trial Registration: (registrar, website, and registration number), where applicable NCT04081740., Competing Interests: Declaration of competing interest Dr. Suehs reports grants from Astra Zeneca, outside the submitted work. Dr Patarin is a full-time employee of Rheonova. Rheonova designed and built the rheometer used in this study. Pr. Molinari reports personal fees from Astra Zeneca, grants from GSK, outside the submitted work. Pr. Bourdin reports grants, personal fees, non-financial support and other from Astra Zeneca, grants, personal fees and other from GSK, grants, personal fees, non-financial support and other from Boeringher Ingelheim, personal fees, non-financial support and other from Novartis, personal fees and other from Teva, personal fees and other from Regeneron, personal fees, non-financial support and other from Chiesi Farmaceuticals, personal fees, non-financial support and other from Actelion, other from Gilead, personal fees and non-financial support from Roche, outside the submitted work. The remaining authors (MV, JV, CH, AP, EML, IV, JC) have no conflicts of interest to disclose., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Chronic Obstructive Pulmonary Disease (COPD), a major cause of mortality and disability, is a complex disease with heterogeneous and ill-understood biological mechanisms. Human induced pluripotent stem cells (hiPSCs) are a promising tool to model human disease, including the impact of genetic susceptibility. Methods: We developed a simple and reliable method for reprogramming peripheral blood mononuclear cells into hiPSCs and to differentiate them into air−liquid interface bronchial epithelium within 45 days. Importantly, this method does not involve any cell sorting step. We reprogrammed blood cells from one healthy control and three patients with very severe COPD. Results: The mean cell purity at the definitive endoderm and ventral anterior foregut endoderm (vAFE) stages was >80%, assessed by quantifying C-X-C Motif Chemokine Receptor 4/SRY-Box Transcription Factor 17 (CXCR4/SOX17) and NK2 Homeobox 1 (NKX2.1) expression, respectively. vAFE cells from all four hiPSC lines differentiated into bronchial epithelium in air−liquid interface conditions, with large zones covered by beating ciliated, basal, goblets, club cells and neuroendocrine cells, as found in vivo. The hiPSC-derived airway epithelium (iALI) from patients with very severe COPD and from the healthy control were undistinguishable. Conclusions: iALI bronchial epithelium is ready for better understanding lung disease pathogenesis and accelerating drug discovery.

Background: The coronavirus disease 2019 (COVID-19) pandemic has put pressure on healthcare services, forcing the reorganisation of traditional care pathways. We investigated how physicians taking care of severe asthma patients in Europe reorganised care, and how these changes affected patient satisfaction, asthma control and future care., Methods: In this European-wide cross-sectional study, patient surveys were sent to patients with a physician-diagnosis of severe asthma, and physician surveys to severe asthma specialists between November 2020 and May 2021., Results: 1101 patients and 268 physicians from 16 European countries contributed to the study. Common physician-reported changes in severe asthma care included use of video/phone consultations (46%), reduced availability of physicians (43%) and change to home-administered biologics (38%). Change to phone/video consultations was reported in 45% of patients, of whom 79% were satisfied or very satisfied with this change. Of 709 patients on biologics, 24% experienced changes in biologic care, of whom 92% were changed to home-administered biologics and of these 62% were satisfied or very satisfied with this change. Only 2% reported worsening asthma symptoms associated with changes in biologic care. Many physicians expect continued implementation of video/phone consultations (41%) and home administration of biologics (52%)., Conclusions: Change to video/phone consultations and home administration of biologics was common in severe asthma care during the COVID-19 pandemic and was associated with high satisfaction levels in most but not all cases. Many physicians expect these changes to continue in future severe asthma care, though satisfaction levels may change after the pandemic., Competing Interests: Conflict of interest: A. Bourdin reports receiving grants or contracts outside the submitted work from AstraZeneca and Boeringher Ingelheim; and payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events as well as support for attending meetings and/or travel from AstraZeneca, Boehringer Ingelheim, GSK, Novartis, Chiesi and Amgen, outside the submitted work. Z. Csoma reports receiving honoraria for presentations from Astra/Zeneca, TEVA and Sanofi/Aventis (Hungary) outside the submitted work. E. Heffler reports receiving payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from AstraZeneca, Sanofi-Genzyme, Regeneron, Novartis, GSK, Circassia; Stallergenes-Greer and Nestlè Purina outside the submitted work. G.W. Canonica reports receiving consulting fees from AstraZeneca GSK, Novartis, Sanofi and Stallergenes Greer; payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from AstraZeneca, GSK, Novartis, Sanofi, Stallergenes Greer, Menarini, Chiesi and Mylan; participation on data safety monitoring or advisory boards for AstraZeneca, GSK, Novartis, Sanofi, Stallergenes Greer and Chiesi; all disclosures made outside the submitted work. G-J. Braunstahl reports grants or contracts from GSK, AstraZeneca and ALK Abello; consulting fees from GSK, Sanofi, ALK Abello, AstraZeneca and Novartis; payment or honoraria for lectures, presentations, speakers' bureaus, manuscript writing or educational events from GSK, Sanofi, ALK Abello, AstraZeneca and Novartis; and is on the scientific board of the Dutch Lung Foundation, task force Asthma NVALT and editorial board NTVAAKI; all disclosures made outside the submitted work. S. van der Sar reports receiving payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from AstraZeneca and GSK; and support received from ALK for attending meetings and/or travel; all disclosures made outside the submitted work. N. Nenasheva reports receiving payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from AstraZeneca plc., Sanofi S.A., Teva Pharmaceuticals, Novartis International AG and Chiesi Farmaceutici S.p.A., outside the submitted work. A. Bossios reports receiving payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from AZ, GSK and Teva; support for attending meetings and/or travel received from Novartis; and participation on a data safety monitoring or advisory boards for AZ, GSK, Novartis, Teva and Sanofi; and is a member of the steering Committee of SHARP, Secretary of Assembly 5 (Airway diseases, asthma, COPD and chronic cough) of the European Respiratory Society and vice-chair of Nordic Severe Asthma Network; all disclosures made outside the submitted work. D. Aronsson reports receiving grants or contracts from ALK-Abello outside the submitted work. A. Egesten reports receiving honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from AstraZeneca, outside the submitted work. L. Ahlbeck reports receiving payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from AstraZeneca; and participation on data safety monitoring or advisory boards for AstraZeneca, Sanofi and GSK; all disclosures made outside the submitted work. S. Škrgat reports receiving honoraria for lectures and educational events supported by GSK, AstraZeneca, Sanofi, Chiesi, Pliva Teva and Medis; and participation on advisory boards of GSK, AstraZeneca, Chiesi and Sanofi; all disclosures made outside the submitted work. N. Edelbaher reports receiving payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from GSK, Astra Zeneca, Chiesi, Pliva Teva, Krka, Novartis, Boehringer Ingelheim and Sanofi; and participation on advisory boards of GSK, Astra Zeneca, Chiesi, Novartis and Boehringer Ingelheim; all disclosures made outside the submitted work. J. Rüdiger reports receiving payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from the Education of Swiss Emergency physicians; and participation on a data safety monitoring board or board for Boehringer Ingelheim; and is a member of the Board of the Swiss Society of Pneumology and President of the Thorax section of the Swiss Ultrasound Society; all disclosures made outside the submitted work. N. Pavlov reports receiving payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from GSK, Novartis and OM Pharma; support for attending meetings and/or travel received from Boehringer Ingelheim; participation on data safety monitoring or advisory boards for AstraZeneca, GSK, Novartis and Sanofi; all disclosures made outside the submitted work. P. Gianella reports participation on an advisory board for Novartis about Xolair for nasal polyps, outside the submitted work. F. Charbonnier reports receiving payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from Sanofi, Novartis, Mundipharma, AstraZeneca and GSK; participation on data safety monitoring or advisory boards for Sanofi, Novartis, Mundipharma, AstraZeneca and GSK; all disclosures made outside the submitted work. R. Chaudhuri reports receiving grants or contracts from AstraZeneca; payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from GSK, AstraZeneca, Teva, Chiesi, Sanofi and Novartis; support for attending meetings and/or travel received from Chiesi, Napp, Sanofi, Boehringer, GSK and AZ; and participation on data safety monitoring or advisory boards for GSK, AstraZeneca, Teva, Chiesi and Novartis; all disclosures made outside the submitted work. S.J. Smith is supported by the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement number 831434 for Taxonomy, Targets, Treatment, and Remission; the JU receives support from the European Union's Horizon 2020 research and innovation programme, and the European Federation of Pharmaceutical Industries and Associates; all disclosures made outside the submitted work. S. Doe reports receiving support for attending meetings and/or travel from GSK and Sanofi, outside the submitted work. L. Heaney reports grants or contracts from Medimmune, Novartis UK, Roche/Genentech Inc., GlaxoSmithKline, Amgen, Genentech/Hoffman la Roche, AstraZeneca, Medimmune, Aerocrine and Vitalograph; payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events for Novartis, Hoffman la Roche/Genentech Inc., Sanofi, GlaxoSmithKline, AstraZeneca, Teva and Circassia; support for attending meetings and/or travel from AstraZeneca, Boehringer Ingelheim, Chiesi, GSK and Napp Pharmaceuticals; participation on data safety monitoring or advisory board for Novartis, Hoffman la Roche/Genentech Inc., Sanofi, Evelo Biosciences, GlaxoSmithKline, AstraZeneca, Teva, Theravance and Circassia; all disclosures made outside the submitted work. M. Masoli reports receiving grants or contracts from ERS SHARP to support the study “The burden of severe asthma on HRQoL across Europe”, outside the submitted work. P. Howarth is an employee of GSK. R. Djukanovic reports support for the present manuscript received from ERS, TEVA, GSK, Novartis, Sanofi and Chiesi; consulting fees from Synairgen for which the author is a co-founder and consultant and owns shares, outside the submitted work; and participation on a data safety monitoring or advisory board for Kymab (Cambridge), outside the submitted work. E. Bel reports support for the present manuscript from the ERS; grants or contracts received from GlaxoSmithKline and Teva, outside the submitted work; consulting fees received from AstraZeneca, GlaxoSmithKline, Sanofi, Sterna and Chiesi, outside the submitted work; honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events received from Teva; participation on a data safety monitoring or advisory board for AstraZeneca, outside the submitted work; and leadership or fiduciary roles, unpaid, for SHARP-CRC and RAPSODI (Dutch registry), outside the submitted work. M. Hyland reports receiving grants or contracts from TEVA; and payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events for GSK; all disclosures made outside the submitted work. The remaining authors have nothing to disclose., (Copyright ©The authors 2022.)

As opposed to surface marker staining, certain cell types can only be recognized by intracellular markers. Intracellular staining for use in cell sorting remains challenging. Fixation and permeabilization steps for intracellular staining and the presence of RNases notably affect preservation of high-quality mRNA. We report the work required for the optimization of a successful protocol for microarray analysis of intracellular target-sorted, formalin-fixed human bronchial club cells. Cells obtained from differentiated air-liquid interface cultures were stained with the most characteristic intracellular markers for club cell (SCGB1A1 + ) sorting. A benchmarked intracellular staining protocol was carried out before flow cytometry. The primary outcome was the extraction of RNA sufficient quality for microarray analysis as assessed by Bioanalyzer System. Fixation with 4% paraformaldehyde coupled with 0.1% Triton/0.1% saponin permeabilization obtained optimal results for SCGB1A1 staining. Addition of RNase inhibitors throughout the protocol and within the appropriate RNA extraction kit (Formalin-Fixed-Paraffin-Embedded) dramatically improved RNA quality, resulting in samples eligible for microarray analysis. The protocol resulted in successful cell sorting according to specific club cell intracellular marker without using cell surface marker. The protocol also preserved RNA of sufficient quality for subsequent microarray transcriptomic analysis, and we were able to generate transcriptomic signature of club cells., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Obstructive lung diseases, such as chronic obstructive pulmonary disease, asthma, or non-cystic fibrosis bronchiectasis, share some major pathophysiological features: small airway involvement, dysregulation of adaptive and innate pulmonary immune homeostasis, mucus hyperproduction, and/or hyperconcentration. Mucus regulation is particularly valuable from a therapeutic perspective given it contributes to airflow obstruction, symptom intensity, disease severity, and to some extent, disease prognosis in these diseases. It is therefore crucial to understand the mucus constitution of our patients, its behavior in a stable state and during exacerbation, and its regulatory mechanisms. These are all elements representing potential therapeutic targets, especially in the era of biologics. Here, we first briefly discuss the composition and characteristics of sputum. We focus on mucus and mucins, and then elaborate on the different sample collection procedures and how their quality is ensured. We then give an overview of the different direct analytical techniques available in both clinical routine and more experimental settings, giving their advantages and limitations. We also report on indirect mucus assessment procedures (questionnaires, high-resolution computed tomography scanning of the chest, lung function tests). Finally, we consider ways of integrating these techniques with current and future therapeutic options. Cystic fibrosis will not be discussed given its monogenic nature.

Mesenchymal cells are an essential cell type because of their role in tissue support, their multilineage differentiation capacities and their potential clinical applications. They play a crucial role during lung development by interacting with airway epithelium, and also during lung regeneration and remodeling after injury. However, much less is known about their function in lung disease. In this review, we discuss the origins of mesenchymal cells during lung development, their crosstalk with the epithelium, and their role in lung diseases, particularly in chronic obstructive pulmonary disease.