LitCovid-sentences  

PMC:7252096 / 10126-56594 JSONTXT 11 Projects

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Id Subject Object Predicate Lexical cue
T56 0-7 Sentence denotes Results
T57 9-108 Sentence denotes Lung Epithelial Cell Expression of Host Factors Used by SARS-CoV-2 in Non-Human Primates and Humans
T58 109-313 Sentence denotes To investigate which cells within human and NHP tissues represent likely SARS-CoV-2 targets, we analyzed new and existing scRNA-seq datasets to assess which cell types express ACE2, alone or with TMPRSS2.
T59 314-659 Sentence denotes In a previously unpublished dataset consisting of NHP (Macaca mulatta) lung tissue collected after necropsy of healthy adult animals and analyzed by using Seq-Well v1 (Gierahn et al., 2017), we recovered at least 17 distinct major cell types, including various lymphoid, myeloid, and stromal populations (Figures 1 A–1C; Table S1; STAR Methods).
T60 660-832 Sentence denotes ACE2 and TMPRSS2 were primarily expressed in epithelial cells, with 6.7% of type II pneumocytes expressing ACE2 and 3.8% co-expressing ACE2 and TMPRSS2 (Figures 1B and 1C).
T61 833-1158 Sentence denotes Notably, the only double-positive cells observed were classified within the type II pneumocyte population; however, we also identified TMPRSS2 expression within club cells, ciliated epithelial cells, and type I pneumocytes, albeit at diminished abundance and frequency compared with type II pneumocytes (Figure 1C; Table S1).
T62 1159-1248 Sentence denotes Figure 1 Expression of ACE2 in Type II Pneumocytes in Healthy Lungs of Non-human Primates
T63 1249-1499 Sentence denotes (A) Schematic of protocol for isolation of lung tissue at necropsy from healthy non-human primates (M. mulatta, n = 3), creation of scRNA-seq libraries by using Seq-Well v1, and computational analysis to identify cell types by using unbiased methods.
T64 1500-1590 Sentence denotes UMAP projection of 3,793 single cells, points colored by cell identity (see STAR Methods).
T65 1591-1784 Sentence denotes (B) Uniform manifold approximation and projection (UMAP) as in (A), points colored by detection of ACE2 (coronavirus receptor, top) or TMPRSS2 (coronavirus S protein priming for entry, bottom).
T66 1785-1853 Sentence denotes Color coding is as follows: black, RNA positive; blue, RNA negative.
T67 1854-1970 Sentence denotes (C) Dot plot of 2 defining genes for each cell type (Table S1) (Bonferroni-adjusted p < 0.001) and ACE2 and TMPRSS2.
T68 1971-2162 Sentence denotes Dot size represents fraction of cells within that type expressing a given gene, and color intensity represents binned count-based expression amount (log(scaled UMI+1)) among expressing cells.
T69 2163-2324 Sentence denotes ACE2 is enriched in type II pneumocytes (6.7% expressing, Bonferroni-adjusted p = 8.62E−33), as is TMPRSS2 (29.5% expressing, Bonferroni-adjusted p = 8.73E−153).
T70 2325-2397 Sentence denotes Of all type II pneumocytes, 3.8% co-express ACE2 and TMPRSS2 (Table S9).
T71 2398-2475 Sentence denotes Red arrow indicates cell type with largest proportion of ACE2+TMPRSS2+ cells.
T72 2476-2671 Sentence denotes (D) Genes differentially expressed among ACE2+ and ACE2− type II pneumocytes. (SCDE package, FDR-adjusted p < 0.05 for IFNGR2, NT5DC1, ARL6IP1, and TRIM27; full results can be found in Table S1).
T73 2672-2690 Sentence denotes See also Table S1.
T74 2691-2902 Sentence denotes Next, we compared ACE2 + with ACE2 − type II pneumocytes to explore broader gene programs that differentiate putative SARS-CoV-2 target cells from cells of a similar phenotype and ontogeny (Figure 1D; Table S1).
T75 2903-3068 Sentence denotes Among genes significantly upregulated in ACE2 + type II pneumocytes, we observed IFNGR2 (false discovery rate [FDR]-adjusted p = 0.022), a receptor for type II IFNs.
T76 3069-3274 Sentence denotes Notably, previous work has demonstrated limited anti-viral potency of IFN-γ for SARS-associated coronaviruses, compared with that of type I IFNs, at least in vitro (Sainz et al., 2004, Zheng et al., 2004).
T77 3275-3593 Sentence denotes Other co-regulated genes of potential interest include TRIM27 (FDR-adjusted p = 0.025), as well as NT5DC1 (FDR-adjusted p = 0.003) and ARL6IP1 (FDR-adjusted p = 0.047), which were upregulated in the A549 adenocarcinoma alveolar basal epithelial cell line after exposure to IFN-α and IFN-γ for 6 h (Sanda et al., 2006).
T78 3594-3859 Sentence denotes We found IFNAR1 consistently expressed among both ACE2 + type II pneumocytes and ACE2 + TMPRSS2 + co-expressing type II pneumocytes, but its level of upregulation compared with all remaining pneumocytes did not meet statistical significance (FDR-adjusted p = 0.11).
T79 3860-4030 Sentence denotes This analysis finds ACE2 + cells enriched within a rare fraction of secretory cells in NHPs and that ACE2 expression is co-regulated with genes involved in IFN responses.
T80 4031-4277 Sentence denotes To assess whether the findings from NHP lung cells were similarly present in humans, we analyzed a previously unpublished scRNA-seq dataset derived from surgical resections of fibrotic lung tissue collected with Seq-Well S3 (Hughes et al., 2019).
T81 4278-4474 Sentence denotes Unsupervised analysis identified multiple cell types and subtypes of immune cells (Figures 2 A–2C; STAR Methods), as defined by the genes displayed in Figure 2C (full lists available in Table S2).
T82 4475-4658 Sentence denotes Here, we found that ACE2 and TMPRSS2 were primarily expressed within type II pneumocytes and ciliated cells, in line with our analysis of the NHP-derived cells (Figures 1 and 2A, 2B).
T83 4659-4913 Sentence denotes In type II pneumocytes (identified by unique expression of surfactant proteins SFTPC, SFTPB, and SFTPA1), we found 1.4% of cells expressing ACE2 (FDR-adjusted p = 1.35E−21), 34.2% expressing TMPRSS2 (FDR-adjusted p < 1E−300), and 0.8% co-expressing both.
T84 4914-5059 Sentence denotes In ciliated cells, we found 7% were ACE2 + (FDR-adjusted p = 5E−64), 24.6% were TMPRSS2 + (FDR-adjusted p = 3.8E−30), and 5.3% co-expressed both.
T85 5060-5208 Sentence denotes Figure 2 Select Lung Epithelial Cells from Control, HIV-1-Infected, and Mycobacterium-tuberculosis-Infected Human Donors Co-Express ACE2 and TMPRSS2
T86 5209-5422 Sentence denotes (A) Schematic of protocol for isolation of human lung tissue from surgical excess, creation of scRNA-seq libraries by using Seq-Well S3, and computational analysis to identify cell types by using unbiased methods.
T87 5423-5551 Sentence denotes Shown on the right is a UMAP projection of 18,915 cells across 8 donors (n = 3 TB+HIV+; n = 3 TB+; n = 2 non-infected patients).
T88 5552-5631 Sentence denotes Cells represented by points, colored according to cell type (see STAR Methods).
T89 5632-5725 Sentence denotes (B) UMAP projection as in (A), points colored by detection of ACE2 (top) or TMPRSS2 (bottom).
T90 5726-5794 Sentence denotes Color coding is as follows: black, RNA positive; blue, RNA negative.
T91 5795-6086 Sentence denotes (C) Dot plot of 2 defining genes for each cell type (FDR-adjusted p < 0.001), and ACE2 and TMPRSS2; dot size represents fraction of cells within cell type expressing a given gene, and color intensity represents binned count-based expression amount (log(scaled UMI+1)) among expressing cells.
T92 6087-6139 Sentence denotes All cluster-defining genes are provided in Table S2.
T93 6140-6218 Sentence denotes Red arrow indicates cell types with largest proportion of ACE2+TMPRSS2+ cells.
T94 6219-6349 Sentence denotes (D) Volcano plot identifying significantly upregulated genes in ACE2+TMPRSS2+ pneumocytes compared with all remaining pneumocytes.
T95 6350-6434 Sentence denotes Red points represent genes with a FDR-adjusted p < 0.05, and log2(fold change) >1.5.
T96 6435-6508 Sentence denotes Text highlighting specific genes; the full list is available in Table S2.
T97 6509-6610 Sentence denotes (E) Expression of ACE2 across human donors by HIV and TB status (p = 0.009 by likelihood-ratio test).
T98 6611-6629 Sentence denotes See also Table S2.
T99 6630-6896 Sentence denotes As above, to assess for cellular pathways significantly co-expressed within putative target cells for SARS-CoV-2, we computed differentially expressed genes between ACE2 + TMPRSS2 + type II pneumocytes and all other type II pneumocytes (Figures 2C and 2D; Table S2).
T100 6897-7100 Sentence denotes We found significant enrichment of BATF among ACE2 + TMPRSS2 + cells (FDR-adjusted p = 3.25E−7), which has been demonstrated previously to be upregulated by type I and type II IFNs (Murphy et al., 2013).
T101 7101-7345 Sentence denotes Of note, we also observed TRIM28 co-expressed with ACE2 and TMPRSS2 among type II pneumocytes in this dataset (FDR-adjusted p = 2.34E−9), which might play a role in potentiating an IFN response in lung epithelial cells (Krischuns et al., 2018).
T102 7346-7558 Sentence denotes Within this cohort of donors, 3 individuals were human immunodeficiency virus (HIV)+ and diagnosed with active tuberculosis, 3 donors had active tuberculosis and were HIV−, and 2 were negative for both pathogens.
T103 7559-7827 Sentence denotes Surprisingly, we found that all of the ACE2 + cells across all cell types were derived from HIV+ Mycobacterium tuberculosis (Mtb)+ donors despite approximately equivalent recovery of epithelial cell types from all donors (likelihood-ratio test, p = 0.009) (Figure 2E).
T104 7828-8370 Sentence denotes Given limited cell and patient numbers combined with potential sampling biases, we caution that this observation requires much broader cohorts to validate a potential role for co-infections; still, we note our observation is suggestive of a role for chronic IFNs in the induction of ACE2, given that HIV infection is associated with persistent upregulation of ISGs, and we observed elevated amounts of IFNAR2, IFI30, and IKBKB (Utay and Douek, 2016) (FDR-adjusted p = 1.1E−6, 8.8E−9, 1.57E−7, respectively; HIV+ versus HIV− epithelial cells).
T105 8371-8668 Sentence denotes Next, using a previously unpublished scRNA-seq dataset consisting of granuloma and adjacent, uninvolved lung samples from Mtb-infected NHPs (Macaca fascicularis) collected with Seq-Well S3, we identified subsets of epithelial cells expressing ACE2 and TMPRSS2 (Figure S1 ; Table S3; STAR Methods).
T106 8669-8938 Sentence denotes The majority of ACE2 + TMPRSS2 + cells were, once again, type II pneumocytes (22%) and type I pneumocytes (9.7%) and were largely enriched within granulomatous regions compared with those in adjacent uninvolved lung (Figures S1B and S1C) (p = 0.006, Fisher Exact Test).
T107 8939-9112 Sentence denotes ACE2 + TMPRSS2 + type II pneumocytes expressed significantly higher amounts of antimicrobial effectors such as LCN2 compared with remaining type II pneumocytes (Figure S1D).
T108 9113-9382 Sentence denotes Cells with club cell/secretory, type I pneumocyte, and ciliated cell types also contained some ACE2 + TMPRSS2 + cells, but we did not have sufficient power to detect significantly differentially expressed genes between these cells and other cells within those clusters.
T109 9383-9599 Sentence denotes Altogether, we identify ACE2 + TMPRSS2 + cells in lower airways of humans and NHPs with consistent cellular phenotypes and evidence supporting a potential role for IFN-associated inflammation in upregulation of ACE2.
T110 9600-9682 Sentence denotes Figure S1 NHP Tuberculosis Infected Lung and Granuloma, Related to Figures 1 and 2
T111 9683-9687 Sentence denotes (A).
T112 9688-9819 Sentence denotes UMAP projection of epithelial cells (1,099 cells) colored by annotated cell type, tissue source, and gating as ACE2+TMPRSS2+ cells.
T113 9820-9943 Sentence denotes ACE2+TMPRSS2+ cells comprise 11% of ciliated cells, 16% of club cells, 10% type I pneumocytes, and 22% type II pneumocytes.
T114 9944-9988 Sentence denotes Data generated using Seq-Well S3 (Table S3).
T115 9989-9993 Sentence denotes (B).
T116 9994-10117 Sentence denotes Number of cells (left) and % (right) ACE2+TMPRSS2+ cells by tissue source (granuloma versus uninvolved lung) and cell type.
T117 10118-10273 Sentence denotes Ciliated cells and club cells were omitted from this analysis as we detected too few cells (< 7 total cells) belonging to these clusters in the granulomas.
T118 10274-10340 Sentence denotes Statistical significance assessed by Fisher Exact Test (Table S3).
T119 10341-10345 Sentence denotes (C).
T120 10346-10436 Sentence denotes Dot plot of top cluster defining genes for each epithelial cell type and ACE2 and TMPRSS2.
T121 10437-10611 Sentence denotes Dot size represents fraction of cells expressing, and color intensity represents average log(normalized UMI + 1) among all cells in each group scaled between 0 and 1 by gene.
T122 10612-10765 Sentence denotes ACE2 expression is enriched in club cells (Bimodal test, Bonferroni-corrected p < 0.001), ciliated cells (p < 0.005), and type I pneumocytes (p < 0.001).
T123 10766-10873 Sentence denotes TMPRSS2 expression is enriched in type I pneumocytes (p < 0.001) and ciliated cells (p < 0.001) (Table S3).
T124 10874-10878 Sentence denotes (D).
T125 10879-11098 Sentence denotes Dot plot of genes differentially expressed between ACE2+TMPRSS2+ epithelial cells versus rest (Bimodal test, Bonferroni-corrected p < 0.01, log fold change > 0.5). (Table S3, c = number of cells, n = number of animals).
T126 11100-11168 Sentence denotes Ileal Absorptive Enterocytes Express Host Factors Used by SARS-CoV-2
T127 11169-11412 Sentence denotes Next, we examined several other tissues for ACE2-expressing cells on the basis of the location of hallmark symptoms of COVID-19, focusing on the gastrointestinal tract due to reports of clinical symptoms and viral shedding (Xiao et al., 2020).
T128 11413-11709 Sentence denotes Leveraging a previously unpublished scRNA-seq atlas of NHP (M. mulatta) tissues collected with Seq-Well v1, we observed that the majority of ACE2 + cells reside in the small intestine, principally within the ileum, jejunum, and, to a lesser extent, the liver and colon (Figure 3 A; STAR Methods).
T129 11710-11946 Sentence denotes Critically, we note that, in this experiment, the dissociation method used on each tissue was optimized to preserve immune cell recovery, and therefore under-sampled stromal and epithelial populations, as well as neurons from the brain.
T130 11947-12238 Sentence denotes Within the ileum, we identified ACE2 + cells as absorptive enterocytes on the basis of specific expression of ACE2 within cells defined by APOA1, SI, FABP6, and ENPEP, among others, by a likelihood-ratio test (Figures 3B and 3C) (p < 1E−300, 62% of all absorptive enterocytes; see Table S4).
T131 12239-12383 Sentence denotes All other epithelial subtypes expressed ACE2 to a lesser extent, and variably co-expressed ACE2 with TMPRSS2 (see Table S4 for full statistics).
T132 12384-12463 Sentence denotes Figure 3 NHP and Human Ileal Absorptive Enterocytes Co-Express ACE2 and TMPRSS2
T133 12464-12553 Sentence denotes (A) Expression ACE2 across diverse tissues in healthy NHPs (n = 3 animals; 52,858 cells).
T134 12554-12736 Sentence denotes (B) Schematic of protocol for isolation of NHP ileum (n = 5) at necropsy for scRNA-seq using Seq-Well v1, and computational pipeline to identify cell types by using unbiased methods.
T135 12737-12813 Sentence denotes Shown on the right is a UMAP projection of 4,515 cells colored by cell type.
T136 12814-12889 Sentence denotes (C) Dot plot of 2 defining genes for each cell type, with ACE2 and TMPRSS2.
T137 12890-13082 Sentence denotes Dot size represents fraction of cells within cell type expressing a given gene, and color intensity represents binned count-based expression amounts (log(scaled UMI+1)) among expressing cells.
T138 13083-13135 Sentence denotes All cluster defining genes are provided in Table S4.
T139 13136-13213 Sentence denotes Red arrow indicates cell type with largest proportion of ACE2+TMPRSS2+ cells.
T140 13214-13339 Sentence denotes (D) Schematic of protocol for isolation of human ileal cells from endoscopic pinch biopsies in non-inflamed regions (n = 13).
T141 13340-13500 Sentence denotes Shown on the right is a tSNE plot of 13,689 epithelial cells selected from original dataset generated by 10x 3′ v2 (see Figure S2), colored by cellular subsets.
T142 13501-13505 Sentence denotes (E).
T143 13506-13577 Sentence denotes Dot plot of 2 defining genes for each cell type, with ACE2 and TMPRSS2.
T144 13578-13770 Sentence denotes Dot size represents fraction of cells within cell type expressing a given gene, and color intensity represents binned count-based expression amounts (log(scaled UMI+1)) among expressing cells.
T145 13771-13823 Sentence denotes All cluster defining genes are provided in Table S5.
T146 13824-13901 Sentence denotes Red arrow indicates cell type with largest proportion of ACE2+TMPRSS2+ cells.
T147 13902-13906 Sentence denotes (F).
T148 13907-14000 Sentence denotes Expression of ACE2 (left) and TMPRSS2 (right) among all epithelial subsets from human donors.
T149 14001-14041 Sentence denotes See also Figure S2 and Tables S4 and S5.
T150 14042-14180 Sentence denotes Persistent viral RNA in rectal swabs has been detected in pediatric infection, even after negative nasopharyngeal tests (Xu et al., 2020).
T151 14181-14565 Sentence denotes In an additional dataset consisting of endoscopic biopsies from the terminal ileum of a human pediatric cohort (n = 13 donors, ranging in age from 10 to 18 years old), collected with 10X 3′ v2, we confirmed a large abundance of ACE2 + cells with selective expression within absorptive enterocytes (29.7% ACE2 +, FDR-adjusted p = 2.46E−100) (Figures 3D and 3E; Table S5; STAR Methods).
T152 14566-14691 Sentence denotes Furthermore, we identified a subset (888 cells, ∼6.5% of all epithelial cells) that co-express both genes (Figures S2 A–S2C).
T153 14692-15041 Sentence denotes We performed differential expression testing and GO-term enrichment using these cells relative to matched non-expressers to highlight putative biological functions enriched within them, such as metabolic processes and catalytic activity, and to identify shared phenotypes of ACE2 + TMPRSS2 + ileal cells across both human and NHP cohorts (Table S5).
T154 15042-15191 Sentence denotes We speculate that viral targeting of these cells, taken from patients without overt clinical viral infection, might help explain intestinal symptoms.
T155 15192-15473 Sentence denotes Finally, we compared ileal absorptive enterocytes from healthy NHPs and NHPs infected with simian-human immunodeficiency virus (SHIV) and then treated for 6 months with anti-retroviral therapy (animal and infection characteristics published in Colonna et al., 2018) (STAR Methods).
T156 15474-15778 Sentence denotes We found significant upregulation of ACE2, STAT1, and IFI6 within the absorptive enterocytes of SHIV-infected animals (which maintain chronically elevated amounts of IFNs and ISGs) compared with those of uninfected controls (FDR-adjusted p < 2E-7) (Figure S2D) (Deeks et al., 2017, Utay and Douek, 2016).
T157 15779-15829 Sentence denotes Figure S2 Human and NHP Ileum, Related to Figure 3
T158 15830-15834 Sentence denotes (A).
T159 15835-15970 Sentence denotes Top: tSNE projection of all cells from healthy pediatric human ileum within a previously-unpublished 10x 3′ v2 dataset (115,569 cells).
T160 15971-16028 Sentence denotes Black: higher expression of ACE2 (left), TMPRSS2 (right).
T161 16029-16036 Sentence denotes Bottom:
T162 16037-16121 Sentence denotes Corresponding violin plots of expression values for ACE2 (left) and TMPRSS2 (right).
T163 16122-16151 Sentence denotes Solid line: epithelial cells.
T164 16152-16156 Sentence denotes (B).
T165 16157-16217 Sentence denotes Co-expression of ACE2 and TMPRSS2 by epithelial cell subset.
T166 16218-16275 Sentence denotes Number indicates % of ACE2+TMPRSS2+ cells by cell subset.
T167 16276-16389 Sentence denotes (C). tSNE projection of 13,689 cells as in Figure 3D, cells colored by co-expression of ACE2 and TMPRSS2 (black).
T168 16390-16394 Sentence denotes (D).
T169 16395-16565 Sentence denotes Expression of ACE2 and canonical interferon-responsive genes among absorptive enterocytes from Healthy (n = 2) and SHIV-infected, anti-retroviral treated animals (n = 3).
T170 16566-16621 Sentence denotes Bonferroni-adjusted p-values by Wilcoxon test (healthy:
T171 16622-16647 Sentence denotes 510 cells, SHIV-infected:
T172 16648-16659 Sentence denotes 636 cells).
T173 16661-16719 Sentence denotes Upper Airway Expression of Host Factors Used by SARS-CoV-2
T174 16720-17180 Sentence denotes To identify potential viral target cells in nasal and sinus tissue, two regions that are frequently primary sites of exposure for coronaviruses, we analyzed existing scRNA-seq datasets from the human upper airway (inferior turbinate and ethmoid sinus mucosa) across a spectrum of healthy donors and individuals with allergic inflammation due to chronic rhinosinusitis (CRS) collected with Seq-Well v1 (Figure 4 A; STAR Methods) (Ordovas-Montanes et al., 2018).
T175 17181-17535 Sentence denotes We had previously noted a significantly enriched IFN-dominated gene signature in inferior turbinate secretory epithelial cells from both healthy and CRS donors compared with CRS samples from the ethmoid sinus, which were significantly enriched for interleukin-4 (IL-4)/IL-13 gene signatures (Giovannini-Chami et al., 2012, Ordovas-Montanes et al., 2018).
T176 17536-17775 Sentence denotes We speculate that these cells, taken from clinically non-virally infected patients, yet constantly exposed to environmental viruses, might provide one of the earliest locations for coronaviruses to infect before spreading to other tissues.
T177 17776-18072 Sentence denotes We observed significant enrichment of ACE2 expression in apical epithelial cells and, to a lesser extent, ciliated cells compared with all cell types recovered from surgically resected mucosa (1% of apical epithelial cells, FDR-adjusted p = 4.55E−6, n.s. in ciliated cells) (Figure 4B; Table S6).
T178 18073-18196 Sentence denotes Figure 4 Healthy and Allergic Inflamed Human Nasal Mucosa Co-Express ACE2 and TMPRSS2 in a Subset of Goblet Secretory Cells
T179 18197-18363 Sentence denotes (A) Schematic for sampling of n = 12 ethmoid sinus surgical samples and n = 9 inferior turbinate nasal scrapings to generate scRNA-seq libraries by using Seq-Well v1.
T180 18364-18400 Sentence denotes See Ordovas-Montanes et al., (2018).
T181 18401-18520 Sentence denotes (B) Dot plot of all cell types from ethmoid-sinus-derived cells (n = 6 non-polyp CRS samples, n = 6 polyp CRS samples).
T182 18521-18628 Sentence denotes Two defining genes for each cell type, in addition to CDHR3 (rhinovirus receptor), ACE2, TMPRSS2, and JAK1.
T183 18629-18861 Sentence denotes Dot size represents fraction of cells within that type expressing a given gene, and color intensity represents binned count-based expression amounts (log(scaled UMI+1)) among expressing cells (see Table S6 for statistics by subset).
T184 18862-18940 Sentence denotes Red arrow indicates cell types with largest proportion of ACE2+TMPRSS2+ cells.
T185 18941-19443 Sentence denotes (C) Dot plot for 2 defining genes for each cell type identified from granular clustering of epithelial cells (18,325 single cells) derived from both ethmoid sinus and inferior turbinate sampling (healthy inferior turbinate [3,681 cells; n = 3 samples], polyp-bearing patient inferior turbinate [1,370 cells; n = 4 samples], non-polyp ethmoid sinus surgical samples [5,928 cells; n = 6 samples], and polyp surgical and scraping samples directly from polyp in ethmoid sinus [7,346 cells; n = 8 samples]).
T186 19444-19521 Sentence denotes Red arrow indicates cell type with largest proportion of ACE2+TMPRSS2+ cells.
T187 19522-19631 Sentence denotes (D) tSNE of 18,325 single epithelial cells from inferior turbinate and ethmoid sinus (omitting immune cells).
T188 19632-19749 Sentence denotes Colored by cell types 3,152 basal, 3,089 differentiating, 8,840 secretory, 1,105 ciliated, and 2,139 glandular cells.
T189 19750-19855 Sentence denotes (E) tSNE as in (D), identifying epithelial cells co-expressing ACE2 and TMPRSS2 (30 cells, black points).
T190 19856-19942 Sentence denotes (F) tSNE as in (D), colored by detailed cell types with higher granularity, as in (C).
T191 19943-20108 Sentence denotes (G) Individual differentially expressed genes between ACE2+TMPRSS2+ cells and all other secretory epithelial cells (see Table S6 for full gene list with statistics).
T192 20109-20184 Sentence denotes Bonferroni-adjusted likelihood-ratio test p < 0.02 for all genes displayed.
T193 20185-20452 Sentence denotes (H) Stacked bar plot of each subset of epithelial cells among all epithelial cells by donor (each bar) and sampling location (noted below graph) (unpaired t test p < 0.00035 for Secretory Goblet 7 inferior turbinate versus ethmoid sinus; see Table S6 for raw values).
T194 20453-20811 Sentence denotes (I) Violin plot of cell clusters in respiratory epithelial cells (from Figures 4C and 4F) ordered by average expression of IFN-α-induced gene signatures, presented as a gene module score; non-normal distribution by Lilliefors test, Mann-Whitney U-test p = 2.2E−16, 1.21 effect size, IFN-α signature for Secretory Goblet Cluster 7 versus all epithelial cells.
T195 20812-20876 Sentence denotes Arrow indicates cluster containing majority ACE2+TMPRSS2+ cells.
T196 20877-20909 Sentence denotes See also Figure S3 and Table S6.
T197 20910-21149 Sentence denotes To better map putative SARS-CoV-2 targets among epithelial subsets, we employed a finer-grained clustering method applied to both ethmoid sinus surgical specimens and scrapings from the inferior turbinate and ethmoid sinus (Figures 4C–4F).
T198 21150-21445 Sentence denotes Once again, we observed selective expression of ACE2 within a minority of cell types, with 1.3% of all secretory cells expressing ACE2 (Figure 4C) (FDR-adjusted p = 0.00023), specifically sub-clusters 7 and 13, which represent two varieties of secretory epithelial cell (Figures 4C, 4F, and 4G).
T199 21446-21739 Sentence denotes Cluster 7 secretory cells are marked by S100P, LYPD2, PSCA, CEACAM5, and STEAP4; encompass some MUC5AC goblet cells; and contain the most significantly enriched ACE2 and TMPRSS2 expression (4% express ACE2, FDR-adjusted p = 7.32E−28; 28% express TMPRSS2, FDR-adjusted p = 2.15E−132; Table S6).
T200 21740-22041 Sentence denotes We next explicitly gated cells by their TMPRSS2 and ACE2 expression, identifying a rare subset that co-expresses both, the majority of which fall within the “Secretory Cluster 7” cell type (Figures 4E and 4F) (30 cells, ∼0.3% of all upper airway secretory cells, 1.6% of goblet “Secretory Cluster 7”).
T201 22042-22265 Sentence denotes These findings are aligned with concurrent work by the HCA Lung Biological Network on human nasal scRNA-seq data, which identified nasal secretory cells to be enriched for ACE2 and TMPRSS2 expression (Sungnak et al., 2020).
T202 22266-22574 Sentence denotes Although we identified co-expression of ACE2 and TMPRSS2 in few airway cells overall, we detected ACE2 and TMPRSS2 single- and double-positive cells in over 20 donors and thus posit that these genes are enriched in secretory cells and are not a product of individual-patient-driven variability (Figure S3 A).
T203 22575-22978 Sentence denotes Inferior turbinate scrapings collected on Seq-Well S3, which increases the resolution of lower-abundance transcripts compared with Seq-Well v1, revealed consistent and specific expression restricted to goblet secretory cells, but at a greater detection frequency in samples from the same donors (Figure S3B) (ACE2 + from 4.7% v1 to 9.8% S3; ACE2 + TMPRSS2 + from 1.9% v1 to 4% S3) (Hughes et al., 2019).
T204 22979-23153 Sentence denotes Using the gated ACE2 + TMPRSS2 + cells, we tested for differentially expressed genes compared to the remaining secretory epithelial cells (full results provided in Table S6).
T205 23154-23349 Sentence denotes Notably, we observed significant upregulation of ADAR, GBP2, OAS1, JAK1, and DUOX2 (FDR adjusted, all p < 0.02) within ACE2 + TMPRSS2 + cells, potentially indicative of IFN signaling (Figure 4G).
T206 23350-23640 Sentence denotes Almost all “Secretory Cluster 7” cells were from inferior turbinate scrapings of healthy and allergically inflamed individuals, few cells were from the ethmoid sinus tissue of patients with chronic rhinosinusitis without nasal polyps, and no cells were detected in polyp tissue (Figure 4H).
T207 23641-23909 Sentence denotes Gene Ontology (GO) analysis of enriched genes in double-positive cells include processes related to intracellular cytoskeleton and macromolecular localization and catabolism, potentially involved in viral particle entry, packaging, and exocytosis (Fung and Liu, 2019).
T208 23910-23970 Sentence denotes Figure S3 Nasal and Sinus Mucosa, Related to Figures 4 and 5
T209 23971-23975 Sentence denotes (A).
T210 23976-24021 Sentence denotes Expression of ACE2 and TMPRSS2 across donors.
T211 24022-24026 Sentence denotes (B).
T212 24027-24119 Sentence denotes Enhanced capture of ACE2 mRNA with second strand synthesis protocol employed in Seq-Well S3.
T213 24120-24169 Sentence denotes Dot size represents fraction of cells expressing.
T214 24170-24174 Sentence denotes (C).
T215 24175-24440 Sentence denotes Cultured human primary basal epithelial cells at confluence were treated with increasing doses (0.1 to 10ng/mL) of IFNα2, IFNγ, IL-4, IL-13, IL-17A, and IL-1B for 12 h and bulk RNA-seq analysis was performed (Replicate experiment using Human Donor 1 as in Figure 5)
T216 24441-24445 Sentence denotes (D).
T217 24446-24487 Sentence denotes ACE2 expression by stimulation condition.
T218 24488-24658 Sentence denotes Wilcoxon test between each cytokine (combined doses) versus rest: IFNα Bonferroni-adjusted p = 4.1E-07; IFNγ Bonferroni-adjusted p = 9.3E-03; all else n.s. ∗∗∗ p < 0.001.
T219 24659-24663 Sentence denotes (E).
T220 24664-24694 Sentence denotes ACE2 expression by IFNα2 dose.
T221 24695-24780 Sentence denotes Bonferroni-corrected t-test compared to 0 ng/mL condition: ∗∗∗ p < 0.001, ∗ p < 0.05.
T222 24781-24785 Sentence denotes (F).
T223 24786-24815 Sentence denotes ACE2 expression by IFNγ dose.
T224 24816-24901 Sentence denotes Bonferroni-corrected t-test compared to 0 ng/mL condition: ∗∗∗ p < 0.001, ∗ p < 0.05.
T225 24902-24906 Sentence denotes (G).
T226 24907-24939 Sentence denotes IFITM1 expression by IFNα2 dose.
T227 24940-25013 Sentence denotes Bonferroni-corrected t-test compared to 0 ng/mL condition: ∗∗∗ p < 0.001.
T228 25014-25018 Sentence denotes (H).
T229 25019-25050 Sentence denotes IFITM1 expression by IFNγ dose.
T230 25051-25124 Sentence denotes Bonferroni-corrected t-test compared to 0 ng/mL condition: ∗∗∗ p < 0.001.
T231 25125-25129 Sentence denotes (I).
T232 25130-25198 Sentence denotes GBP5 expression among cultured human primary basal epithelial cells.
T233 25199-25302 Sentence denotes Wilcoxon test: IFNα versus IFNγ Bonferroni-adjusted p = 2.94E-07; IFNγ Bonferroni-adjusted p = 9.3E-03.
T234 25303-25354 Sentence denotes TP10K: transcripts per 10,000 reads. ∗∗∗ p < 0.001.
T235 25355-25359 Sentence denotes (J).
T236 25360-25390 Sentence denotes GBP5 expression by IFNα2 dose.
T237 25391-25464 Sentence denotes Bonferroni-corrected t-test compared to 0 ng/mL condition: ∗∗∗ p < 0.001.
T238 25465-25469 Sentence denotes (K).
T239 25470-25499 Sentence denotes GBP5 expression by IFNγ dose.
T240 25500-25573 Sentence denotes Bonferroni-corrected t-test compared to 0 ng/mL condition: ∗∗∗ p < 0.001.
T241 25574-25959 Sentence denotes We next utilized IFN-inducible gene sets of relevance to human airway epithelial cells, which we derived from a prior study by performing differential expression on a published dataset where air-liquid interface cultures from primary human nasal epithelial cells were treated with IFN-αA/D, IFN-β1a, IFN-γ, IL-4, or IL-13 (Giovannini-Chami et al., 2012, Ordovas-Montanes et al., 2018).
T242 25960-26223 Sentence denotes Using these gene lists, we scored the human nasal epithelial cells analyzed by scRNA-seq described in Figures 4C and 4F and found significant concomitant upregulation of the IFN-α-stimulated gene set within ACE2 + TMPRSS2 + secretory goblet cluster 7 (Figure 4I).
T243 26225-26311 Sentence denotes Type I Interferon IFN-α Drives ACE2 Expression in Primary Human Nasal Epithelial Cells
T244 26312-26471 Sentence denotes The meta-analysis described above consistently identified an association between ACE2 expression and canonical ISGs or components of the IFN-signaling pathway.
T245 26472-26731 Sentence denotes This prompted us to investigate whether IFNs might play an active role in regulating ACE2 expression levels in specific target cell subsets, thus potentially allowing for a tissue-protective host response or increased viral binding of SARS-CoV-2 through ACE2.
T246 26732-26960 Sentence denotes Our initial literature search indicated that IFN-γ and IL-4 downregulate the SARS-CoV receptor ACE2 in Vero E6 cells (African green monkey kidney epithelial cells [de Lang et al., 2006]), appearing to invalidate this hypothesis.
T247 26961-27159 Sentence denotes Relatedly, in vitro stimulation of A549 cells, a commonly used cell line model for lung epithelia, with IFN-α, IFN-γ, and IFN-α+IFN-γ for 24 h did not identify ACE2 as an ISG (Russell et al., 2018).
T248 27160-27456 Sentence denotes This is potentially explained by recent work that aimed to understand SARS-CoV-2 receptor usage by performing screening studies within cell line models and found that A549 cells did not express ACE2 and therefore represents a poor model to understand regulation of this gene (Letko et al., 2020).
T249 27457-27774 Sentence denotes While conducting experiments to directly test the hypothesis that ACE2 is an ISG, we noted in our own gene lists used for scoring from Ordovas-Montanes et al., 2018 and in a supplementary extended table available from Giovannini-Chami et al., 2012 that ACE2 was in upregulated gene lists after exposure to Type I IFN.
T250 27775-27886 Sentence denotes We directly tested whether IFN-α induces ACE2 in primary human upper airway epithelial cells in greater detail.
T251 27887-28129 Sentence denotes We cultured human primary basal (stem and progenitors) epithelial cells to confluence and treated them with increasing doses (0.1–10 ng/mL) of IFN-α2, IFN-γ, IL-4, IL-13, IL-17A, or IL-1β for 12 h and then performed bulk RNA-seq (Figure S3C).
T252 28130-28476 Sentence denotes Only IFN-α2 and IFN-γ led to upregulation of ACE2 over the time period tested, and compared with all other cytokines, IFN-α2 lead to greater and more significant upregulation over all doses tested (Figure S3D,Wilcoxon test: IFN-α2 FDR-adjusted p = 4.1E−07; IFN-γ p = 9.3E-03,Figures S3E and S3F, all statistical tests compared with 0 ng/mL dose).
T253 28477-28624 Sentence denotes We confirmed substantial and dose-dependent induction of canonical members of the interferon response after IFN-α2 and IFN-γ (Figures S3G and S3H).
T254 28625-28977 Sentence denotes Conversely, we found that IFN-γ, relative to IFN-α2, induced potent upregulation of GBP5, a GTPase-like protein thought to act as a viral restriction factor through inhibiting furin-mediated protease activity, which could limit viral processing from infected cells, whereas IFN-α2 more robustly induced IFITM1 (Figure S3G–S3K) (Braun and Sauter, 2019).
T255 28978-29208 Sentence denotes To further extend and substantiate these findings, as above, we stimulated primary mouse tracheal basal cells, the commonly used human bronchial cell line BEAS-2B, and upper airway basal cells from two human donors (Figure 5 A-D).
T256 29209-29403 Sentence denotes We confirmed appropriate induction of an IFN response in each cell type by performing differential expression testing between untreated cells and IFN-treated cells for each condition (Table S7).
T257 29404-29608 Sentence denotes Within each cell type, stimulation with IFN-α2, IFN-γ, or IFN-β resulted in dose-dependent upregulation of canonical ISGs, including STAT1/Stat1, BST2/Bst2, XAF1/Xaf1, IFI35/Ifi35, MX1/Mx1, and GBP2/Gbp2.
T258 29609-29764 Sentence denotes Notably, Ace2 expression was not robustly induced in basal cells derived from healthy mouse trachea under any interferon stimulation condition (Figure 5A).
T259 29765-30038 Sentence denotes The magnitude of ACE2 upregulation was diminished in BEAS-2B cells compared to that in our original findings in primary human upper airway epithelial cells, but reached statistical significance compared with that of the untreated condition after IFN-γ exposure (Figure 5B).
T260 30039-30348 Sentence denotes In primary basal cells derived from healthy nasal mucosa, we confirmed significant induction of ACE2 after IFN-α2 stimulation and, to a lesser extent, after stimulation with IFN-γ (IFN-α2-stimulated: both Bonferroni-adjusted p < 0.001; IFN-γ-stimulated: both Bonferroni-adjusted p < 0.05) (Figures 5C and 5D).
T261 30349-30544 Sentence denotes Expression of ACE2 was significantly correlated with expression of STAT1 in all human cell types, with a larger effect size and correlation coefficient in primary human basal cells (Figure 5E-H).
T262 30545-30720 Sentence denotes These experiments support a relationship between induction of the canonical IFN response, including key transcription factors and transcriptional regulation of the ACE2 locus.
T263 30721-30960 Sentence denotes Finally, among primary human samples, we confirmed the dose-dependence of ACE2 upregulation after IFN-α2 or IFN-γ treatment and significant induction of ACE2 after IFN-α2 stimulation at concentrations as low as 0.1–0.5 ng/mL (Figure 5I-L).
T264 30961-31056 Sentence denotes Figure 5 ACE2 is an Interferon-Stimulated Gene in Primary Human Barrier Tissue Epithelial Cells
T265 31057-31279 Sentence denotes (A–D) Basal epithelial cells from distinct sources were cultured to confluence and treated with increasing doses (0.1–10 ng/mL) of IFN-α2, IFN-γ, IL-4, IL-17A, and/or IFN-β for 12 h and bulk RNA-seq analysis was performed.
T266 31280-31606 Sentence denotes Expression of ACE2 (human) or Ace2 (mouse) by cell type and stimulation condition. (A) Primary mouse basal cells from tracheal epithelium are shown. (B) BEAS-2B human bronchial cell line is shown. (C) Primary human basal cells from nasal scraping, Donor 1, is shown. (D) Primary human basal cells from nasal scraping, Donor 2.
T267 31607-31771 Sentence denotes Abbreviation is as follows: TP10K, transcripts per 10,000 reads. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05, Bonferroni-corrected t test compared with untreated condition.
T268 31772-32089 Sentence denotes (E–H) Co-expression of STAT1/Stat1 and ACE2/Ace2 by cell type. (E) Primary mouse basal cells from tracheal epithelium are shown. (F) BEAS-2B human bronchial cell line is shown. (G) Primary human basal cells from nasal scraping, Donor 1, are shown. (H) Primary human basal cells from nasal scraping, Donor 2 are shown.
T269 32090-32154 Sentence denotes Abbreviation is as follows: TP10K, transcripts per 10,000 reads.
T270 32155-32220 Sentence denotes Statistical significance assessed by Spearman’s rank correlation.
T271 32221-32636 Sentence denotes (I–L) Expression of ACE2 in primary human basal cells from nasal scrapings across a range of concentrations of IFN-γ or IFN-α2. (I) IFN-α2 dose response in Donor 1 (p < 0.001 by one-way ANOVA) is shown. (J) IFN-γ dose response in Donor 1 (p < 0.01 by one-way ANOVA) is shown. (K) IFN-α2 dose response in Donor 2 (p < 0.001 by one-way ANOVA) is shown. (L) IFN-γ dose response in Donor 2 (p < 0.001 by one-way ANOVA).
T272 32637-32809 Sentence denotes Abbreviation is as follows: TP10K, transcripts per 10,000 reads. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05, Bonferroni-corrected post hoc testing compared with 0 ng/mL condition.
T273 32810-32850 Sentence denotes See also Figures S3 and S4 and Table S7.
T274 32851-33193 Sentence denotes Next, using a publicly available resource (interferome.org) that hosts genomic and transcriptomic data from cells or tissues treated with IFN, we queried ACE2 expression within human and mouse cells, searching for datasets with a log2-fold-change of >1 or < −1 compared with untreated samples, including all IFN types (Rusinova et al., 2013).
T275 33194-33371 Sentence denotes We recovered 21 datasets spanning 8 distinct primary tissues or cell lines with non-trivial changes in ACE2 expression after both type I and type II IFN treatment (Figure S4 A).
T276 33372-33642 Sentence denotes We observed substantial upregulation of ACE2 in primary skin and primary bronchial cells treated with either type I or type II IFN (> 5-fold upregulation compared with that in untreated cells), in strong support of our in vitro data (Figures 5C, 5D, 5G–5L, and S3D–S3F).
T277 33643-34013 Sentence denotes Immune cell types, such as CD4 T cells and macrophages, were noticeably absent from datasets with a significant change in ACE2 expression after IFN stimulation or were even found to downregulate ACE2 (e.g., primary CD4 T cells + type I IFN) (Figure S4A, and in our analysis of scRNA-seq peripheral blood mononuclear cell data from Butler et al., (2018); data not shown).
T278 34014-34112 Sentence denotes Figure S4 Published Studies of Epithelial Cells Following Interferon Treatment Related to Figure 5
T279 34113-34117 Sentence denotes (A).
T280 34118-34258 Sentence denotes Fold change of ACE2 expression among human or mouse datasets following Type I or Type II interferon treatment compared to untreated control.
T281 34259-34336 Sentence denotes Generated from publicly available microarray data curated at interferome.org.
T282 34337-34384 Sentence denotes Includes all studies with abs(fold-change) > 1.
T283 34385-34389 Sentence denotes (B).
T284 34390-34548 Sentence denotes Location of transcription factors binding regions spanning −1500 bp to +500 bp from the transcription start site of ACE2 (human, top) or Ace2 (mouse, bottom).
T285 34549-34657 Sentence denotes Generated from TRANSFAC data using the interferome.org database (Matys et al., 2003, Rusinova et al., 2013).
T286 34658-34887 Sentence denotes Given that the majority of cells robustly upregulating ACE2 were epithelial, this observation potentially explains why previous analyses to define canonical ISGs within immune populations did not identify ACE2 as an induced gene.
T287 34888-35352 Sentence denotes Furthermore, using both Transcription Factor database (TRANSFAC) data hosted by the interferome database, as well as chromatin immunoprecipitation sequencing (ChIP-seq) data (provided by the ENCODE Factorbook repository), we found evidence for STAT1, STAT3, IRF8, and IRF1 binding sites within −1500–500 bp of the transcription start site of ACE2 (all in human studies, Figure S4B) (Gerstein et al., 2012, Matys et al., 2003, Wang et al., 2012, Wang et al., 2013).
T288 35353-35501 Sentence denotes This finding is supportive of our current hypothesis that ACE2 represents a previously unappreciated ISG in epithelial cells within barrier tissues.
T289 35502-35647 Sentence denotes Given minimal upregulation of Ace2 among primary mouse basal cells in vitro, we were curious as to whether Ace2 represented a murine ISG in vivo.
T290 35648-35776 Sentence denotes We treated two mice intranasally with saline and two mice intranasally with 10,000 units of IFN-α (Guerrero-Plata et al., 2005).
T291 35777-35964 Sentence denotes After 12 h, we isolated the nasal mucosa, consisting of both respiratory and olfactory epithelium, with underlying lamina propria, and performed scRNA-seq using Seq-Well S3 (Figure S5 A).
T292 35965-36072 Sentence denotes We collected from both tissue sites because of early reports of anosmia in COVID-19 (Lechien et al., 2020).
T293 36073-36284 Sentence denotes We recovered 11,358 single cells, including epithelial, stromal, neuronal, and immune cell types, generating the largest single-cell atlas of mouse respiratory and olfactory mucosa to date (Figures 6 A and S5B).
T294 36285-36504 Sentence denotes We annotated all 36 clusters, focusing our attention on epithelial cell clusters, given that we noted enrichment for Ace2 and Tmprss2 within epithelial cell subsets, consistent with our human and NHP results (Table S8).
T295 36505-36802 Sentence denotes Specifically, we found Ace2 enriched within olfactory epithelial gland cells, Muc5b + Scgb1c1 + goblet cells, basal epithelial cells, and myofibroblasts/pericytes (Bonferroni-corrected p < 0.01) (Figures 6B and S5B) (Brann et al., 2020, Dear et al., 1991, Montoro et al., 2018, Tepe et al., 2018).
T296 36803-36882 Sentence denotes Notably, Furin was enriched within olfactory epithelial gland cells (Table S8).
T297 36883-36966 Sentence denotes Next, we asked whether a 12 h stimulation with IFN-α would upregulate Ace2 in vivo.
T298 36967-37220 Sentence denotes Focusing on basal epithelial cells, which contain the highest abundance of Ace2 + cells, we found that despite robust upregulation of canonical murine ISGs, Ace2 expression was only slightly elevated after IFN-α treatment (Figures 6C, 6D, S5C, and S5D).
T299 37221-37305 Sentence denotes Figure S5 Mouse Nasal Epithelium Following Interferon-α Exposure Related to Figure 6
T300 37306-37310 Sentence denotes (A).
T301 37311-37419 Sentence denotes Schematic: mice were exposed to 10,000 units of IFN-α or saline by intranasal application (n = 2 per group).
T302 37420-37539 Sentence denotes After 12 h, animals were sacrificed and nasal epithelium was dissected and dissociated for scRNA-seq using Seq-Well S3.
T303 37540-37544 Sentence denotes (B).
T304 37545-37624 Sentence denotes Dot plot of 2 defining genes for each cell type, with Ace2, Tmprss2, and Cdhr3.
T305 37625-37791 Sentence denotes Dot size represents fraction of cells within cell type expressing, and color intensity binned count-based expression level (log(scaled UMI+1)) among expressing cells.
T306 37792-37844 Sentence denotes All cluster defining genes are provided in Table S8.
T307 37845-37907 Sentence denotes Red arrows: cell types with largest proportion of Ace2+ cells.
T308 37908-38005 Sentence denotes Dendrogram (left) by person correlation over differentially expressed genes with Ward clustering.
T309 38006-38010 Sentence denotes (C).
T310 38011-38068 Sentence denotes UMAP of Basal Epithelial Cells (380 cells) across 4 mice.
T311 38069-38075 Sentence denotes Black:
T312 38076-38117 Sentence denotes Saline-treated mouse; red: IFN-α treated.
T313 38118-38122 Sentence denotes (D).
T314 38123-38199 Sentence denotes UMAP of Basal Epithelial Cells as in C, points colored by detection of Ace2.
T315 38200-38346 Sentence denotes Black: RNA positive, blue: RNA negative (6.6% Ace2+, Bonferroni-adjusted p = 1.1E-10 for Basal Epithelial Cell expression versus all other cells).
T316 38347-38351 Sentence denotes (E).
T317 38352-38488 Sentence denotes Schematic: wildtype (WT) and IFNγ-receptor knockout (IFNγR−/−) mice were infected intranasally with murine gamma-herpesvirus-68 (MHV68).
T318 38489-38584 Sentence denotes Cells from whole lung were digested for scRNA-seq using Drop-seq (yielding 5,558 Epcam+ cells).
T319 38585-38589 Sentence denotes (F).
T320 38590-38654 Sentence denotes Expression of Ace2 by epithelial cell type, wild type (WT) mice.
T321 38655-38733 Sentence denotes Statistical significance by Wilcoxon rank sum test with Bonferroni correction.
T322 38734-38738 Sentence denotes (G).
T323 38739-38822 Sentence denotes Expression of Ace2 among type II pneumocytes binned by infection status in WT mice.
T324 38823-38901 Sentence denotes All pairwise comparisons non-significant (p > 0.05) by Wilcoxon rank sum test.
T325 38902-38906 Sentence denotes (H).
T326 38907-39060 Sentence denotes Percent of Ace2+ cells by infection condition (uninfected, bystander cells in MHV68-infected mouse, MHV68 RNA+ cells) and mouse genotype (WT, IFNγR −/−).
T327 39061-39072 Sentence denotes Black bars:
T328 39073-39106 Sentence denotes Ace2+ positive cells; white bars:
T329 39107-39119 Sentence denotes Ace2- cells.
T330 39120-39124 Sentence denotes (I).
T331 39125-39279 Sentence denotes Schematic of RNA-Seq data from (Matos et al., 2019) of human lung explants (n = 5 donors) exposed to influenza A virus (IAV, H3N2) at 24 h post infection.
T332 39280-39284 Sentence denotes (J).
T333 39285-39407 Sentence denotes Expression of SFTPC (surfactant protein C, a marker of type II pneumocytes) versus ACE2 among mock-infected lung explants.
T334 39408-39488 Sentence denotes Statistical significance assessed by Pearson’s correlation, r = 0.93, p = 0.021.
T335 39489-39518 Sentence denotes TPM: transcripts per million.
T336 39519-39523 Sentence denotes (K).
T337 39524-39603 Sentence denotes SFTPC expression among matched donors following mock or IAV infection for 24 h.
T338 39604-39671 Sentence denotes Statistical significance assessed by ratio paired t test, p = 0.86.
T339 39672-39676 Sentence denotes (L).
T340 39677-39755 Sentence denotes ACE2 expression among matched donors following mock or IAV infection for 24 h.
T341 39756-39825 Sentence denotes Statistical significance assessed by ratio paired t test, p = 0.0054.
T342 39826-39830 Sentence denotes (M).
T343 39831-39959 Sentence denotes Western blot of fully-differentiated air-liquid interface cultures from bronchial cells derived from 4 human donors with asthma.
T344 39960-40070 Sentence denotes Cells from each donor were treated with 10 ng/mL IFNγ for 24 h, and compared to a matched untreated condition.
T345 40071-40097 Sentence denotes ACE2 protein: AF933 (R&D).
T346 40098-40212 Sentence denotes Fold changes quantified for IFNγ treated versus untreated for each patient donor following normalization to GAPDH.
T347 40213-40368 Sentence denotes Figure 6 In Vivo Administration of Interferons in Mice Does Not Induce Ace2, and ACE2 Is Induced in Goblet Secretory Cells during Human Influenza Infection
T348 40369-40437 Sentence denotes (A) UMAP of 11,358 single cells from mouse nasal epithelium (n = 4).
T349 40438-40535 Sentence denotes (B) UMAP projection as in (A), points colored by detection of Ace2 (SARS-CoV-2 receptor homolog).
T350 40536-40604 Sentence denotes Color coding is as follows: black, RNA positive; blue, RNA negative.
T351 40605-40708 Sentence denotes (C) Percent of Ace2+ cells by treatment condition (n = 4 arrays per condition; n = 2 arrays per mouse).
T352 40709-40803 Sentence denotes Black bars indicate Ace2+ cells; white bars indicate Ace2− cells. p = 0.4 by Student’s t test.
T353 40804-41049 Sentence denotes (D) Heatmap of cell-type-defining genes (Trp63 and Krt17), interferon-induced genes (Irf7, Stat1, Irf9, and Oasl2), and Ace2 among basal epithelial cells, separated by cells derived from saline-treated mice (left) and IFN-α-treated mice (right).
T354 41050-41136 Sentence denotes Statistical significance by likelihood-ratio test with Bonferroni correction is shown.
T355 41137-41208 Sentence denotes A full list of differentially expressed genes can be found in Table S8.
T356 41209-41381 Sentence denotes (E) Schematic for sampling cells derived from nasal washes of n = 18 human donors with and without current influenza A or B infection for Seq-Well v1 (35,840 single cells).
T357 41382-41405 Sentence denotes See Cao et al., (2020).
T358 41406-41498 Sentence denotes (F and G) ACE2 expression among goblet cells (F) and squamous cells (G) by infection status.
T359 41499-41776 Sentence denotes Shown are Healthy Donor cells from influenza-negative donors (white); Bystander Cells from influenza A (IAV)- or influenza B (IBV)-infected donors, no intracellular viral RNA detected (black); Flu Viral RNA+ Cells with detectable intracellular influenza A or B viral RNA (red).
T360 41777-41888 Sentence denotes Statistical significance by Wilcoxon test with Bonferroni correction, n.s. for Bystander versus Flu Viral RNA+.
T361 41889-41929 Sentence denotes See also Figure S5 and Tables S6 and S8.
T362 41930-42132 Sentence denotes This observation was supported by analysis of scRNA-seq data from 5,558 epithelial cells from the lungs of mice 3–6 days after intranasal infection with murine gamma herpesvirus-68 (MHV68) (Figure S5E).
T363 42133-42276 Sentence denotes Here, we found significant enrichment of Ace2 + cells within type II pneumocytes, in line with our data from NHP and human lungs (Figures S5F).
T364 42277-42602 Sentence denotes We did not observe changes in Ace2 expression among viral-transcript-positive cells or “bystander” type II pneumocytes (those without detectable cell-associated viral RNA in MHV68-infected animals), nor did we see significant alterations in Ace2 + cell abundance among MHV68-infected mice lacking IFN-γR (Figure S5G and S5H).
T365 42603-42701 Sentence denotes These observations were in agreement with our in vitro murine basal cell assay (Figure 5A and 5E).
T366 42702-43022 Sentence denotes Finally, we sought to validate our hypothesis that ACE2 is upregulated in human epithelial cells during upper airway viral infections, which are known to induce a robust IFN response (Bailey et al., 2014, Everitt et al., 2012, Iwasaki and Pillai, 2014, Jewell et al., 2010, Russell et al., 2018, Steuerman et al., 2018).
T367 43023-43197 Sentence denotes We re-analyzed a publicly available dataset of RNA-seq from human lung explants isolated after surgical resections that were infected with influenza A virus ex vivo for 24 h.
T368 43198-43411 Sentence denotes Here, we found that ACE2 expression was significantly correlated with that of SFTPC, supporting our hypothesis that ACE2 is expressed within type II pneumocytes (Figures 1C, 2C, S5I, and S5J) (Matos et al., 2019).
T369 43412-43642 Sentence denotes Furthermore, although the abundance of SFTPC was not significantly altered by influenza A virus infection, ACE2 expression was significantly upregulated after viral exposure (p = 0.0054, ratio paired t test) (Figures S5K and S5L).
T370 43643-43716 Sentence denotes This suggests that influenza A virus infection increases ACE2 expression.
T371 43717-43908 Sentence denotes Nevertheless, these population-level analyses are not able to definitively resolve specific cell subsets of relevance, nor whether they are directly infected cells or bystanders of infection.
T372 43909-44243 Sentence denotes In order to address these questions, we leveraged an ongoing scRNA-seq study of nasal washes from 18 individuals with confirmed influenza A virus or influenza B virus infection or healthy controls collected with Seq-Well v1, which yielded 35,840 cells resolved into 17 distinct cell types (Figure 6E; STAR Methods) (Cao et al., 2020).
T373 44244-44385 Sentence denotes We investigated the cell types with greatest enrichment for ACE2 and TMPRSS2 in non-infected controls and individuals with influenza A and B.
T374 44386-44570 Sentence denotes Strikingly, ACE2 was most upregulated in samples from influenza-virus-infected individuals within bystander goblet or squamous cells not directly infected by virus (Figures 6F and 6G).
T375 44571-44935 Sentence denotes ACE2 + TMPRSS2 + goblet cells during influenza infection exhibited enrichment for canonical ISGs such as the CXCL9/CXCL10/CXCL11 gene cluster; correspondence with ACE2 + TMPRSS2 + goblet cells in healthy and allergic nasal scrapings; and a shared overlap in ISGs including GBP2, ZNFX1, ADAR, and ACE2 (significantly differentially expressed gene lists) (Table S6).
T376 44936-45181 Sentence denotes Together, our data suggest that ACE2 is an ISG in vitro and in vivo in human primary upper airway epithelial basal cells, but that the murine homolog Ace2 is not in airway epithelial basal cells or pulmonary epithelial cells in vitro or in vivo.
T377 45182-45398 Sentence denotes Collectively, our findings suggest that careful considerations of animal and cellular models will be needed for assessing therapeutic interventions targeting the IFN system when studying ACE2/Ace2-associated biology.
T378 45399-45685 Sentence denotes Finally, because our in vivo and in vitro work indicate that IFN might promote human cellular targets for SARS-CoV-2 infection in the human upper airway by inducing ACE2, we attempted to extend our transcriptomic data on IFN-driven expression of ACE2 to protein-level induction of ACE2.
T379 45686-46002 Sentence denotes As testing of various commercially available polyclonal antibody preparations found broad evidence for non-specific or inconclusive staining in histological immunofluorescent based readouts (data not shown), we assessed whether IFN-γ-stimulated human bronchial air-liquid interface cultures induced ACE2 within 24 h.
T380 46003-46197 Sentence denotes Our results show that cells from one patient robustly induced ACE2 (+2.02x), cells from another mildly induced ACE2 (+1.21x) and two patient’s cells showed minor changes (+/−1.12x) (Figure S5M).
T381 46198-46331 Sentence denotes We provide a note of caution as these cells were derived from asthmatic patients, and the overall changes did not reach significance.
T382 46332-46468 Sentence denotes Furthermore, we could not determine cell surface localization of ACE2 but do note that these results align with our transcriptomic data.