Soluble ACE2 correlates with severe COVID-19 and can impair antibody responses.

Identifying immune modulators that impact neutralizing antibody responses against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is of great relevance. We postulated that high serum concentrations of soluble angiotensin-converting enzyme 2 (sACE2) might mask the spike and interfere with antibody maturation toward the SARS-CoV-2-receptor-binding motif (RBM). We tested 717 longitudinal samples from 295 COVID-19 patients and showed a 2- to 10-fold increase of enzymatically active sACE2 (a-sACE2), with up to 1 µg/mL total sACE2 in moderate and severe patients. Fifty percent of COVID-19 sera inhibited ACE2 activity, in contrast to 1.3% of healthy donors and 4% of non-COVID-19 pneumonia patients. A mild inverse correlation of a-sACE2 with RBM-directed serum antibodies was observed. In silico, we show that sACE2 concentrations measured in COVID-19 sera can disrupt germinal center formation and inhibit timely production of high-affinity antibodies. We suggest that sACE2 is a biomarker for COVID-19 and that soluble receptors may contribute to immune suppression informing vaccine design.

Comparable CD8+ T-cell responses to SARS-CoV-2 vaccination in single-cell transcriptomics of recently allogeneic transplanted patients and healthy individuals.

Despite extensive research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination responses in healthy individuals, there is comparatively little known beyond antibody titers and T-cell responses in the vulnerable cohort of patients after allogeneic hematopoietic stem cell transplantation (ASCT). In this study, we assessed the serological response and performed longitudinal multimodal analyses including T-cell functionality and single-cell RNA sequencing combined with T cell receptor (TCR)/B cell receptor (BCR) profiling in the context of BNT162b2 vaccination in ASCT patients. In addition, these data were compared to publicly available data sets of healthy vaccinees. Protective antibody titers were achieved in 40% of patients. We identified a distorted B- and T-cell distribution, a reduced TCR diversity, and increased levels of exhaustion marker expression as possible causes for the poorer vaccine response rates in ASCT patients. Immunoglobulin heavy chain gene rearrangement after vaccination proved to be highly variable in ASCT patients. Changes in TCRα and TCRß gene rearrangement after vaccination differed from patterns observed in healthy vaccinees. Crucially, ASCT patients elicited comparable proportions of SARS-CoV-2 vaccine-induced (VI) CD8+ T-cells, characterized by a distinct gene expression pattern that is associated with SARS-CoV-2 specificity in healthy individuals. Our study underlines the impaired immune system and thus the lower vaccine response rates in ASCT patients. However, since protective vaccine responses and VI CD8+ T-cells can be induced in part of ASCT patients, our data advocate early posttransplant vaccination due to the high risk of infection in this vulnerable group.

Partial Resistance to Thyroid Hormone-Induced Tachycardia and Cardiac Hypertrophy in Mice Lacking Thyroid Hormone Receptor ß.

Background: Thyroid hormones regulate cardiac functions mainly through direct actions in the heart and by binding to the thyroid hormone receptor (TR) isoforms α1 and ß. While the role of the most abundantly expressed isoform, TRα1, is widely studied and well characterized, the role of TRß in regulating heart functions is still poorly understood, primarily due to the accompanying elevation of circulating thyroid hormone in TRß knockout mice (TRß-KO). However, their hyperthyroidism is ameliorated at thermoneutrality, which allows studying the role of TRß without this confounding factor. Methods: Here, we noninvasively monitored heart rate in TRß-KO mice over several days using radiotelemetry at different housing temperatures (22°C and 30°C) and upon 3,3',5-triiodothyronine (T3) administration in comparison to wild-type animals. Results: TRß-KO mice displayed normal average heart rate at both 22°C and 30°C with only minor changes in heart rate frequency distribution, which was confirmed by independent electrocardiogram recordings in freely-moving conscious mice. Parasympathetic nerve activity was, however, impaired in TRß-KO mice at 22°C, and only partly rescued at 30°C. As expected, oral treatment with pharmacological doses of T3 at 30°C led to tachycardia in wild-types, accompanied by broader heart rate frequency distribution and increased heart weight. The TRß-KO mice, in contrast, showed blunted tachycardia, as well as resistance to changes in heart rate frequency distribution and heart weight. At the molecular level, these observations were paralleled by a blunted cardiac mRNA induction of several important genes, including the pacemaker channels Hcn2 and Hcn4, as well as Kcna7. Conclusions: The phenotyping of TRß-KO mice conducted at thermoneutrality allows novel insights on the role of TRß in cardiac functions in the absence of the usual confounding hyperthyroidism. Even though TRß is expressed at lower levels than TRα1 in the heart, our findings demonstrate an important role for this isoform in the cardiac response to thyroid hormones.

Prdm16 mutation determines sex-specific cardiac metabolism and identifies two novel cardiac metabolic regulators.

AIMS: Mutation of the PRDM16 gene causes human dilated and non-compaction cardiomyopathy. The PRDM16 protein is a transcriptional regulator that affects cardiac development via Tbx5 and Hand1, thus regulating myocardial structure. The biallelic inactivation of Prdm16 induces severe cardiac dysfunction with post-natal lethality and hypertrophy in mice. The early pathological events that occur upon Prdm16 inactivation have not been explored. METHODS AND RESULTS: This study performed in-depth pathophysiological and molecular analyses of male and female Prdm16csp1/wt mice that carry systemic, monoallelic Prdm16 gene inactivation. We systematically assessed early molecular changes through transcriptomics, proteomics, and metabolomics. Kinetic modelling of cardiac metabolism was performed in silico with CARDIOKIN. Prdm16csp1/wt mice are viable up to 8 months, develop hypoplastic hearts, and diminished systolic performance that is more pronounced in female mice. Prdm16csp1/wt cardiac tissue of both sexes showed reductions in metabolites associated with amino acid as well as glycerol metabolism, glycolysis, and the tricarboxylic acid cycle. Prdm16csp1/wt cardiac tissue revealed diminished glutathione (GSH) and increased inosine monophosphate (IMP) levels indicating oxidative stress and a dysregulated energetics, respectively. An accumulation of triacylglycerides exclusively in male Prdm16csp1/wt hearts suggests a sex-specific metabolic adaptation. Metabolic modelling using CARDIOKIN identified a reduction in fatty acid utilization in males as well as lower glucose utilization in female Prdm16csp1/wt cardiac tissue. On the level of transcripts and protein expression, Prdm16csp1/wt hearts demonstrate an up-regulation of pyridine nucleotide-disulphide oxidoreductase domain 2 (Pyroxd2) and the transcriptional regulator pre-B-cell leukaemia transcription factor interacting protein 1 (Pbxip1). The strongest concordant transcriptional up-regulation was detected for Prdm16 itself, probably through an autoregulatory mechanism. CONCLUSIONS: Monoallelic, global Prdm16 mutation diminishes cardiac performance in Prdm16csp1/wt mice. Metabolic alterations and transcriptional dysregulation in Prdm16csp1/wt affect cardiac tissue. Female Prdm16csp1/wt mice develop a more pronounced phenotype, indicating sexual dimorphism at this early pathological window. This study suggests that metabolic dysregulation is an early event in the PRDM16 associated cardiac pathology.

Correction: State-of-the-art analytical methods of viral infections in human lung organoids.

[This corrects the article DOI: 10.1371/journal.pone.0276115.].

The pleiotropic functions of Pri smORF peptides synchronize leg development regulators.

The last decade witnesses the emergence of the abundant family of smORF peptides, encoded by small ORF (<100 codons), whose biological functions remain largely unexplored. Bioinformatic analyses here identify hundreds of putative smORF peptides expressed in Drosophila imaginal leg discs. Thanks to a functional screen in leg, we found smORF peptides involved in morphogenesis, including the pioneer smORF peptides Pri. Since we identified its target Ubr3 in the epidermis and pri was known to control leg development through poorly understood mechanisms, we investigated the role of Ubr3 in mediating pri function in leg. We found that pri plays several roles during leg development both in patterning and in cell survival. During larval stage, pri activates independently of Ubr3 tarsal transcriptional programs and Notch and EGFR signaling pathways, whereas at larval pupal transition, Pri peptides cooperate with Ubr3 to insure cell survival and leg morphogenesis. Our results highlight Ubr3 dependent and independent functions of Pri peptides and their pleiotropy. Moreover, we reveal that the smORF peptide family is a reservoir of overlooked developmental regulators, displaying distinct molecular functions and orchestrating leg development.

Early-set POMC methylation variability is accompanied by increased risk for obesity and is addressable by MC4R agonist treatment.

Increasing evidence points toward epigenetic variants as a risk factor for developing obesity. We analyzed DNA methylation of the POMC (pro-opiomelanocortin) gene, which is pivotal for satiety regulation. We identified sex-specific and nongenetically determined POMC hypermethylation associated with a 1.4-fold (confidence interval, 1.03 to 2.04) increased individual risk of developing obesity. To investigate the early embryonic establishment of POMC methylation states, we established a human embryonic stem cell (hESC) model. Here, hESCs (WA01) were transferred into a naïve state, which was associated with a reduction of DNA methylation. Naïve hESCs were differentiated via a formative state into POMC-expressing hypothalamic neurons, which was accompanied by re-establishment of DNA methylation patterning. We observed that reduced POMC gene expression was associated with increased POMC methylation in POMC-expressing neurons. On the basis of these findings, we treated POMC-hypermethylated obese individuals (n = 5) with an MC4R agonist and observed a body weight reduction of 4.66 ± 2.16% (means ± SD) over a mean treatment duration of 38.4 ± 26.0 weeks. In summary, we identified an epigenetic obesity risk variant at the POMC gene fulfilling the criteria for a metastable epiallele established in early embryonic development that may be addressable by MC4R agonist treatment to reduce body weight.

CD4+ T cell calibration of antigen-presenting cells optimizes antiviral CD8+ T cell immunity.

Antiviral CD8+ T cell immunity depends on the integration of various contextual cues, but how antigen-presenting cells (APCs) consolidate these signals for decoding by T cells remains unclear. Here, we describe gradual interferon-α/interferon-ß (IFNα/ß)-induced transcriptional adaptations that endow APCs with the capacity to rapidly activate the transcriptional regulators p65, IRF1 and FOS after CD4+ T cell-mediated CD40 stimulation. While these responses operate through broadly used signaling components, they induce a unique set of co-stimulatory molecules and soluble mediators that cannot be elicited by IFNα/ß or CD40 alone. These responses are critical for the acquisition of antiviral CD8+ T cell effector function, and their activity in APCs from individuals infected with severe acute respiratory syndrome coronavirus 2 correlates with milder disease. These observations uncover a sequential integration process whereby APCs rely on CD4+ T cells to select the innate circuits that guide antiviral CD8+ T cell responses.

Single-cell clonal tracking of persistent T-cells in allogeneic hematopoietic stem cell transplantation.

The critical balance between intended and adverse effects in allogeneic hematopoietic stem cell transplantation (alloHSCT) depends on the fate of individual donor T-cells. To this end, we tracked αßT-cell clonotypes during stem cell mobilization treatment with granulocyte-colony stimulating factor (G-CSF) in healthy donors and for six months during immune reconstitution after transfer to transplant recipients. More than 250 αßT-cell clonotypes were tracked from donor to recipient. These clonotypes consisted almost exclusively of CD8+ effector memory T cells (CD8TEM), which exhibited a different transcriptional signature with enhanced effector and cytotoxic functions compared to other CD8TEM. Importantly, these distinct and persisting clonotypes could already be delineated in the donor. We confirmed these phenotypes on the protein level and their potential for selection from the graft. Thus, we identified a transcriptional signature associated with persistence and expansion of donor T-cell clonotypes after alloHSCT that may be exploited for personalized graft manipulation strategies in future studies.

Distinct tissue niches direct lung immunopathology via CCL18 and CCL21 in severe COVID-19.

Prolonged lung pathology has been associated with COVID-19, yet the cellular and molecular mechanisms behind this chronic inflammatory disease are poorly understood. In this study, we combine advanced imaging and spatial transcriptomics to shed light on the local immune response in severe COVID-19. We show that activated adventitial niches are crucial microenvironments contributing to the orchestration of prolonged lung immunopathology. Up-regulation of the chemokines CCL21 and CCL18 associates to endothelial-to-mesenchymal transition and tissue fibrosis within these niches. CCL21 over-expression additionally links to the local accumulation of T cells expressing the cognate receptor CCR7. These T cells are imprinted with an exhausted phenotype and form lymphoid aggregates that can organize in ectopic lymphoid structures. Our work proposes immune-stromal interaction mechanisms promoting a self-sustained and non-resolving local immune response that extends beyond active viral infection and perpetuates tissue remodeling.

State-of-the-art analytical methods of viral infections in human lung organoids.

Human-based organ models can provide strong predictive value to investigate the tropism, virulence, and replication kinetics of viral pathogens. Currently, such models have received widespread attention in the study of SARS-CoV-2 causing the COVID-19 pandemic. Applicable to a large set of organoid models and viruses, we provide a step-by-step work instruction for the infection of human alveolar-like organoids with SARS-CoV-2 in this protocol collection. We also prepared a detailed description on state-of-the-art methodologies to assess the infection impact and the analysis of relevant host factors in organoids. This protocol collection consists of five different sets of protocols. Set 1 describes the protein extraction from human alveolar-like organoids and the determination of protein expression of angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and FURIN as exemplary host factors of SARS-CoV-2. Set 2 provides detailed guidance on the extraction of RNA from human alveolar-like organoids and the subsequent qPCR to quantify the expression level of ACE2, TMPRSS2, and FURIN as host factors of SARS-CoV-2 on the mRNA level. Protocol set 3 contains an in-depth explanation on how to infect human alveolar-like organoids with SARS-CoV-2 and how to quantify the viral replication by plaque assay and viral E gene-based RT-qPCR. Set 4 provides a step-by-step protocol for the isolation of single cells from infected human alveolar-like organoids for further processing in single-cell RNA sequencing or flow cytometry. Set 5 presents a detailed protocol on how to perform the fixation of human alveolar-like organoids and guides through all steps of immunohistochemistry and in situ hybridization to visualize SARS-CoV-2 and its host factors. The infection and all subsequent analytical methods have been successfully validated by biological replications with human alveolar-like organoids based on material from different donors.

Placental Transcriptome Profiling in Subtypes of Diabetic Pregnancies Is Strongly Confounded by Fetal Sex.

The placenta is a temporary organ with a unique structure and function to ensure healthy fetal development. Placental dysfunction is involved in pre-eclampsia (PE), fetal growth restriction, preterm birth, and gestational diabetes mellitus (GDM). A diabetic state affects maternal and fetal health and may lead to functional alterations of placental metabolism, inflammation, hypoxia, and weight, amplifying the fetal stress. The placental molecular adaptations to the diabetic environment and the adaptive spatio-temporal consequences to elevated glucose or insulin are largely unknown (2). We aimed to identify gene expression signatures related to the diabetic placental pathology of placentas from women with diabetes mellitus. Human placenta samples (n = 77) consisting of healthy controls, women with either gestational diabetes mellitus (GDM), type 1 or type 2 diabetes, and women with GDM, type 1 or type 2 diabetes and superimposed PE were collected. Interestingly, gene expression differences quantified by total RNA sequencing were mainly driven by fetal sex rather than clinical diagnosis. Association of the principal components with a full set of clinical patient data identified fetal sex as the single main explanatory variable. Accordingly, placentas complicated by type 1 and type 2 diabetes showed only few differentially expressed genes, while possible effects of GDM and diabetic pregnancy complicated by PE were not identifiable in this cohort. We conclude that fetal sex has a prominent effect on the placental transcriptome, dominating and confounding gene expression signatures resulting from diabetes mellitus in settings of well-controlled diabetic disease. Our results support the notion of placenta as a sexual dimorphic organ.

Transcriptomic comparison of primary human lung cells with lung tissue samples and the human A549 lung cell line highlights cell type specific responses during infections with influenza A virus.

Influenza A virus (IAV) causes pandemics and annual epidemics of severe respiratory infections. A better understanding of the molecular regulation in tissue and cells upon IAV infection is needed to thoroughly understand pathogenesis. We analyzed IAV replication and gene expression induced by IAV strain H3N2 Panama in isolated primary human alveolar epithelial type II cells (AECIIs), the permanent A549 adenocarcinoma cell line, alveolar macrophages (AMs) and explanted human lung tissue by bulk RNA sequencing. Primary AECII exhibit in comparison to AM a broad set of strongly induced genes related to RIG-I and interferon (IFN) signaling. The response of AECII was partly mirrored in A549 cells. In human lung tissue, we observed induction of genes unlike in isolated cells. Viral RNA was used to correlate host cell gene expression changes with viral burden. While relative induction of key genes was similar, gene abundance was highest in AECII cells and AM, while weaker in the human lung (due to less IAV replication) and A549 cells (pointing to their limited suitability as a model). Correlation of host gene induction with viral burden allows a better understanding of the cell-type specific induction of pathways and a possible role of cellular crosstalk requiring intact tissue.

Single-cell transcriptomics reveals common epithelial response patterns in human acute kidney injury.

BACKGROUND: Acute kidney injury (AKI) occurs frequently in critically ill patients and is associated with adverse outcomes. Cellular mechanisms underlying AKI and kidney cell responses to injury remain incompletely understood. METHODS: We performed single-nuclei transcriptomics, bulk transcriptomics, molecular imaging studies, and conventional histology on kidney tissues from 8 individuals with severe AKI (stage 2 or 3 according to Kidney Disease: Improving Global Outcomes (KDIGO) criteria). Specimens were obtained within 1-2 h after individuals had succumbed to critical illness associated with respiratory infections, with 4 of 8 individuals diagnosed with COVID-19. Control kidney tissues were obtained post-mortem or after nephrectomy from individuals without AKI. RESULTS: High-depth single cell-resolved gene expression data of human kidneys affected by AKI revealed enrichment of novel injury-associated cell states within the major cell types of the tubular epithelium, in particular in proximal tubules, thick ascending limbs, and distal convoluted tubules. Four distinct, hierarchically interconnected injured cell states were distinguishable and characterized by transcriptome patterns associated with oxidative stress, hypoxia, interferon response, and epithelial-to-mesenchymal transition, respectively. Transcriptome differences between individuals with AKI were driven primarily by the cell type-specific abundance of these four injury subtypes rather than by private molecular responses. AKI-associated changes in gene expression between individuals with and without COVID-19 were similar. CONCLUSIONS: The study provides an extensive resource of the cell type-specific transcriptomic responses associated with critical illness-associated AKI in humans, highlighting recurrent disease-associated signatures and inter-individual heterogeneity. Personalized molecular disease assessment in human AKI may foster the development of tailored therapies.

C/EBPß regulates lipid metabolism and Pparg isoform 2 expression in alveolar macrophages.

Pulmonary alveolar proteinosis (PAP) is a syndrome characterized by accumulation of surfactant lipoproteins within the lung alveoli. Alveolar macrophages (AMs) are crucial for surfactant clearance, and their differentiation depends on colony-stimulating factor 2 (CSF2), which regulates the establishment of an AM-characteristic gene regulatory network. Here, we report that the transcription factor CCAAT/enhancer binding protein ß (C/EBPß) is essential for the development of the AM identity, as demonstrated by transcriptome and chromatin accessibility analysis. Furthermore, C/EBPß-deficient AMs showed severe defects in proliferation, phagocytosis, and lipid metabolism, collectively resulting in a PAP-like syndrome. Mechanistically, the long C/EBPß protein variants LAP* and LAP together with CSF2 signaling induced the expression of Pparg isoform 2 but not Pparg isoform 1, a molecular regulatory mechanism that was also observed in other CSF2-primed macrophages. These results uncover C/EBPß as a key regulator of AM cell fate and shed light on the molecular networks controlling lipid metabolism in macrophages.

Human alveolar progenitors generate dual lineage bronchioalveolar organoids.

Mechanisms of epithelial renewal in the alveolar compartment remain incompletely understood. To this end, we aimed to characterize alveolar progenitors. Single-cell RNA-sequencing (scRNA-seq) analysis of the HTII-280+/EpCAM+ population from adult human lung revealed subclusters enriched for adult stem cell signature (ASCS) genes. We found that alveolar progenitors in organoid culture in vitro show phenotypic lineage plasticity as they can yield alveolar or bronchial cell-type progeny. The direction of the differentiation is dependent on the presence of the GSK-3ß inhibitor, CHIR99021. By RNA-seq profiling of GSK-3ß knockdown organoids we identified additional candidate target genes of the inhibitor, among others FOXM1 and EGF. This gives evidence of Wnt pathway independent regulatory mechanisms of alveolar specification. Following influenza A virus (IAV) infection organoids showed a similar response as lung tissue explants which confirms their suitability for studies of sequelae of pathogen-host interaction.

A pulmonologist's guide to perform and analyse cross-species single lung cell transcriptomics.

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.

Spermidine reduces neuroinflammation and soluble amyloid beta in an Alzheimer's disease mouse model.

BACKGROUND: Deposition of amyloid beta (Aß) and hyperphosphorylated tau along with glial cell-mediated neuroinflammation are prominent pathogenic hallmarks of Alzheimer's disease (AD). In recent years, impairment of autophagy has been identified as another important feature contributing to AD progression. Therefore, the potential of the autophagy activator spermidine, a small body-endogenous polyamine often used as dietary supplement, was assessed on Aß pathology and glial cell-mediated neuroinflammation. RESULTS: Oral treatment of the amyloid prone AD-like APPPS1 mice with spermidine reduced neurotoxic soluble Aß and decreased AD-associated neuroinflammation. Mechanistically, single nuclei sequencing revealed AD-associated microglia to be the main target of spermidine. This microglia population was characterized by increased AXL levels and expression of genes implicated in cell migration and phagocytosis. A subsequent proteome analysis of isolated microglia confirmed the anti-inflammatory and cytoskeletal effects of spermidine in APPPS1 mice. In primary microglia and astrocytes, spermidine-induced autophagy subsequently affected TLR3- and TLR4-mediated inflammatory processes, phagocytosis of Aß and motility. Interestingly, spermidine regulated the neuroinflammatory response of microglia beyond transcriptional control by interfering with the assembly of the inflammasome. CONCLUSIONS: Our data highlight that the autophagy activator spermidine holds the potential to enhance Aß degradation and to counteract glia-mediated neuroinflammation in AD pathology.

Human lungs show limited permissiveness for SARS-CoV-2 due to scarce ACE2 levels but virus-induced expansion of inflammatory macrophages.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilises the angiotensin-converting enzyme 2 (ACE2) transmembrane peptidase as cellular entry receptor. However, whether SARS-CoV-2 in the alveolar compartment is strictly ACE2-dependent and to what extent virus-induced tissue damage and/or direct immune activation determines early pathogenesis is still elusive. METHODS: Spectral microscopy, single-cell/-nucleus RNA sequencing or ACE2 "gain-of-function" experiments were applied to infected human lung explants and adult stem cell derived human lung organoids to correlate ACE2 and related host factors with SARS-CoV-2 tropism, propagation, virulence and immune activation compared to SARS-CoV, influenza and Middle East respiratory syndrome coronavirus (MERS-CoV). Coronavirus disease 2019 (COVID-19) autopsy material was used to validate ex vivo results. RESULTS: We provide evidence that alveolar ACE2 expression must be considered scarce, thereby limiting SARS-CoV-2 propagation and virus-induced tissue damage in the human alveolus. Instead, ex vivo infected human lungs and COVID-19 autopsy samples showed that alveolar macrophages were frequently positive for SARS-CoV-2. Single-cell/-nucleus transcriptomics further revealed nonproductive virus uptake and a related inflammatory and anti-viral activation, especially in "inflammatory alveolar macrophages", comparable to those induced by SARS-CoV and MERS-CoV, but different from NL63 or influenza virus infection. CONCLUSIONS: Collectively, our findings indicate that severe lung injury in COVID-19 probably results from a macrophage-triggered immune activation rather than direct viral damage of the alveolar compartment.

Venn Diagrams May Indicate Erroneous Statistical Reasoning in Transcriptomics.

A common application of differential expression analysis is finding genes that are differentially expressed upon treatment in only one out of several groups of samples. One of the approaches is to test for significant difference in expression between treatment and control separately in the two groups, and then select genes that show statistical significance in one group only. This approach is then often combined with a gene set enrichment analysis to find pathways and gene sets regulated by treatment in only this group. Here we show that this procedure is statistically incorrect and that the interaction between treatment and group should be tested instead. Moreover, we show that gene set enrichment analysis applied to such incorrectly defined genes group-specific genes may result in misleading artifacts. Due to the presence of false negatives, genes significant in one, but not the other group are enriched in gene sets which correspond to the overall effect of the treatment. Thus, the results appear related to the problem at hand, but do not reflect the group-specific effect of a treatment. A literature search revealed that more than a quarter of papers which used a Venn diagram to illustrate the results of separate differential analysis have also applied this incorrect reasoning.