Publications by authors named "Susanne Herold"

128 Publications

Nucleic Acid-based Testing for Noninfluenza Viral Pathogens in Adults with Suspected Community-acquired Pneumonia. An Official American Thoracic Society Clinical Practice Guideline.

Am J Respir Crit Care Med 2021 05;203(9):1070-1087

This document provides evidence-based clinical practice guidelines on the diagnostic utility of nucleic acid-based testing of respiratory samples for viral pathogens other than influenza in adults with suspected community-acquired pneumonia (CAP). A multidisciplinary panel developed a Population-Intervention-Comparison-Outcome question, conducted a pragmatic systematic review, and applied Grading of Recommendations, Assessment, Development, and Evaluation methodology for clinical recommendations. The panel evaluated the literature to develop recommendations regarding whether routine diagnostics should include nucleic acid-based testing of respiratory samples for viral pathogens other than influenza in suspected CAP. The evidence addressing this topic was generally adjudicated to be of very low quality because of risk of bias and imprecision. Furthermore, there was little direct evidence supporting a role for routine nucleic acid-based testing of respiratory samples in improving critical outcomes such as overall survival or antibiotic use patterns. However, on the basis of direct and indirect evidence, recommendations were made for both outpatient and hospitalized patients with suspected CAP. Testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was not addressed in the literature at the time of the evidence review. The panel formulated and provided their rationale for recommendations on nucleic acid-based diagnostics for viral pathogens other than influenza for patients with suspected CAP.
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http://dx.doi.org/10.1164/rccm.202102-0498STDOI Listing
May 2021

Identification of a novel subset of alveolar type 2 cells enriched in PD-L1 and expanded following pneumonectomy.

Eur Respir J 2021 Apr 16. Epub 2021 Apr 16.

Key Laboratory of Interventional Pulmonology of Zhejiang Province, Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China

Alveolar type 2 (AT2) cells are heterogeneous cells; where specialised AT2 subpopulations within this lineage exhibit stem cell properties. However, the existence of quiescent, immature cells within the AT2 lineage, which are activated during lung regeneration, is unknown. mice were used for the labelling of AT2 cells and labeled subpopulations were analysed by flow cytometry, qPCR, ATAC-seq, gene arrays, pneumonectomy, and culture of precision-cut lung slides. ScRNA-seq data from human lungs were analysed.In mice, we detected two distinct AT2 subpopulations with low tdTomato level (Tom) and high tdTomato level (Tom). Tom express lower level of AT2 differentiation markers, and , while Tom, as mature AT2 cells, show higher levels of , , , and expression. ATAC-seq analysis indicates that Tom and Tom constitute two distinct cell populations with specific silencing of , and cell cycle gene loci in Tom Upon pneumonectomy, the number of Tom but not Tom cells increases and Tom upregulate the expression of , , , and compared to sham. Tom cells overexpress PD-L1, an immune inhibitory membrane receptor ligand, which is used by flow cytometry to differentially isolate these two sub-populations. In the human lung, data mining of a recent scRNA-seq AT2 dataset demonstrates the existence of a population. Therefore, we have identified a novel population of AT2 quiescent, immature progenitor cells in mouse that expand upon pneumonectomy and provided evidence for the existence of such cells in human.
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http://dx.doi.org/10.1183/13993003.04168-2020DOI Listing
April 2021

P-Glycoprotein Inhibitors Differently Affect , and Proliferation in Bovine Primary Endothelial Cells.

Pathogens 2021 Mar 25;10(4). Epub 2021 Mar 25.

Biomedical Research Center Seltersberg, Institute of Parasitology, Justus Liebig University Giessen, 35392 Giessen, Germany.

Apicomplexan parasites are obligatory intracellular protozoa. In the case of , or , to ensure proper tachyzoite production, they need nutrients and cell building blocks. However, apicomplexans are auxotrophic for cholesterol, which is required for membrane biosynthesis. P-glycoprotein (P-gp) is a transmembrane transporter involved in xenobiotic efflux. However, the physiological role of P-gp in cholesterol metabolism is unclear. Here, we analyzed its impact on parasite proliferation in -, - and -infected primary endothelial cells by applying different generations of P-gp inhibitors. Host cell treatment with verapamil and valspodar significantly diminished tachyzoite production in all three parasite species, whereas tariquidar treatment affected proliferation only in . 3D-holotomographic analyses illustrated impaired meront development driven by valspodar treatment being accompanied by swollen parasitophorous vacuoles in the case of . Tachyzoite and host cell pre-treatment with valspodar affected infection rates in all parasites. Flow cytometric analyses revealed verapamil treatment to induce neutral lipid accumulation. The absence of a pronounced anti-parasitic impact of tariquidar, which represents here the most selective P-gp inhibitor, suggests that the observed effects of verapamil and valspodar are associated with mechanisms independent of P-gp. Out of the three species tested here, this compound affected only proliferation and its effect was much milder as compared to verapamil and valspodar.
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http://dx.doi.org/10.3390/pathogens10040395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8065907PMC
March 2021

From Clones to Buds and Branches: The Use of Lung Organoids to Model Branching Morphogenesis .

Front Cell Dev Biol 2021 4;9:631579. Epub 2021 Mar 4.

Department of Internal Medicine II, Cardio-Pulmonary Institute, Universities of Giessen and Marburg Lung Center, Giessen, Germany.

Three-dimensional (3D) organoid culture systems have rapidly emerged as powerful tools to study organ development and disease. The lung is a complex and highly specialized organ that comprises more than 40 cell types that offer several region-specific roles. During organogenesis, the lung goes through sequential and morphologically distinctive stages to assume its mature form, both structurally and functionally. As branching takes place, multipotent epithelial progenitors at the distal tips of the growing/bifurcating epithelial tubes progressively become lineage-restricted, giving rise to more differentiated and specialized cell types. Although many cellular and molecular mechanisms leading to branching morphogenesis have been explored, deeper understanding of biological processes governing cell-fate decisions and lung patterning is still needed. Given that these distinct processes cannot be easily analyzed , 3D culture systems have become a valuable platform to study organogenesis . This minireview focuses on the current lung organoid systems that recapitulate developmental events occurring before and during branching morphogenesis. In addition, we also discuss their limitations and future directions.
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http://dx.doi.org/10.3389/fcell.2021.631579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7969706PMC
March 2021

WASP: a versatile, web-accessible single cell RNA-Seq processing platform.

BMC Genomics 2021 Mar 18;22(1):195. Epub 2021 Mar 18.

Bioinformatics and Systems Biology, Justus Liebig University Giessen, 35392, Giessen, Germany.

Background: The technology of single cell RNA sequencing (scRNA-seq) has gained massively in popularity as it allows unprecedented insights into cellular heterogeneity as well as identification and characterization of (sub-)cellular populations. Furthermore, scRNA-seq is almost ubiquitously applicable in medical and biological research. However, these new opportunities are accompanied by additional challenges for researchers regarding data analysis, as advanced technical expertise is required in using bioinformatic software.

Results: Here we present WASP, a software for the processing of Drop-Seq-based scRNA-Seq data. Our software facilitates the initial processing of raw reads generated with the ddSEQ or 10x protocol and generates demultiplexed gene expression matrices including quality metrics. The processing pipeline is realized as a Snakemake workflow, while an R Shiny application is provided for interactive result visualization. WASP supports comprehensive analysis of gene expression matrices, including detection of differentially expressed genes, clustering of cellular populations and interactive graphical visualization of the results. The R Shiny application can be used with gene expression matrices generated by the WASP pipeline, as well as with externally provided data from other sources.

Conclusions: With WASP we provide an intuitive and easy-to-use tool to process and explore scRNA-seq data. To the best of our knowledge, it is currently the only freely available software package that combines pre- and post-processing of ddSEQ- and 10x-based data. Due to its modular design, it is possible to use any gene expression matrix with WASP's post-processing R Shiny application. To simplify usage, WASP is provided as a Docker container. Alternatively, pre-processing can be accomplished via Conda, and a standalone version for Windows is available for post-processing, requiring only a web browser.
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http://dx.doi.org/10.1186/s12864-021-07469-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7977290PMC
March 2021

A comparison of airway pressures for inflation fixation of developing mouse lungs for stereological analyses.

Histochem Cell Biol 2021 Feb 29;155(2):203-214. Epub 2020 Dec 29.

Department of Lung Development and Remodelling, Max Planck Institute for Heart and Lung Research, member of the German Center for Lung Research (DZL), Parkstrasse 1, 60231, Bad Nauheim, Germany.

The morphometric analysis of lung structure using the principles of stereology has emerged as a powerful tool to describe the structural changes in lung architecture that accompany the development of lung disease that is experimentally modelled in adult mice. These stereological principles are now being applied to the study of the evolution of the lung architecture over the course of prenatal and postnatal lung development in mouse neonates and adolescents. The immature lung is structurally and functionally distinct from the adult lung, and has a smaller volume than does the adult lung. These differences have raised concerns about whether the inflation fixation of neonatal mouse lungs with the airway pressure (P) used for the inflation fixation of adult mouse lungs may cause distortion of the neonatal mouse lung structure, leading to the generation of artefacts in subsequent analyses. The objective of this study was to examine the impact of a P of 10, 20 and 30 cmHO on the estimation of lung volumes and stereologically assessed parameters that describe the lung structure in developing mouse lungs. The data presented demonstrate that low P (10 cmHO) leads to heterogeneity in the unfolding of alveolar structures within the lungs, and that high P (30 cmHO) leads to an overestimation of the lung volume, and thus, affects the estimation of volume-dependent parameters, such as total alveoli number and gas-exchange surface area. Thus, these data support the use of a P of 20 cmHO for inflation fixation in morphometric studies on neonatal mouse lungs.
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http://dx.doi.org/10.1007/s00418-020-01951-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7910376PMC
February 2021

Identification of a Repair-Supportive Mesenchymal Cell Population during Airway Epithelial Regeneration.

Cell Rep 2020 Dec;33(12):108549

Key Laboratory of Interventional Pulmonology of Zhejiang Province, Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325015, China; Department of Internal Medicine, Universities of Giessen and Marburg Lung Center (UGMLC), Cardio-Pulmonary Institute (CPI), Member of the German Center for Lung Research (DZL), Justus-Liebig University Giessen, 35392 Giessen, Germany; Institute for Lung Health (ILH), 35392 Giessen, Germany. Electronic address:

Tissue regeneration requires coordinated and dynamic remodeling of stem and progenitor cells and the surrounding niche. Although the plasticity of epithelial cells has been well explored in many tissues, the dynamic changes occurring in niche cells remain elusive. Here, we show that, during lung repair after naphthalene injury, a population of PDGFRα cells emerges in the non-cartilaginous conducting airway niche, which is normally populated by airway smooth muscle cells (ASMCs). This cell population, which we term "repair-supportive mesenchymal cells" (RSMCs), is distinct from conventional ASMCs, which have previously been shown to contribute to epithelial repair. Gene expression analysis on sorted lineage-labeled cells shows that RSMCs express low levels of ASMC markers, but high levels of the pro-regenerative marker Fgf10. Organoid co-cultures demonstrate an enhanced ability for RSMCs in supporting club-cell growth. Our study highlights the dynamics of mesenchymal cells in the airway niche and has implications for chronic airway-injury-associated diseases.
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http://dx.doi.org/10.1016/j.celrep.2020.108549DOI Listing
December 2020

The HS-generating enzyme 3-mercaptopyruvate sulfurtransferase regulates pulmonary vascular smooth muscle cell migration and proliferation but does not impact normal or aberrant lung development.

Nitric Oxide 2021 02 15;107:31-45. Epub 2020 Dec 15.

Department of Lung Development and Remodelling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Member of the German Center for Lung Research (DZL), Parkstrasse 1, 60231, Bad Nauheim, Germany; Department of Internal Medicine (Pulmonology), University of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Justus Liebig University, Aulweg 123, 35392, Giessen, Germany; CardioPulmonary Institute, Justus Liebig University Giessen, Klinikstrasse 33, Giessen, Germany. Electronic address:

Along with nitric oxide (NO), the gasotransmitters carbon monoxide (CO) and hydrogen sulfide (HS) are emerging as potentially important players in newborn physiology, as mediators of newborn disease, and as new therapeutic modalities. Several recent studies have addressed HS in particular in animal models of bronchopulmonary dysplasia (BPD), a common complication of preterm birth where oxygen toxicity stunts lung development. In those studies, exogenous HS attenuated the impact of oxygen toxicity on lung development, and two HS-generating enzymes were documented to affect pulmonary vascular development. HS is directly generated endogenously by three enzymes, one of which, 3-mercaptopyruvate sulfurtransferase (MPST), has not been studied in the lung. In a hyperoxia-based animal model of BPD, oxygen exposure deregulated MPST expression during post-natal lung development, where MPST was localized to the smooth muscle layer of the pulmonary vessels in developing lungs. siRNA-mediated abrogation of MPST expression in human pulmonary artery smooth muscle cells in vitro limited baseline cell migration and cell proliferation, without affecting apoptosis or cell viability. In vivo, MPST was dispensable for normal lung development in Mpstmice, and MPST did not contribute to stunted lung development driven by hyperoxia exposure, assessed by design-based stereology. These data demonstrate novel roles for MPST in pulmonary vascular smooth muscle cell physiology. The potential caveats of using Mpst mice to study normal and aberrant lung development are also discussed, highlighting the possible confounding, compensatory effects of other HS-generating enzymes that are present alongside MPST in the smooth muscle compartment of developing pulmonary vessels.
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http://dx.doi.org/10.1016/j.niox.2020.12.002DOI Listing
February 2021

Extracorporeal Carbon Dioxide Removal Using a Renal Replacement Therapy Platform to Enhance Lung-Protective Ventilation in Hypercapnic Patients With Coronavirus Disease 2019-Associated Acute Respiratory Distress Syndrome.

Front Med (Lausanne) 2020 12;7:598379. Epub 2020 Nov 12.

Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine II, University Hospital Giessen and Marburg, Justus Liebig University Giessen, Giessen, Germany.

Coronavirus disease 2019 (COVID-19)-associated acute respiratory distress syndrome (ARDS) is associated with high mortality. Lung-protective ventilation is the current standard of care in patients with ARDS, but it might lead to hypercapnia, which is independently associated with worse outcomes. Extracorporeal carbon dioxide removal (ECCOR) has been proposed as an adjuvant therapy to avoid progression of clinical severity and limit further ventilator-induced lung injury, but its use in COVID-19 has not been described yet. Acute kidney injury requiring renal replacement therapy (RRT) is common among critically ill COVID-19 patients. In centers with available dialysis, low-flow ECCOR (<500 mL/min) using RRT platforms could be carried out by dialysis specialists and might be an option to efficiently allocate resources during the COVID-19 pandemic for patients with hypercapnia as the main indication. Here, we report the feasibility, safety, and efficacy of ECCOR using an RRT platform to provide either standalone ECCOR or ECCOR combined with RRT in four hypercapnic patients with moderate ARDS. A randomized clinical trial is required to assess the overall benefit and harm. ClinicalTrials.gov. Unique identifier: NCT04351906.
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http://dx.doi.org/10.3389/fmed.2020.598379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693445PMC
November 2020

Immunoglobulin deficiency as an indicator of disease severity in patients with COVID-19.

Am J Physiol Lung Cell Mol Physiol 2021 04 25;320(4):L590-L599. Epub 2020 Nov 25.

Department of Nephrology and Hypertension, Hannover Medical School, Hannover, Germany.

Despite the pandemic status of COVID-19, there is limited information about host risk factors and treatment beyond supportive care. Immunoglobulin G (IgG) could be a potential treatment target. Our aim was to determine the incidence of IgG deficiency and associated risk factors in a cohort of 62 critically ill patients with COVID-19 admitted to two German ICUs (72.6% male, median age: 61 yr). Thirteen (21.0%) of the patients displayed IgG deficiency (IgG < 7 g/L) at baseline (predominant for the IgG1, IgG2, and IgG4 subclasses). Patients who were IgG-deficient had worse measures of clinical disease severity than those with normal IgG levels (shorter duration from disease onset to ICU admission, lower ratio of [Formula: see text] to [Formula: see text], higher Sequential Organ Failure Assessment score, and higher levels of ferritin, neutrophil-to-lymphocyte ratio, and serum creatinine). Patients who were IgG-deficient were also more likely to have sustained lower levels of lymphocyte counts and higher levels of ferritin throughout the hospital stay. Furthermore, patients who were IgG-deficient compared with those with normal IgG levels displayed higher rates of acute kidney injury (76.9% vs. 26.5%; = 0.001) and death (46.2% vs. 14.3%; = 0.012), longer ICU [28 (6-48) vs. 12 (3-18) days; = 0.012] and hospital length of stay [30 (22-50) vs. 18 (9-24) days; = 0.004]. Univariable logistic regression showed increasing odds of 90-day overall mortality associated with IgG-deficiency (odds ratio 5.14, 95% confidence interval 1.3-19.9; = 0.018). IgG deficiency might be common in patients with COVID-19 who are critically ill, and warrants investigation as both a marker of disease severity as well as a potential therapeutic target.
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http://dx.doi.org/10.1152/ajplung.00359.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8057306PMC
April 2021

Group 3 Innate Lymphoid Cells Program a Distinct Subset of IL-22BP-Producing Dendritic Cells Demarcating Solitary Intestinal Lymphoid Tissues.

Immunity 2020 11;53(5):1015-1032.e8

Laboratory of Innate Immunity, Department of Microbiology, Infectious Diseases and Immunology, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12203 Berlin, Germany; Berlin Institute of Health (BIH), Anna-Louisa-Karsch Strasse 2, 10117 Berlin, Germany; Mucosal and Developmental Immunology, Deutsches Rheuma-Forschungszentrum (DRFZ), an institute of the Leibniz Association, 10117 Berlin, Germany. Electronic address:

Solitary intestinal lymphoid tissues such as cryptopatches (CPs) and isolated lymphoid follicles (ILFs) constitute steady-state activation hubs containing group 3 innate lymphoid cells (ILC3) that continuously produce interleukin (IL)-22. The outer surface of CPs and ILFs is demarcated by a poorly characterized population of CD11c cells. Using genome-wide single-cell transcriptional profiling of intestinal mononuclear phagocytes and multidimensional flow cytometry, we found that CP- and ILF-associated CD11c cells were a transcriptionally distinct subset of intestinal cDCs, which we term CIA-DCs. CIA-DCs required programming by CP- and ILF-resident CCR6 ILC3 via lymphotoxin-β receptor signaling in cDCs. CIA-DCs differentially expressed genes associated with immunoregulation and were the major cellular source of IL-22 binding protein (IL-22BP) at steady state. Mice lacking CIA-DC-derived IL-22BP exhibited diminished expression of epithelial lipid transporters, reduced lipid resorption, and changes in body fat homeostasis. Our findings provide insight into the design principles of an immunoregulatory checkpoint controlling nutrient absorption.
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http://dx.doi.org/10.1016/j.immuni.2020.10.012DOI Listing
November 2020

Severe organising pneumonia following COVID-19.

Thorax 2020 Nov 11. Epub 2020 Nov 11.

Department of Internal Medicine, Faculty of Medicine, Justus Liebig University Giessen, Giessen, Hessen, Germany.

Various forms of diffuse parenchymal lung disease have been proposed as potential consequences of severe COVID‑19. We describe the clinical, radiological and histological findings of patients with COVID‑19-associated acute respiratory distress syndrome who later developed severe organising pneumonia including longitudinal follow-up. Our findings may have important implications for the therapeutic modalities in the late-phase of severe COVID‑19 and might partially explain why a subgroup of COVID‑19 patients benefits from systemic corticosteroids.
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http://dx.doi.org/10.1136/thoraxjnl-2020-216088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7661377PMC
November 2020

infections induce G cell cycle arrest and a senescence-like phenotype in endothelial host cells.

Parasitology 2021 Mar 26;148(3):341-353. Epub 2020 Oct 26.

Institute of Parasitology, Biomedical Research Center Seltersberg, Justus Liebig University Giessen, Giessen, Germany.

Apicomplexan parasites are well-known to modulate their host cells at diverse functional levels. As such, apicomplexan-induced alteration of host cellular cell cycle was described and appeared dependent on both, parasite species and host cell type. As a striking evidence of species-specific reactions, we here show that Eimeria bovis drives primary bovine umbilical vein endothelial cells (BUVECs) into a senescence-like phenotype during merogony I. In line with senescence characteristics, E. bovis induces a phenotypic change in host cell nuclei being characterized by nucleolar fusion and heterochromatin-enriched peripheries. By fibrillarin staining we confirm nucleoli sizes to be increased and their number per nucleus to be reduced in E. bovis-infected BUVECs. Additionally, nuclei of E. bovis-infected BUVECs showed enhanced signals for HH3K9me2 as heterochromatin marker thereby indicating an infection-induced change in heterochromatin transition. Furthermore, E. bovis-infected BUVECs show an enhanced β-galactosidase activity, which is a well-known marker of senescence. Referring to cell cycle progression, protein abundance profiles in E. bovis-infected endothelial cells revealed an up-regulation of cyclin E1 thereby indicating a cell cycle arrest at G1/S transition, signifying a senescence key feature. Similarly, abundance of G2 phase-specific cyclin B1 was found to be downregulated at the late phase of macromeront formation. Overall, these data indicate that the slow proliferative intracellular parasite E. bovis drives its host endothelial cells in a senescence-like status. So far, it remains to be elucidated whether this phenomenon indeed reflects an intentionally induced mechanism to profit from host cell-derived energy and metabolites present in a non-dividing cellular status.
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http://dx.doi.org/10.1017/S0031182020002097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7890351PMC
March 2021

Minoxidil Cannot Be Used To Target Lysyl Hydroxylases during Postnatal Mouse Lung Development: A Cautionary Note.

J Pharmacol Exp Ther 2020 12 5;375(3):478-487. Epub 2020 Oct 5.

Department of Lung Development and Remodelling, Max Planck Institute for Heart and Lung Research, member of the German Center for Lung Research (DZL), Bad Nauheim, Germany (T.P., E.L., I.M., D.E.S.S., D.M., W.S., R.E.M.); Department of Internal Medicine (Pulmonology), University of Giessen and Marburg Lung Center (UGMLC), member of the German Center for Lung Research (DZL), Giessen, Germany (T.P., E.L., I.M., D.E.S.S., D.M., I.V., S.H., W.S., R.E.M.); Division of Regenerative Medicine, Department of Plastic and Reconstructive Surgery, St. Marianna University School of Medicine, Kawasaki, Japan (H.I.); and Institute of Virology and Cell Biology (H.S., J.B.) and Department of Dermatology (J.B.), University of Lübeck, Lübeck, Germany,

The lysyl hydroxylases (procollagen-lysine 5-dioxygenases) PLOD1, PLOD2, and PLOD3 have been proposed as pathogenic mediators of stunted lung development in bronchopulmonary dysplasia (BPD), a common complication of preterm birth. In affected infants, pulmonary oxygen toxicity stunts lung development. Mice lacking exhibit 15% mortality, and mice lacking or exhibit embryonic lethality. Therefore, to address any pathogenic role of lysyl hydroxylases in stunted lung development associated with BPD, minoxidil was administered to newborn mice in an oxygen toxicity-based BPD animal model. Minoxidil, which has attracted much interest in the management of systemic hypertension and androgenetic alopecia, can also be used to reduce lysyl hydroxylase activity in cultured cells. An in vivo pilot dosing study established 50 mg⋅kg⋅day as the maximum possible minoxidil dose for intraperitoneal administration in newborn mouse pups. When administered at 50 mg⋅kg⋅day to newborn mouse pups, minoxidil was detected in the lungs but did not impact lysine hydroxylation, collagen crosslinking, or lysyl hydroxylase expression in the lungs. Consistent with no impact on mouse lung extracellular matrix structures, minoxidil administration did not alter the course of normal or stunted lung development in newborn mice. At doses of up to 50 mg⋅kg⋅day, pharmacologically active concentrations of minoxidil were not achieved in neonatal mouse lung tissue; thus, minoxidil cannot be used to attenuate lysyl hydroxylase expression or activity during mouse lung development. These data also highlight the need for new and specific lysyl hydroxylase inhibitors. SIGNIFICANCE STATEMENT: Extracellular matrix crosslinking is mediated by lysyl hydroxylases, which generate hydroxylated lysyl residues in procollagen peptides. Deregulated collagen crosslinking is a pathogenic component of a spectrum of diseases, and thus, there is interest in validating lysyl hydroxylases as pathogenic mediators of disease and potential "druggable" targets. Minoxidil, administered at the maximum possible dose, did not inhibit lysyl hydroxylation in newborn mouse lungs, suggesting that minoxidil was unlikely to be of use in studies that pharmacologically target lysyl hydroxylation in vivo.
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http://dx.doi.org/10.1124/jpet.120.000138DOI Listing
December 2020

Multilineage murine stem cells generate complex organoids to model distal lung development and disease.

EMBO J 2020 11 28;39(21):e103476. Epub 2020 Sep 28.

Department of Internal Medicine II and Cardio-Pulmonary Institute (CPI), Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL) and The Institute of Lung Health (ILH), Giessen, Germany.

Organoids derived from mouse and human stem cells have recently emerged as a powerful tool to study organ development and disease. We here established a three-dimensional (3D) murine bronchioalveolar lung organoid (BALO) model that allows clonal expansion and self-organization of FACS-sorted bronchioalveolar stem cells (BASCs) upon co-culture with lung-resident mesenchymal cells. BALOs yield a highly branched 3D structure within 21 days of culture, mimicking the cellular composition of the bronchioalveolar compartment as defined by single-cell RNA sequencing and fluorescence as well as electron microscopic phenotyping. Additionally, BALOs support engraftment and maintenance of the cellular phenotype of injected tissue-resident macrophages. We also demonstrate that BALOs recapitulate lung developmental defects after knockdown of a critical regulatory gene, and permit modeling of viral infection. We conclude that the BALO model enables reconstruction of the epithelial-mesenchymal-myeloid unit of the distal lung, thereby opening numerous new avenues to study lung development, infection, and regenerative processes in vitro.
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http://dx.doi.org/10.15252/embj.2019103476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604576PMC
November 2020

Elevated FiO increases SARS-CoV-2 co-receptor expression in respiratory tract epithelium.

Am J Physiol Lung Cell Mol Physiol 2020 10 2;319(4):L670-L674. Epub 2020 Sep 2.

Department of Lung Development and Remodelling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany.

The severity of coronavirus disease 2019 (COVID-19) is linked to an increasing number of risk factors, including exogenous (environmental) stimuli such as air pollution, nicotine, and cigarette smoke. These three factors increase the expression of angiotensin I converting enzyme 2 (ACE2), a key receptor involved in the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the etiological agent of COVID-19-into respiratory tract epithelial cells. Patients with severe COVID-19 are managed with oxygen support, as are at-risk individuals with chronic lung disease. To date, no study has examined whether an increased fraction of inspired oxygen (FiO) may affect the expression of SARS-CoV-2 entry receptors and co-receptors, including ACE2 and the transmembrane serine proteases TMPRSS1, TMPRSS2, and TMPRSS11D. To address this, steady-state mRNA levels for genes encoding these SARS-CoV-2 receptors were assessed in the lungs of mouse pups chronically exposed to elevated FiO, and in the lungs of preterm-born human infants chronically managed with an elevated FiO. These two scenarios served as models of chronic elevated FiO exposure. Additionally, SARS-CoV-2 receptor expression was assessed in primary human nasal, tracheal, esophageal, bronchial, and alveolar epithelial cells, as well as primary mouse alveolar type II cells exposed to elevated oxygen concentrations. While gene expression of was unaffected, gene and protein expression of was consistently upregulated by exposure to an elevated FiO. These data highlight the need for further studies that examine the relative contribution of the various viral co-receptors on the infection cycle, and point to oxygen supplementation as a potential risk factor for COVID-19.
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http://dx.doi.org/10.1152/ajplung.00345.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7516381PMC
October 2020

Commercially available transfection reagents and negative control siRNA are not inert.

Anal Biochem 2020 10 1;606:113828. Epub 2020 Aug 1.

Department of Lung Development and Remodelling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Member of the German Center for Lung Research (DZL), Parkstrasse 1, 60231, Bad Nauheim, Germany; Department of Internal Medicine (Pulmonology), University of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Justus Liebig University, Aulweg 123, 35392, Giessen, Germany. Electronic address:

The transfection of synthetic small interfering (si)RNA into cultured cells forms the basis of studies that use RNA interference (commonly referred to as "gene knockdown") to study the impact of loss of gene or protein expression on a biological pathway or process. In these studies, mock transfections (with transfection reagents alone), and the use of synthetic negative control (apparently inert) siRNA are both essential negative controls. This report reveals that three widely-used transfection reagents (X-tremeGENE™, HiPerFect, and Lipofectamine® 2000) and five commercially-available control siRNA (from Ambion, Sigma, Santa Cruz, Cell Signaling Technology, and Qiagen) are not inert in cell-culture studies. Both transfection reagents and control siRNA perturbed steady-state mRNA and protein levels in primary mouse lung fibroblasts and in NIH/3T3 cells (a widely-used mouse embryonic fibroblast cell-line), using components of the canonical transforming growth factor-β signaling machinery as a model system. Furthermore, transfection reagents and control siRNA reduced the viability and proliferation of both lung fibroblasts and NIH/3T3 cells. These data collectively provide a cautionary note to investigators to carefully consider the impact of control interventions, such as mock transfections and control siRNA, in RNA interference studies with synthetic siRNA.
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http://dx.doi.org/10.1016/j.ab.2020.113828DOI Listing
October 2020

Impact of litter size on survival, growth and lung alveolarization of newborn mouse pups.

Ann Anat 2020 Nov 18;232:151579. Epub 2020 Jul 18.

Department of Lung Development and Remodelling, Max Planck Institute for Heart and Lung Research, Parkstrasse 1, 60231 Bad Nauheim, Germany; Department of Internal Medicine (Pulmonology), University of Giessen and Marburg Lung Center (UGMLC), Justus Liebig University, Aulweg 123, 35392 Giessen, Germany. Electronic address:

Background: Lung alveolarization, the development of the alveoli, is disturbed in preterm infants with bronchopulmonary dysplasia (BPD), the most common complication of preterm birth. Animal models based on oxygen toxicity to the developing mouse lung are used to understand the mechanisms of stunted alveolarization in BPD, and to develop new medical management strategies for affected infants. The toxicity of genetic and pharmacological interventions, together with maternal cannibalism, reduce mouse litter sizes in experimental studies. The impact of litter size on normal and stunted lung alveolarization is unknown, but may influence data interpretation. The aim of the study was to assess the impact of litter size on normal and oxygen-stunted lung alveolarization in mice.

Methods: BPD was experimentally modelled in newborn C57BL/6J mice by exposure to 85% O in the inspired air for the first 14 days of post-natal life. Perturbations to mouse lung architecture were assessed by design-based stereology, in which the alveolar density, total number of alveoli, gas-exchange surface area, and the septal thickness were estimated.

Results: Litter sizes of a single mouse were not viable to post-natal day 14. Normal lung alveolarization was comparable in mouse pups in litters of 2, 4, 6, and 8 pups per litter. Hyperoxia was equally effective at stunting lung alveolarization in mouse pups in litters of 2, 4, 6, and 8 pups per litter.

Conclusions: Studies on normal lung alveolarization as well as alveolarization stunted by oxygen toxicity can be undertaken in mouse litters as small as two pups, and as large as eight pups. There is no evidence to suggest that data cannot be compared within and between litters of two to eight mouse pups.
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http://dx.doi.org/10.1016/j.aanat.2020.151579DOI Listing
November 2020

Infection in a young immunocompetent male caused by Streptobacillus felis, a putative zoonotic microorganism transmitted by cats.

Clin Infect Dis 2020 Jul 13. Epub 2020 Jul 13.

Hessian State Laboratory, Giessen, Germany.

Rat bite fever (RBF) is predominantly caused by Streptobacillus moniliformis. We report a human infection with Streptobacillus felis. Clinical presentation was consistent with RBF, but serologic testing was negative for S. moniliformis. Eventually, S. felis specific sequences were detected in skin lesions of the patient and in the oropharynx of local cats.
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http://dx.doi.org/10.1093/cid/ciaa968DOI Listing
July 2020

Cyclophilin inhibitors restrict Middle East respiratory syndrome coronavirus interferon-λ and in mice.

Eur Respir J 2020 Nov 26;56(5). Epub 2020 Nov 26.

Dept of Internal Medicine II, University of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Giessen, Germany.

While severe coronavirus infections, including Middle East respiratory syndrome coronavirus (MERS-CoV), cause lung injury with high mortality rates, protective treatment strategies are not approved for clinical use.We elucidated the molecular mechanisms by which the cyclophilin inhibitors cyclosporin A (CsA) and alisporivir (ALV) restrict MERS-CoV to validate their suitability as readily available therapy in MERS-CoV infection.Calu-3 cells and primary human alveolar epithelial cells (hAECs) were infected with MERS-CoV and treated with CsA or ALV or inhibitors targeting cyclophilin inhibitor-regulated molecules including calcineurin, nuclear factor of activated T-cells (NFATs) or mitogen-activated protein kinases. Novel CsA-induced pathways were identified by RNA sequencing and manipulated by gene knockdown or neutralising antibodies. Viral replication was quantified by quantitative real-time PCR and 50% tissue culture infective dose. Data were validated in a murine MERS-CoV infection model.Both CsA and ALV reduced MERS-CoV titres and viral RNA replication in Calu-3 cells and hAECs, improving epithelial integrity. While neither calcineurin nor NFAT inhibition reduced MERS-CoV propagation, blockade of c-Jun N-terminal kinase diminished infectious viral particle release but not RNA accumulation. Importantly, CsA induced interferon regulatory factor 1 (IRF1), a pronounced type III interferon (IFNλ) response and expression of antiviral genes. Downregulation of IRF1 or IFNλ increased MERS-CoV propagation in the presence of CsA. Importantly, oral application of CsA reduced MERS-CoV replication , correlating with elevated lung IFNλ levels and improved outcome.We provide evidence that cyclophilin inhibitors efficiently decrease MERS-CoV replication and upregulation of inflammatory antiviral cell responses, in particular IFNλ. CsA might therefore represent a promising candidate for treating MERS-CoV infection.
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http://dx.doi.org/10.1183/13993003.01826-2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7331652PMC
November 2020

Besnoitia besnoiti-driven endothelial host cell cycle alteration.

Parasitol Res 2020 Aug 17;119(8):2563-2577. Epub 2020 Jun 17.

Institute of Parasitology, Biomedical Research Center Seltersberg, Justus Liebig University, Schubertstr. 81, 35392, Giessen, Germany.

Besnoitia besnoiti is an important obligate intracellular parasite of cattle which primarily infects host endothelial cells of blood vessels during the acute phase of infection. Similar to the closely related parasite Toxoplasma gondii, B. besnoiti has fast proliferating properties leading to rapid host cell lysis within 24-30 h p.i. in vitro. Some apicomplexan parasites were demonstrated to modulate the host cellular cell cycle to successfully perform their intracellular development. As such, we recently demonstrated that T. gondii tachyzoites induce G2/M arrest accompanied by chromosome missegregation, cell spindle alteration, formation of supernumerary centrosomes, and cytokinesis impairment when infecting primary bovine umbilical vein endothelial cells (BUVEC). Here, we follow a comparative approach by using the same host endothelial cell system for B. besnoiti infections. The current data showed that-in terms of host cell cycle modulation-infections of BUVEC by B. besnoiti tachyzoites indeed differ significantly from those by T. gondii. As such, cyclin expression patterns demonstrated a significant upregulation of cyclin E1 in B. besnoiti-infected BUVEC, thereby indicating parasite-driven host cell stasis at G1-to-S phase transition. In line, the mitotic phase of host cell cycle was not influenced since alterations of chromosome segregation, mitotic spindle formation, and cytokinesis were not observed. In contrast to respective T. gondii-related data, we furthermore found a significant upregulation of histone H3 (S10) phosphorylation in B. besnoiti-infected BUVEC, thereby indicating enhanced chromosome condensation to occur in these cells. In line to altered G1/S-transition, we here additionally showed that subcellular abundance of proliferating cell nuclear antigen (PCNA), a marker for G1 and S phase sub-stages, was affected by B. besnoiti since infected cells showed increased nuclear PCNA levels when compared with that of control cells.
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http://dx.doi.org/10.1007/s00436-020-06744-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7366594PMC
August 2020

Evidence for Overlapping and Distinct Biological Activities and Transcriptional Targets Triggered by Fibroblast Growth Factor Receptor 2b Signaling between Mid- and Early Pseudoglandular Stages of Mouse Lung Development.

Cells 2020 05 21;9(5). Epub 2020 May 21.

Key Laboratory of Interventional Pulmonology of Zhejiang Province, Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325035, China.

Branching morphogenesis is the basic developmental mode common to organs such as the lungs that undergo a process of ramification from a rudimentary tree. However, the precise molecular and cellular bases underlying the formation of branching organs are still unclear. As inactivation of fibroblast growth factor receptor 2b (Fgfr2b) signaling during early development leads to lung agenesis, thereby preventing the analysis of this pathway at later developmental stages, we used transgenic mice to induce expression of a soluble form of Fgfr2b to inactivate Fgfr2b ligands at embryonic day (E) 14.5, corresponding to the mid-pseudoglandular stage of lung development. We identified an Fgfr2b signaling signature comprised of 46 genes enriched in the epithelium, some of which were common to, but most of them distinct from, the previously identified Fgfr2b signaling signature at E12.5. Our results indicate that Fgfr2b signaling at E14.5 controls mostly proliferation and alveolar type 2 cell (AT2) differentiation. In addition, inhibition of Fgfr2b signaling at E14.5 leads to morphological and cellular impairment at E18.5, with defective alveolar lineage formation. Further studies will have to be conducted to elucidate the role of Fgfr2b signaling at successive stages (canalicular/saccular/alveolar) of lung development as well as during homeostasis and regeneration and repair after injury.
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http://dx.doi.org/10.3390/cells9051274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290466PMC
May 2020

Hypercapnia Impairs Na,K-ATPase Function by Inducing Endoplasmic Reticulum Retention of the β-Subunit of the Enzyme in Alveolar Epithelial Cells.

Int J Mol Sci 2020 Feb 21;21(4). Epub 2020 Feb 21.

Department of Internal Medicine, Justus Liebig University, Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), 35392 Giessen, Germany.

Alveolar edema, impaired alveolar fluid clearance, and elevated CO levels (hypercapnia) are hallmarks of the acute respiratory distress syndrome (ARDS). This study investigated how hypercapnia affects maturation of the Na,K-ATPase (NKA), a key membrane transporter, and a cell adhesion molecule involved in the resolution of alveolar edema in the endoplasmic reticulum (ER). Exposure of human alveolar epithelial cells to elevated CO concentrations caused a significant retention of NKA-β in the ER and, thus, decreased levels of the transporter in the Golgi apparatus. These effects were associated with a marked reduction of the plasma membrane (PM) abundance of the NKA-α/β complex as well as a decreased total and ouabain-sensitive ATPase activity. Furthermore, our study revealed that the ER-retained NKA-β subunits were only partially assembled with NKA α-subunits, which suggests that hypercapnia modifies the ER folding environment. Moreover, we observed that elevated CO levels decreased intracellular ATP production and increased ER protein and, particularly, NKA-β oxidation. Treatment with α-ketoglutaric acid (α-KG), which is a metabolite that has been shown to increase ATP levels and rescue mitochondrial function in hypercapnia-exposed cells, attenuated the deleterious effects of elevated CO concentrations and restored NKA PM abundance and function. Taken together, our findings provide new insights into the regulation of NKA in alveolar epithelial cells by elevated CO levels, which may lead to the development of new therapeutic approaches for patients with ARDS and hypercapnia.
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http://dx.doi.org/10.3390/ijms21041467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073107PMC
February 2020

Toward a universal flu vaccine.

Science 2020 02;367(6480):852-853

Department of Infectious Diseases and Pulmonary Medicine, Charité-Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, Berlin, Germany.

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http://dx.doi.org/10.1126/science.aba2754DOI Listing
February 2020

Improving the Quality and Reproducibility of Flow Cytometry in the Lung. An Official American Thoracic Society Workshop Report.

Am J Respir Cell Mol Biol 2019 08;61(2):150-161

Defining responses of the structural and immune cells in biologic systems is critically important to understanding disease states and responses to injury. This requires accurate and sensitive methods to define cell types in organ systems. The principal method to delineate the cell populations involved in these processes is flow cytometry. Although researchers increasingly use flow cytometry, technical challenges can affect its accuracy and reproducibility, thus significantly limiting scientific advancements. This challenge is particularly critical to lung immunology, as the lung is readily accessible and therefore used in preclinical and clinical studies to define potential therapeutics. Given the importance of flow cytometry in pulmonary research, the American Thoracic Society convened a working group to highlight issues and technical challenges to the performance of high-quality pulmonary flow cytometry, with a goal of improving its quality and reproducibility.
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http://dx.doi.org/10.1165/rcmb.2019-0191STDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6670040PMC
August 2019

Metformin induces lipogenic differentiation in myofibroblasts to reverse lung fibrosis.

Nat Commun 2019 07 5;10(1):2987. Epub 2019 Jul 5.

Key Laboratory of Interventional Pulmonology of Zhejiang Province, Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Wenzhou Medical University, 325035, Wenzhou, China.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease in which the intricate alveolar network of the lung is progressively replaced by fibrotic scars. Myofibroblasts are the effector cells that excessively deposit extracellular matrix proteins thus compromising lung structure and function. Emerging literature suggests a correlation between fibrosis and metabolic alterations in IPF. In this study, we show that the first-line antidiabetic drug metformin exerts potent antifibrotic effects in the lung by modulating metabolic pathways, inhibiting TGFβ1 action, suppressing collagen formation, activating PPARγ signaling and inducing lipogenic differentiation in lung fibroblasts derived from IPF patients. Using genetic lineage tracing in a murine model of lung fibrosis, we show that metformin alters the fate of myofibroblasts and accelerates fibrosis resolution by inducing myofibroblast-to-lipofibroblast transdifferentiation. Detailed pathway analysis revealed a two-arm mechanism by which metformin accelerates fibrosis resolution. Our data report an antifibrotic role for metformin in the lung, thus warranting further therapeutic evaluation.
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http://dx.doi.org/10.1038/s41467-019-10839-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6611870PMC
July 2019