Rationale:Basal cells (BC) appear ectopically within the lung parenchyma of interstitial lung disease (ILD) patients, potentially through migration of airway BC or though trans-differentiation of alveolar epithelial type 2 (AT2) cells. The exact origin and function of these ectopic alveolar BC remains elusive. By comparing ectopic alveolar to "classical" airway BC, we aimed to get a better understanding of the origin and characteristics of alveolar BC in ILD. Methods:Alveolar and airway BC were isolated from transbronchial and airway mucosal biopsies, respectively, from the same ILD patients and expanded in culture. Samples were analysed by single-cell RNA sequencing (scRNA-seq), TaqMan real-time PCR and immunochemistry. Results:scRNA-seq analysis revealed several differences in gene expression that suggested a shift to a more mesenchymal-like phenotype and a decrease in keratinisation genes in alveolar compared to airway BC. Specific AT2 cell marker genes were not expressed in either BC type. While the morphology, wound repair and proliferation capacities of BC from both origins were not significantly different, alveolar BC formed significantly fewer organoids, expressing more MUC5B. After instillation into bleomycin-injured mice, alveolar and airway BC showed similar engraftment, differentiation capacity and effects on fibrosis. Conclusion:Despite similar overall functionality in vitro and after instillation into bleomycin-injured mice, alveolar and airway BC differed in their transcriptomes and in their capacities to form and to differentiate in organoids. Our data provide no evidence to support their potential derivation from AT2 cells.

INTRODUCTION:

Idiopathic pulmonary fibrosis (IPF) represents a terminal, age-related disease characterized by a complex pathophysiology. Recent studies have reported the presence of basal cells in IPF lungs, yet their specific role and impact on other cell types within the fibrotic lung remains unclear. Lung resident mesenchymal stem cells (MSCs) are a crucial resident stem cell population essential for maintaining lung homeostasis; MSCs exhaustion, a hallmark of aging, has been implicated in IPF through poorly understood mechanisms. In this study, we investigated the effects of hepatocyte growth factor (HGF), an anti-fibrotic factor, on MSCs quiescence using a bleomycin-induced lung injury model. RNA-sequencing analysis of basal cells isolated from IPF lungs identified upregulation of matrix metalloproteinase 24 (MMP24) in IPF (2.45±0.53) compared to non-IPF basal cells (1.12±0.2) (p˂0.05). Moreover, in vivo experiments indicated increased MMP24 levels in bleomycin injured lung homogenates compared to healthy lungs (6.89±0.78 vs 3.52±0.65 ng/mL) p<0.01, and decreased frequencies of activated lung MSCs in bleomycin-injured lungs compared to healthy lungs (21.8±6.2% vs 51.0±9.3%) p<0.01. Consistently, in vivo overexpression of MMP24 resulted in enhanced quiescence of lung MSCs and increased fibrosis. Conversely, in vivo HGF gene transfer increased frequencies of activated lung MSCs (27.5±3.2%, p<0.05) and reduced MMP24 levels in lung homogenates (4.39±0.38 ng/mL, p<0.001). Mechanistically, HGF reduced MMP24 expression through the YAP-1 pathway. Collectively, our findings uncover MMP24 as a pro-fibrotic mediator in IPF patients and in bleomycin injured lung and identify HGF-YAP1-MMP24 as a novel therapeutic axis in lung fibrosis.

Background Honeycomb cysts (HC) within the alveolar region are distinct histopathological features in the lungs of idiopathic pulmonary fibrosis (IPF) patients. HC are lined with a single-or stratified layer of basal cells (BC), or with a bronchiolar-like epithelium composed of basal-, ciliated- and secretory epithelial cells. By using cultured IPF patient-derived alveolar BC, we aimed to establish an in vitro- and in vivo model to mimic HC formation in IPF. We (1) optimized conditions to culture and propagate IPF patient-derived alveolar BC, (2) cultured the cells on an air liquid interface (ALI) or in a three dimensional (3D) organoid model, and (3) investigated the cells` behavior after instillation into bleomycin-challenged mice. Methods Alveolar BC were cultured from peripheral IPF lung tissue and grown on tissue-culture treated plastic, an ALI, or in a 3D organoid model. Furthermore, cells were instilled into bleomycin-challenged NRG mice. Samples were analyzed by TaqMan RT-PCR, immunoblotting, immunocytochemistry/immunofluorescence (ICC/IF), or immunohistochemistry (IHC)/IF. Mann–Whitney tests were performed using GraphPad Prism software. Results Cultured alveolar BC showed high expression of canonical basal cell markers (TP63, keratin (KRT)5, KRT14, KRT17), robust proliferation, and wound closure capacity. The cells could be cryopreserved and propagated for up to four passages without a significant loss of basal cell markers. When cultured on an ALI or in a 3D organoid model, alveolar BC differentiated to ciliated- and secretory epithelial cells. When instilled into bleomycin-challenged mice, human alveolar BC cells formed HC-like structures composed of human basal-, and secretory epithelial cells within the mouse parenchyma. Conclusion IPF patient-derived alveolar BC on an ALI, in 3D organoids or after instillation into bleomycin-challenged mice form HC-like structures that closely resemble HC within the IPF lung. These models therefore represent powerful tools to study honeycomb formation, and its potential therapeutic inhibition in IPF.

Introduction: Long-term survival after allogeneic hematopoietic cell transplantation (alloHCT) is limited by chronic pulmonary graft-versus-host disease (cGvHD) which comprise classical bronchiolitis obliterans (BO), but also various forms of restrictive disease. Histological confirmation of BO by transbronchial forceps biopsy has a low sensitivity and surgical lung biopsy is gold standard. The clinical diagnosis of BO syndrome (BOS) is based on lung function and radiological data. However, sensitivity of BOS-criteria for biopsy-proven BO is unsatisfactory. Aim: Retrospective study to investigate the diagnostic value of transbronchial cryobiopsy in alloHCT-patients with suspicion of pulmonary cGvHD not fulfilling BOS-criteria. Histology was integrated into the multidisciplinary discussion (MDD) and diagnostic consensus was sought. Results: 20 patients underwent cryobiopsy. In 13/20 patients (65%) cryobiopsies revealed findings leading to a specific diagnosis: obliterative/lymphocytic bronchiolitis in four (20%), distinct parenchymal abnormalities leading to the diagnosis of interstitial lung disease (ILD) or cGvHD-associated ILD in 9/20 patients (45%). In 7/20 patients (35%), cryobiopsies showed normal lung parenchyma. In 4/7, a consensus diagnosis of BOS was reached after MDD. Complications of cryobiopsy included pneumothorax (25%) and locally controlled bleeding (20%). Conclusion: In alloHCT-patients with suspicion of pulmonary cGvHD not fulfilling BOS-criteria, transbronchial cryobiopsy led to a histology-based diagnosis in two-thirds of patients, and – embedded in an MDD - to a final diagnosis in 90% of patients.

Background High bacterial burden in the lung microbiota predicts progression of idiopathic pulmonary fibrosis (IPF). Azithromycin (AZT) is a macrolide antibiotic known to alter the lung microbiota in several chronic pulmonary diseases, and observational studies have shown a positive effect of AZT on mortality and hospitalisation rate in IPF. However, the effect of AZT on the lung microbiota in IPF remains unknown. Methods We sought to determine the impact of a 3-month course of AZT on the lung microbiota in IPF. We assessed sputum and oropharyngeal swab specimens from 24 adults with IPF included in a randomised controlled crossover trial of oral AZT 500 mg 3 times per week. 16S rRNA gene amplicon sequencing and quantitative PCR (qPCR) were performed to assess bacterial communities. Antibiotic resistance genes (ARGs) were assessed using real-time qPCR. Results AZT significantly decreased community diversity with a stronger and more persistent effect in the lower airways (sputum). AZT treatment altered the temporal kinetics of the upper (oropharyngeal swab) and lower airway microbiota, increasing community similarity between the two sites for 1 month after macrolide cessation. Patients with an increase in ARG carriage had lower bacterial density and enrichment of the genus Streptococcus. In contrast, patients with more stable ARG carriage had higher bacterial density and enrichment in Prevotella. Conclusions AZT caused sustained changes in the diversity and composition of the upper and lower airway microbiota in IPF, with effects on the temporal and spatial dynamics between the two sites.

Introduction: Honeycomb cysts (HC) within the alveolar region are distinct histopathological features in the lungs of idiopathic pulmonary fibrosis (IPF) patients. HC are lined with a bronchiolar-like epithelium consisting of basal cells (BC), and differentiated ciliated- and secretory epithelial cells. By using primary IPF-derived alveolar BC, we here aimed to establish a 3D organoid model that mimics HC in IPF. Methods: IPF alveolar BC were embedded in Matrigel and cultured in differentiation medium supplemented with ROCK inhibitor (Y-27632), TGF-ß inhibitor (A83-01), DCI (dexamethasone, 8-Bromo-cAMP, IBMX), fibroblast growth factor (FGF)-2, FGF-10, and epithelial growth factor (EGF) for 21 days. Colony forming efficiency (CFE) was determined and cell marker expression analyzed by TaqMan RT-PCR and immunofluorescence. Results: Alveolar BC expand in numbers and start to self-assemble into clusters at day 3. Organoids first formed after 7 days (>50µm diameter) and continued to grow over a period of 21 days. A polarized lumen was present in 40% of the organoids after 21 days. Beating cilia and mucus secretion was observed between 10-15 days. After 20 days, a 3-10% CFE of organoids with a diameter of 50-280µm was calculated. After 21 days, organoids displayed a heterogenous cell population expressing basal (KRT5, KRT17, TP63)-, ciliated (AcTub)-, and secretory (SCGB1A1, MUC5AC, MUC5B) epithelial cell markers. Conclusion: Organoids reconstituted functional and morphological properties of the in vivo HC in IPF lungs and therefore represents a valuable in vitro tool to study honeycomb formation, and its potential therapeutic inhibition.

aLung Center, Cantonal Hospital St. Gallen, St. Gallen, Switzerland; bDepartment of Pulmonary Medicine, Inselspital, University Hospital of Berne, Berne, Switzerland; cClinics of Respiratory Medicine, University Hospital Basel, Basel, Switzerland; dDepartement of Pulmonary Medicine, University Hospital of Zurich, Zurich, Switzerland; eDepartement of Pulmonary Medicine, Réseau Hospitalier Neuchâtelois, Pourtalès Hospital, Neuchâtel, Switzerland; fDepartement of Pulmonary Medicine, Lucerne Cantonal Hospital, Lucerne, Switzerland; gDivision of Pneumology, Geneva University Hospitals, Geneva, Switzerland; hDepartement of Pulmonology, City Hospital Triemli, Zurich, Switzerland; iDepartement of Pneumology, Hospital of Bellinzona, Bellinzona, Switzerland

Rationale: Directed trans-differentiation of ectopic alveolar basal cells (ABC) to alveolar epithelial type (AT)2 cells may facilitate normal lung regeneration and inhibit pathological bronchiolization in idiopathic pulmonary fibrosis (IPF). To induce ABC to AT2 trans-differentiation, we treated cultured ABC with KF-DCI (KGF, FGF-10, dexamethasone, 8-Bromo-cAMP, IBMX) and determined AT2-, ciliated-, and secretory epithelial cell marker expression. Furthermore, we examined what factors in KF-DCI were essential for its effects. Methods: IPF ABC were cultured on plastic or on an air-liquid interface and maintained in growth medium, or in differentiation medium +/- KF-DCI (KGF, FGF-10 (both 10 ng/ml), dexamethasone (50 nM), 8-Bromo-cAMP (0.1 mM), IBMX (0.1 mM)). Cell marker expression was analyzed by TaqMan RT-PCR and immunofluorescence. Results: Differentiation medium strongly induced the expression of secretory (SCGB1A1, MUC5AC, MUC5B)-, and ciliated (FOXJ1)- epithelial cell markers, and weakly that of surfactant protein (SP)-B in ABC. Addition of KF-DCI inhibited the expression of SCGB1A1, MUC5AC, MUC5B and FOXJ1 and further up-regulated SP-B. SP-A, C and D were not induced by KF-DCI in ABC. Removing the cAMP elevating compounds IBMX or 8-Bromo-cAMP from KF-DCI, reversed all KF-DCI effects on ABC. Conclusion: KF-DCI inhibits ABC differentiation towards ciliated- or secretory epithelial cells and directs it towards SP-B expressing cells in a cAMP-dependent manner. Increasing cellular cAMP represents an attractive pharmacological intervention that may reduce bronchiolization and facilitate normal lung regeneration in IPF.

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In idiopathic pulmonary fibrosis (IPF), basal-like cells are atypically present in the alveolar region, where they may affect adjacent stromal cells by paracrine mechanisms. We here aimed to confirm the presence of basal-like cells in peripheral IPF lung tissue in vivo, to culture and characterize the cells in vitro, and to investigate their paracrine effects on IPF fibroblasts in vitro and in bleomycin-injured rats in vivo. Basal-like cells are mainly localized in areas of pathological bronchiolization or honeycomb cysts in peripheral IPF lung tissue. Single-cell RNA sequencing (scRNA-seq) demonstrated an overall homogeneity, the expression of the basal cell markers cytokeratin KRT5 and KRT17, and close transcriptomic similarities to basal cells in the majority of cells cultured in vitro. Basal-like cells secreted significant levels of prostaglandin E2 (PGE2), and their conditioned medium (CM) inhibited alpha-smooth muscle actin (α-SMA) and collagen 1A1 (Col1A1) and upregulated matrix metalloproteinase-1 (MMP-1) and hepatocyte growth factor (HGF) by IPF fibroblasts in vitro. The instillation of CM in bleomycin-injured rat lungs resulted in reduced collagen content, improved lung architecture, and reduced α-SMA-positive cells. Our data suggested that basal-like cells may limit aberrant fibroblast activation and differentiation in IPF through paracrine mechanisms.

Sarcoidosis is a systemic inflammatory disease, characterised by granuloma formation upon an unknown trigger in genetically predisposed individuals. The inflammation is characterised by an activation of both the innate immune system, with macrophages differentiating into epitheloid cells and dendritic cells, and the adaptive immune system, particularly T helper (Th) 1 and Th17 cells. Since all organs can be affected to varying extents, clinical presentation is often diverse. Most commonly, the lungs, lymph nodes, skin and eyes are involved, whereas cardiac, renal and neurological manifestations are less common but associated with higher morbidity. Depending on the clinical symptoms, a detailed evaluation including thorough clinical examination, imaging and laboratory tests should explore all possible organ involvements. In some patients, fatigue manifests as a para-sarcoidosis symptom impacting quality of life, even if sarcoidosis is in remission. Some acute syndromic presentations, such as Löfgren's syndrome, have a good prognosis and are commonly self-limiting. If possible, a topical treatment, for example for cutaneous sarcoidosis or bronchial involvement, should be applied. Treatment of severe cases with persisting disease activity necessitates long-term immunosuppressive drugs, with glucocorticoids as the first-line option. Steroid-sparing and second-line drugs include methotrexate, azathioprine, mycophenolate mofetil and immunomodulators such hydroxychloroquine, with the latter being first-line therapy in cutaneous sarcoidosis. Tumour necrosis factor-alpha inhibitors (particularly adalimumab and infliximab) are used as third-line agents but are administered earlier in cases of persistent disease activity, severe organ-involvement or intolerance to conventional drugs. Treatment decisions should be based on a multidisciplinary approach, depending on organ involvement and treatment tolerability. Para-sarcoidosis manifestations, particularly fatigue, should also be carefully addressed, where the patient could also be enrolled in multidimensional rehabilitation programmes. With various organ involvement and different phenotypes, larger studies including real-world data from registries are necessary to evaluate different sarcoidosis endotypes and preferential treatment pathways.

In idiopathic pulmonary fibrosis (IPF), keratin (KRT)17+/KRT5+ basal and KRT17+/KRT5− aberrant basaloid cells are atypically present within the alveolar space. We previously described the fibrosis-enriched outgrowth of alveolar basal cells from peripheral fibrotic lung tissue. Using single cell RNA sequencing (scRNA-seq), we here characterize the transcriptome of these cultured alveolar basal cells under different culture conditions. Methods: Fibrotic peripheral lung tissue pieces were placed in DMEM growth medium. Outgrown cells were analysed by scRNA-seq, TaqMan-PCR or immunofluorescence (IF) either directly or after medium change to an epithelial cell specific medium (Cnt-PR-A). Results: A fraction of alveolar basal cells cultured in DMEM growth medium showed close transcriptomic similarities to IPF basal cells. However, although they expressed KRT5, the transcriptome of the majority of cells matched best to the transcriptome of recently described KRT17+/KRT5− aberrant basaloid cells, co-expressing the canonical basal cell marker KRT17 and mesenchymal cell marker (VIM, FN1). A smaller fraction of cells matched best to secretory epithelial cells. Two differentiation gradients from basal to aberrant basaloid-like cells and basal to secretory epithelial-like cells were apparent. Interestingly, these differentiation paths seemed reversed when the cell culture medium was changed to Cnt-PR-A. Conclusions: Our results suggest that cultured alveolar basal cells have the capacity to differentiate towards secretory epithelial-like cells and to aberrant basaloid-like cells. However, due to the persistent expression of KRT5, a complete differentiation towards aberrant basaloid cells did not seem to be achieved in our culture conditions. Importantly, differentiation seemed reversible by changing the cells microenvironment. Determining specific factors influencing these differentiation paths may help to define novel drug targets for IPF therapy.

Rational: Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease and is associated with high mortality due to a lack of effective treatment. Excessive deposition of the extracellular matrix by activated myofibroblasts in the alveolar space leads to scar formation that hinders gas exchange. Therefore, selectively removing activated myofibroblasts with the aim to repair and remodel fibrotic lungs is a promising approach. Stromal-derived growth factor (SDF-1) is known to stimulate cellular signals which attract stem cells to the site of injury for tissue repair and remodeling. Here, we investigate the effect of overexpression of SDF-1β on lung structure using the bleomycin-injured rat lung model. Methods: Intratracheal administration of bleomycin was performed in adult male rats (F344). Seven days later, in vivo electroporation-mediated gene transfer of either SDF-1β or the empty vector was performed. Animals were sacrificed seven days after gene transfer and histology, design-based stereology, flow cytometry, and collagen measurement were performed on the tissue collected. For in vitro experiments, lung fibroblasts obtained from IPF patients were used. Results: Seven days after SDF-1β gene transfer to bleomycin-injured rat lungs, reduced total collagen, reduced collagen fibrils, improved histology and induced apoptosis of myofibroblasts were observed. Furthermore, it was revealed that TNF-α mediates SDF-1β-induced apoptosis of myofibroblasts; moreover, SDF-1β overexpression increased alveolar epithelial cell numbers and proliferation in vivo and also induced their migration in vitro. Conclusions: Our study demonstrates a new antifibrotic mechanism of SDF-1β overexpression and suggests SDF-1β as a potential new approach for the treatment of lung fibrosis.