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Background: Translating findings from animal models to human disease is essential for dissecting disease mechanisms, developing and testing precise therapeutic strategies. The coronavirus disease 2019 (COVID-19) pandemic has highlighted this need, particularly for models showing disease severity-dependent immune responses., Methods: Single-cell transcriptomics (scRNAseq) is well poised to reveal similarities and differences between species at the molecular and cellular level with unprecedented resolution. However, computational methods enabling detailed matching are still scarce. Here, we provide a structured scRNAseq-based approach that we applied to scRNAseq from blood leukocytes originating from humans and hamsters affected with moderate or severe COVID-19., Findings: Integration of data from patients with COVID-19 with two hamster models that develop moderate (Syrian hamster, Mesocricetus auratus) or severe (Roborovski hamster, Phodopus roborovskii) disease revealed that most cellular states are shared across species. A neural network-based analysis using variational autoencoders quantified the overall transcriptomic similarity across species and severity levels, showing highest similarity between neutrophils of Roborovski hamsters and patients with severe COVID-19, while Syrian hamsters better matched patients with moderate disease, particularly in classical monocytes. We further used transcriptome-wide differential expression analysis to identify which disease stages and cell types display strongest transcriptional changes., Interpretation: Consistently, hamsters' response to COVID-19 was most similar to humans in monocytes and neutrophils. Disease-linked pathways found in all species specifically related to interferon response or inhibition of viral replication. Analysis of candidate genes and signatures supported the results. Our structured neural network-supported workflow could be applied to other diseases, allowing better identification of suitable animal models with similar pathomechanisms across species., Funding: This work was supported by German Federal Ministry of Education and Research, (BMBF) grant IDs: 01ZX1304B, 01ZX1604B, 01ZX1906A, 01ZX1906B, 01KI2124, 01IS18026B and German Research Foundation (DFG) grant IDs: 14933180, 431232613., Competing Interests: Declaration of interests MS received funding from Pfizer Inc. for a project related to pneumococcal vaccination. MS receives funding from Owkin for a project not related to this research. MW reports grants and personal fees from Biotest, grants and personal fees from Pantherna, grants and personal fees from Vaxxilon, personal fees from Aptarion, personal fees from Astra Zeneca, personal fees from Chiesi, personal fees from Insmed, personal fees from Gilead, outside the submitted work. GN reports grants from Biotest AG outside the submitted work. Unrelated to this work, Freie Universität Berlin has filed a patent application (PCT/EP2022/051215) for SARS-CoV-2 vaccines. JT is named as inventor on this application and receives remuneration in accordance with German law (“Gesetz über Arbeitnehmererfindungen”). Freie Universität Berlin is collaborating with RocketVax Inc. for further development of SARS-CoV-2 vaccines and receives funding for research. The other authors declare no competing interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

In vitro lung research requires appropriate cell culture models that adequately mimic in vivo structure and function. Previously, researchers extensively used commercially available and easily expandable A549 and NCI-H441 cells, which replicate some but not all features of alveolar epithelial cells. Specifically, these cells are often restricted by terminally altered expression while lacking important alveolar epithelial characteristics. Of late, human primary alveolar epithelial cells (hPAEpCs) have become commercially available but are so far poorly specified. Here, we applied a comprehensive set of technologies to characterize their morphology, surface marker expression, transcriptomic profile, and functional properties. At optimized seeding numbers of 7,500 cells per square centimeter and growth at a gas-liquid interface, hPAEpCs formed regular monolayers with tight junctions and amiloride-sensitive transepithelial ion transport. Electron microscopy revealed lamellar body and microvilli formation characteristic for alveolar type II cells. Protein and single-cell transcriptomic analyses revealed expression of alveolar type I and type II cell markers; yet, transcriptomic data failed to detect NKX2-1, an important transcriptional regulator of alveolar cell differentiation. With increasing passage number, hPAEpCs transdifferentiated toward alveolar-basal intermediates characterized as SFTPC - , KRT8 high , and KRT5 - cells. In spite of marked changes in the transcriptome as a function of passaging, Uniform Manifold Approximation and Projection plots did not reveal major shifts in cell clusters, and epithelial permeability was unaffected. The present work delineates optimized culture conditions, cellular characteristics, and functional properties of commercially available hPAEpCs. hPAEpCs may provide a useful model system for studies on drug delivery, barrier function, and transepithelial ion transport in vitro .

Community-acquired pneumonia remains a major contributor to global communicable disease-mediated mortality. Neutrophils play a leading role in trying to contain bacterial lung infection, but they also drive detrimental pulmonary inflammation, when dysregulated. Here we aimed at understanding the role of microRNA-223 in orchestrating pulmonary inflammation during pneumococcal pneumonia. Serum microRNA-223 was measured in patients with pneumococcal pneumonia and in healthy subjects. Pulmonary inflammation in wild-type and microRNA-223-knockout mice was assessed in terms of disease course, histopathology, cellular recruitment and evaluation of inflammatory protein and gene signatures following pneumococcal infection. Low levels of serum microRNA-223 correlated with increased disease severity in pneumococcal pneumonia patients. Prolonged neutrophilic influx into the lungs and alveolar spaces was detected in pneumococci-infected microRNA-223-knockout mice, possibly accounting for aggravated histopathology and acute lung injury. Expression of microRNA-223 in wild-type mice was induced by pneumococcal infection in a time-dependent manner in whole lungs and lung neutrophils. Single-cell transcriptome analyses of murine lungs revealed a unique profile of antimicrobial and cellular maturation genes that are dysregulated in neutrophils lacking microRNA-223. Taken together, low levels of microRNA-223 in human pneumonia patient serum were associated with increased disease severity, whilst its absence provoked dysregulation of the neutrophil transcriptome in murine pneumococcal pneumonia.

Single-cell ribonucleic acid sequencing is becoming widely employed to study biological processes at a novel resolution depth. The ability to analyse transcriptomes of multiple heterogeneous cell types in parallel is especially valuable for cell-focused lung research where a variety of resident and recruited cells are essential for maintaining organ functionality. We compared the single-cell transcriptomes from publicly available and unpublished datasets of the lungs in six different species: human ( Homo sapiens ), African green monkey ( Chlorocebus sabaeus ), pig ( Sus domesticus ), hamster ( Mesocricetus auratus ), rat ( Rattus norvegicus ) and mouse ( Mus musculus ) by employing RNA velocity and intercellular communication based on ligand-receptor co-expression, among other techniques. Specifically, we demonstrated a workflow for interspecies data integration, applied a single unified gene nomenclature, performed cell-specific clustering and identified marker genes for each species. Overall, integrative approaches combining newly sequenced as well as publicly available datasets could help identify species-specific transcriptomic signatures in both healthy and diseased lung tissue and select appropriate models for future respiratory research., Competing Interests: Conflict of interest: H. Kirsten reports support for the present manuscript from German Federal Ministry of Education and Research (BMBF) grants e:Med CAPSyS (01ZX1304A) and e:Med SYMPATH (01ZX1906B). E. Wyler has received payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from Podcast Gegenblende by DGB, outside the submitted work. M. Landthaler reports support for the present manuscript from Berlin Institute of Health. M. Scholz has received grants or contracts from Pfizer Inc. for a project not related to this research. W.M. Kuebler reports support for the present manuscript from German Research Foundation (KU 1218/9-1, KU 1218/11-1, CRC TR84 A02, CRC TR84 C09, CRC 1449 B01), Germany Ministry for Research and Education (SYMPATH, PROVID consortia), German Center for Cardiovascular Research (Partner site project Berlin) and Berlin Institute of Health (Focus Area Vascular Biology). M. Witzenrath has received grants or contracts from Deutsche Forschungsgemeinschaft, Bundesministerium für Bildung und Forschung, Deutsche Gesellschaft für Pneumologie, European Respiratory Society, Marie Curie Foundation, Else Kröner Fresenius Stiftung, Capnetz Stiftung, International Max Planck Research School, Quark Pharma, Takeda Pharma, Noxxon, Pantherna, Silence Therapeutics, Vaxxilon, Actelion, Bayer Health Care, Biotest and Boehringer Ingelheim, outside the submitted work. M. Witzenrath has received personal fees for consulting from Noxxon, Pantherna, Silence Therapeutics, Vaxxilon, Aptarion, GlaxoSmithKline, Sinoxa and Biotest, and for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from AstraZeneca, Berlin Chemie, Chiesi, Novartis, Teva, Actelion, Boehringer Ingelheim, GlaxoSmithKline, Biotest and Bayer Health Care, outside the submitted work. M. Witzenrath has the following patents planned, issued or pending: EPO 12181535.1: IL-27 for modulation of immune response in acute lung injury (issued: 2012); WO/2010/094491: Means for inhibiting the expression of Ang-2 (issued: 2010); and DE 102020116249.9: Camostat/Niclosamide cotreatment in SARS-CoV-2 infected human lung cells (issued: 2020/21). G. Nouailles receives funding from Biotest AG for a project not related to this work. All other authors have nothing to disclose., (Copyright ©The authors 2022.)