Background In patients with asthma, respiratory syncytial virus (RSV) infections can cause disease exacerbation by infecting the epithelial layer of the airways, inducing subsequent immune response. The type I interferon antiviral response of epithelial cells upon RSV infection is found to be reduced in asthma in most-but not all-studies. Moreover, the molecular mechanisms causing the differences in the asthmatic bronchial epithelium in response to viral infection are poorly understood. Methods Here, we investigated the transcriptional response to RSV infection of primary bronchial epithelial cells (pBECs) from patients with asthma (n=8) and healthy donors (n=8). The pBECs obtained from bronchial brushes were differentiated in air-liquid interface conditions and infected with RSV. After 3 days, cells were processed for single-cell RNA sequencing. Results A strong antiviral response to RSV was observed for all cell types, for all samples (p<1e-48). Most (1045) differentially regulated genes following RSV infection were found in cells transitioning to secretory cells. Goblet cells from patients with asthma showed lower expression of genes involved in the interferon response (false discovery rate <0.05), including OASL, ICAM1 and TNFAIP3. In multiciliated cells, an impairment of the signalling pathways involved in the response to RSV in asthma was observed. Conclusion Our results highlight that the response to RSV infection of the bronchial epithelium in asthma and healthy airways was largely similar. However, in asthma, the response of goblet and multiciliated cells is impaired, highlighting the need for studying airway epithelial cells at high resolution in the context of asthma exacerbation.

Asthma is a chronic inflammatory disease of the airways characterized by wheezing, dyspnea, and shortness of breath. Asthma is a complex and heterogeneous disease, caused by mechanisms that are not fully known. To better characterize the cellular mechanisms in asthma, we performed single-cell RNA sequencing (scRNA-seq) analysis on bronchial biopsies from patients with asthma and healthy controls. We recruited 29 controls and 26 patients with asthma for bronchoscopy and scRNA-seq analysis. We performed quality control, then integrated and annotated the data using consensus labels from the Human Lung Cell Atlas. We compared composition, transcriptional phenotypes, trajectories of cell-state transitions and cell-cell communication patterns between the airway wall of patients with asthma and controls. Ex vivo cultured primary bronchial epithelial cells (PBECs) were used to further study mechanisms of epithelial differentiation. We find that composition of the airway epithelium and immune cell subsets such as myeloid and T cells is altered in patients with asthma. The airway epithelial changes reflect an altered transition from basal cells into club/goblet and multiciliated cells. Ex vivo PBEC cultures show that these changes are cell-autonomous. Differential gene expression analysis reveals cell-type specific changes in immune and epithelial cells, including a more activated state of innate and adaptive immune cell subsets, and the response of the epithelium to their mediators. In conclusion, we report extensive changes in the airway epithelium and the tissue-resident immune cell subsets in the airways of patients with asthma, including altered cell-cell communication patterns.

ABSTRACTOrgan- and body-scale cell atlases have the potential to transform our understanding of human biology. To capture the variability present in the population, these atlases must include diverse demographics such as age and ethnicity from both healthy and diseased individuals. The growth in both size and number of single-cell datasets, combined with recent advances in computational techniques, for the first time makes it possible to generate such comprehensive large-scale atlases through integration of multiple datasets. Here, we present the integrated Human Lung Cell Atlas (HLCA) combining 46 datasets of the human respiratory system into a single atlas spanning over 2.2 million cells from 444 individuals across health and disease. The HLCA contains a consensus re-annotation of published and newly generated datasets, resolving under- or misannotation of 59% of cells in the original datasets. The HLCA enables recovery of rare cell types, provides consensus marker genes for each cell type, and uncovers gene modules associated with demographic covariates and anatomical location within the respiratory system. To facilitate the use of the HLCA as a reference for single-cell lung research and allow rapid analysis of new data, we provide an interactive web portal to project datasets onto the HLCA. Finally, we demonstrate the value of the HLCA reference for interpreting disease-associated changes. Thus, the HLCA outlines a roadmap for the development and use of organ-scale cell atlases within the Human Cell Atlas.

Rationale: The airway epithelium is altered in asthma, characterized by goblet cells hyperplasia, loss of ciliated cells and increases in basal cells. It is yet unclear whether these changes are a consequence of the disease process, or whether the cells are inherently different. Here we aim to explore differentiation in air-liquid interface (ALI) and 3D organoid cultures of primary bronchial epithelial cells (PBECs) from patients with asthma and matched controls. Methods: Results: The scRNAseq data show the presence of basal and secretory cells in both types of culture models and at all time points. PBECs grown in spheroid cultures show an increase in the proportion of secretory cells over basal cells in time. However, no ciliated cell differentiation is observed, contrary to the PBECs grown in ALI cultures. Directed single-cell fate mapping reveals two differentiation trajectories from suprabasal cells to secretory cells, with PBECs derived from asthma patients being more prone to differentiate towards squamous-like cells. Gene ontology analysis of the genes driving the differentiation towards club cells suggest higher metabolic activity in the healthy PBECs. Conclusion: Taken together, our results suggest that the differentiation process of the airway epithelium is altered in asthma, with cell fate decisions being inherently different from PBECs derived from healthy subjects.

Antiviral therapies are urgently needed to treat and limit the development of severe COVID-19 disease. Ivermectin, a broad-spectrum anti-parasitic agent, has been shown to have anti-SARS-CoV-2 activity in Vero cells at a concentration of 5 μM.

The current COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has an enormous impact on human health and economy. In search for therapeutic options, researchers have proposed resveratrol, a food supplement with known antiviral, anti-inflammatory, and antioxidant properties as an advantageous antiviral therapy for SARS-CoV-2 infection. Here, we provide evidence that both resveratrol and its metabolically more stable structural analog, pterostilbene, exhibit potent antiviral properties against SARS-CoV-2 in vitro. First, we show that resveratrol and pterostilbene antiviral activity in African green monkey kidney cells. Both compounds actively inhibit virus replication within infected cells as reduced virus progeny production was observed when the compound was added at post-inoculation conditions. Without replenishment of the compound, antiviral activity was observed up to roughly five rounds of replication, demonstrating the long-lasting effect of these compounds. Second, as the upper respiratory tract represents the initial site of SARS-CoV-2 replication, we also assessed antiviral activity in air–liquid interface (ALI) cultured human primary bronchial epithelial cells, isolated from healthy volunteers. Resveratrol and pterostilbene showed a strong antiviral effect in these cells up to 48 h post-infection. Collectively, our data indicate that resveratrol and pterostilbene are promising antiviral compounds to inhibit SARS-CoV-2 infection. Because these results represent laboratory findings in cells, we advocate evaluation of these compounds in clinical trials before statements are made whether these drugs are advantageous for COVID-19 treatment.

This protocol enables the dissociation of human bronchial biopsies into a single-cell suspension starting from fresh materials. These single cells are suitable for use in 10x chromium kits for single-cell RNA sequencing.

The current COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has an enormous impact on human health and economy. In search for therapeutic options, researchers have proposed resveratrol, a food supplement with known antiviral, anti-inflammatory and anti-oxidant properties as an advantageous antiviral therapy for SARS-CoV-2 infection. Here, we provide evidence that both resveratrol and its metabolically more stable structural analog, pterostilbene, exhibit potent antiviral properties against SARS-CoV-2 in vitro . Resveratrol and pterostilbene showed antiviral activity in African green monkey kidney cells and in human primary bronchial epithelial cells cultured in an air-liquid interface system. Both compounds actively inhibit virus replication within infected cells as reduced virus progeny production was observed when the compound was added at post-inoculation conditions. Without replenishment of the compound, antiviral activity was observed up to roughly 5 rounds of replication, demonstrating the long-lasting effect of these compounds. Collectively, our data indicate that resveratrol and pterostilbene are promising antiviral compounds to treat SARS-CoV-2 infection. Because these results represent laboratory findings in cells, we advocate evaluation of these compounds in clinical trials before statements are made whether or not these drugs are advantageous for COVID-19 treatment.### Competing Interest StatementThe authors have declared no competing interest.