Angiotensin-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.
We report on the quantitative proteomic analysis of single mammalian cells. Fluorescence-activated cell sorting was employed to deposit cells into a newly developed nanodroplet sample processing chip, after which samples were analyzed by ultrasensitive nanoLC-MS. An average of circa 670 protein groups were confidently identified from single HeLa cells, which is a far greater level of proteome coverage for single cells than has been previously reported. We demonstrate that the single-cell proteomics platform can be used to differentiate cell types from enzyme-dissociated human lung primary cells and identify specific protein markers for epithelial and mesenchymal cells.
Respiratory failure associated with COVID-19 has placed focus on the lungs. Here, we present single-nucleus accessible chromatin profiles of 90,980 nuclei and matched single-nucleus transcriptomes of 46,500 nuclei in non-diseased lungs from donors of ~30 weeks gestation,~3 years and ~30 years. We mapped candidate cis-regulatory elements (cCREs) and linked them to putative target genes. We identified distal cCREs with age-increased activity linked to SARS-CoV-2 host entry gene TMPRSS2 in alveolar type 2 cells, which had immune regulatory signatures and harbored variants associated with respiratory traits. At the 3p21.31 COVID-19 risk locus, a candidate variant overlapped a distal cCRE linked to SLC6A20, a gene expressed in alveolar cells and with known functional association with the SARS-CoV-2 receptor ACE2. Our findings provide insight into regulatory logic underlying genes implicated in COVID-19 in individual lung cell types across age. More broadly, these datasets will facilitate interpretation of risk loci for lung diseases.
‘LungGENS’, our previously developed web tool for mapping single-cell gene expression in the developing lung, has been well received by the pulmonary research community. With continued support from the ‘LungMAP’ consortium, we extended the scope of the LungGENS database to accommodate transcriptomics data from pulmonary tissues and cells from human and mouse at different stages of lung development. Lung Gene Expression Analysis (LGEA) web portal is an extended version of LungGENS useful for the analysis, display and interpretation of gene expression patterns obtained from single cells, sorted cell populations and whole lung tissues. The LGEA web portal is freely available at http://research.cchmc.org/pbge/lunggens/mainportal.html.
Although viral infections elicit robust interferon-g (IFN-g) and long-lived antibody-secreting cell (ASC) responses, the roles for IFN-g and IFN-g-induced transcription factors (TFs) in ASC development are unclear. We showed that B cell intrinsic expression of IFN-gR and the IFN-g-induced TF T-bet were required for T-helper 1 cell-induced differentiation of B cells into ASCs. IFN-gR signaling induced Blimp1 expression in B cells but also initiated an inflammatory gene program that, if not restrained, prevented ASC formation. T-bet did not affect Blimp1 upregulation in IFN-g-activated B cells but instead regulated chromatin accessibility within the Ifng and Ifngr2 loci and repressed the IFN-g-induced inflammatory gene program. Consistent with this, B cell intrinsic T-bet was required for formation of long-lived ASCs and secondary ASCs following viral, but not nematode, infection. Therefore, T-bet facilitates differentiation of IFN-g-activated inflammatory effector B cells into ASCs in the setting of IFN-g-, but not IL-4-, induced inflammatory responses.
Immunity to heterosubtypic strains of influenza is thought to be mediated primarily by memory T cells, which recognize epitopes in conserved proteins. However, the involvement of B cells in this process is controversial. We show in this study that influenza-specific memory T cells are insufficient to protect mice against a lethal challenge with a virulent strain of influenza in the absence of B cells. B cells contribute to protection in multiple ways. First, although non-neutralizing Abs by themselves do not provide any protection to challenge infection, they do reduce weight loss, lower viral titers, and promote recovery of mice challenged with a virulent heterosubtypic virus in the presence of memory T cells. Non-neutralizing Abs also facilitate the expansion of responding memory CD8 T cells. Furthermore, in cooperation with memory T cells, naive B cells also promote recovery from infection with a virulent heterosubtypic virus by generating new neutralizing Abs. These data demonstrate that B cells use multiple mechanisms to promote resistance to heterosubtypic strains of influenza and suggest that vaccines that elicit both memory T cells and Abs to conserved epitopes of influenza may be an effective defense against a wide range of influenza serotypes.
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