Theodoros Kapellos

-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2 + TMPRSS2 + cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention.

ABSTRACT The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, creates an urgent need for identifying molecular mechanisms that mediate viral entry, propagation, and tissue pathology. Cell membrane bound angiotensin-converting enzyme 2 (ACE2) and associated proteases, transmembrane protease serine 2 (TMPRSS2) and Cathepsin L (CTSL), were previously identified as mediators of SARS-CoV2 cellular entry. Here, we assess the cell type-specific RNA expression of ACE2 , TMPRSS2 , and CTSL through an integrated analysis of 107 single-cell and single-nucleus RNA-Seq studies, including 22 lung and airways datasets (16 unpublished), and 85 datasets from other diverse organs. Joint expression of ACE2 and the accessory proteases identifies specific subsets of respiratory epithelial cells as putative targets of viral infection in the nasal passages, airways, and alveoli. Cells that co-express ACE2 and proteases are also identified in cells from other organs, some of which have been associated with COVID-19 transmission or pathology, including gut enterocytes, corneal epithelial cells, cardiomyocytes, heart pericytes, olfactory sustentacular cells, and renal epithelial cells. Performing the first meta-analyses of scRNA-seq studies, we analyzed 1,176,683 cells from 282 nasal, airway, and lung parenchyma samples from 164 donors spanning fetal, childhood, adult, and elderly age groups, associate increased levels of ACE2 , TMPRSS2 , and CTSL in specific cell types with increasing age, male gender, and smoking, all of which are epidemiologically linked to COVID-19 susceptibility and outcomes. Notably, there was a particularly low expression of ACE2 in the few young pediatric samples in the analysis. Further analysis reveals a gene expression program shared by ACE2 + TMPRSS2 + cells in nasal, lung and gut tissues, including genes that may mediate viral entry, subtend key immune functions, and mediate epithelial-macrophage cross-talk. Amongst these are IL6, its receptor and co-receptor, IL1R , TNF response pathways, and complement genes. Cell type specificity in the lung and airways and smoking effects were conserved in mice. Our analyses suggest that differences in the cell type-specific expression of mediators of SARS-CoV-2 viral entry may be responsible for aspects of COVID-19 epidemiology and clinical course, and point to putative molecular pathways involved in disease susceptibility and pathogenesis.

ABSTRACT Organ- 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.

Infectious respiratory diseases comprise the 4th most fatal group of diseases worldwide. Most studies on COVID-19 lack appropriate comparison to other viral pneumonias with similar severity. Here, we leverage the SCRIPT cohort at NWU Chicago for longitudinal proteomic profiling of bronchoalveolar lavage fluid (BALF) and patient plasma to analyze pathogen-specific differences during disease progression. We used mass spectrometry to analyze BALF and matched plasma from COVID-19 (n=14), bacterial pneumonia (n=8), influenza (n=8) patients, and non-pneumonia controls (n=8) at up to five time points after intubation in the intensive care unit. BALF of COVID-19 patients was specifically enriched in immunoglobulins, blood clotting proteins, and collagens, suggesting increased fibrogenesis and B-cell immunity already at the time of intubation compared to other bacterial or viral types of pneumonia. We employed our recently developed Differential Antigen Capture assay to analyze the binding specificities of plasma antibodies in all patients and identified potential autoantibody responses in COVID-19. Bacterial pneumonia was rich in neutrophil degranulation proteins, whereas influenza samples contained high levels of mucins. Computational deconvolution of the proteomics samples using scRNA-seq-derived marker gene sets predicted increased plasma cell and myofibroblast frequency in COVID-19, and neutrophilia in bacterial pneumonia. Importantly, the differential signatures in BALF were partially conserved in patient plasma. In summary, we identified specific proteomic signatures of COVID-19 compared to influenza and bacteria-driven pneumonia, suggesting increased early onset local B-cell immunity and fibrogenesis in COVID-19.

Cannabinoid receptor 2 (CB2R) is a G protein-coupled receptor (GPCR) expressed in the periphery by immune cells, such as B cells, natural killer (NK) cells, neutrophils, macrophages and T cells. It belongs to the endocannabinoid system which also comprises CB1R, endogenous lipophilic agonists called endocannabinoids and the enzymes responsible for their synthesis and degradation. During inflammatory diseases the endocannabinoid system is dysregulated and increased expression levels of endocannabinoids and cannabinoid receptors are observed. Therefore there is a continuing interest to target the endocannabinoid system to restore homeostasis in patients who suffer from a number of inflammatory diseases, such as multiple sclerosis, atherosclerosis, neuropathic pain and others. However, to date there has been no good anti-inflammatory drug in the market or clinical trials that acts via CB2R. The main aim of this DPhil thesis was to critically assess the role of this receptor in health and inflammation with a particular focus on innate immune cell recruitment. Here I describe my findings regarding the immunophenotype of a CB2R knockout (KO) mouse strain at steady state and under acute systemic inflammation. I showed that CB2R KO mice display a trend in neutrophil accumulation and in monocyte egress in the bone marrow, as well as higher myeloperoxidase activity in the liver. These findings support the view that this cannabinoid receptor plays a pivotal role in innate immune cell trafficking during homeostasis. Furthermore, I discovered that CB2R KO animals display exacerbated immune cell recruitment to peripheral tissues in a model of low dose endotoxemia. In particular, I found more neutrophils in the lungs and spleens of CB2R KO mice at the model’s peak time point. These observations are consistent with a model in which CB2R plays a non-redundant role in the inhibition of neutrophil migration to injured tissues after an inflammatory insult. My <em>in vitro</em> experiments showed that CB2R-selective agonists had no anti-inflammatory effects during macrophage activation. Looking for a mechanistic explanation for this result, I found that the expression levels of the receptor are severely downregulated upon challenge with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) which suggests that it is possible that CB2R is differentially regulated in immune cells during inflammation. Collectively, the data presented in this thesis support the view that CB2R plays an important role in health and disease in pre-clinical models, but further studies are required to make more soluble CB2R-selective agonists and to understand which immune cells we should aim to target therapeutically.