Journal: Developmental Cell

Article Title: Cigarette Smoke Exposure and Inflammatory Signaling Increase the Expression of the SARS-CoV-2 Receptor ACE2 in the Respiratory Tract

doi: 10.1016/j.devcel.2020.05.012

Figure Lengend Snippet: ACE2 Expression in the Lung Is Uncorrelated with Age or Sex (A) ACE2 expression in the lungs of young mice (<26 weeks old) and old mice (>78 weeks old). (B) ACE2 expression in the lungs of young rats (6 weeks old) and old rats (104 weeks old). (C) ACE2 expression in the lungs of female mice and male mice. (D) ACE2 expression in the lungs of female rats and male rats. (E) ACE2 expression in lungs from the GTEx cohort by age. (F) ACE2 expression in lungs from the GTEx cohort by sex. (G) ACE2 expression in lungs from a cohort of organ donors by age. (H) ACE2 expression in lungs from a cohort of organ donors by sex. (I) ACE2 expression in pathologically normal lung tissue from patients from TCGA by age. (J) ACE2 expression in pathologically normal lung tissue from patients from TCGA by sex. Each panel displays log 2 -normalized ACE2 expression relative to a control group. Data analyzed in (A) and (C) were from GSE132040 . Data analyzed in (B) and (D) were from GSE53960 . Data analyzed in (E) and (F) were from www.gtexportal.org . Data analyzed in (G) and (H) were from GSE1643 . Data analyzed in (I) and (J) were from https://gdac.broadinstitute.org/ . Additional information on the data sources and sample sizes is included in .

Article Snippet: Recombinant Anti-ACE2 antibody [ERP4435(2)] , Abcam , ab108252.

Techniques: Expressing

Journal: Developmental Cell

Article Title: Cigarette Smoke Exposure and Inflammatory Signaling Increase the Expression of the SARS-CoV-2 Receptor ACE2 in the Respiratory Tract

doi: 10.1016/j.devcel.2020.05.012

Figure Lengend Snippet: Cigarette Smoke Increases the Expression of ACE2 in Mouse and Human Lungs (A) ACE2 expression in the lungs of mice that were sham-treated or that were exposed to diluted cigarette smoke for 2, 3, or 4 h a day (low, medium, and high smoke exposure, respectively). (B) A diagram showing the approximate locations of tissue samples used in this analysis. Tracheal, large airway epithelial, and small airway epithelial specimens were collected by fiberoptic bronchoscopy as described in their respective publications. Lung resections were collected surgically from various locations. (C) ACE2 expression in human tracheal epithelia analyzed according to smoking history. (D) ACE2 expression in human large airway epithelia analyzed according to smoking history. (E) ACE2 expression in human small airway epithelia analyzed according to smoking history. (F) A volcano plot comparing gene expression in the respiratory epithelia of current smokers and never smokers from (C)–(E). The dotted lines indicate various p value thresholds (e.g., genes located above the 10th percentile have a combined p value greater than 90% of the genes included in the meta-analysis). The location of ACE2 is indicated with a red star. (G) ACE2 expression in the lungs of a cohort of patients undergoing thoracic surgery analyzed according to the number of pack years each patient smoked. (H) ACE2 expression in the lungs of TCGA patients analyzed according to the number of pack years each patient smoked. (I) ACE2 expression in respiratory epithelia collected by fiberoptic bronchoscopy among either current smokers or former smokers. (J) A volcano plot comparing gene expression between current smokers and former smokers. The dotted lines indicate various p value thresholds (e.g., genes located above the 10 th percentile have a combined p value greater than 90% of the genes included in the meta-analysis). The location of ACE2 is indicated with a red star. Each panel displays log 2 -normalized ACE2 expression relative to a control group. Data analyzed in (A) were from GSE18344 . Data analyzed in (C) were from GSE13933 . Data analyzed in (D) were from GSE22047 . Data analyzed in (E) were from GSE64614 . Data analyzed in (G) were from GSE76925 . Data analyzed in (H) were from https://gdac.broadinstitute.org/ . Data analyzed in (I) were from GSE79209 . Additional information on the data sources and sample sizes are included in . ∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.0005 (Student’s t test).

Article Snippet: Recombinant Anti-ACE2 antibody [ERP4435(2)] , Abcam , ab108252.

Techniques: Expressing

Journal: Developmental Cell

Article Title: Cigarette Smoke Exposure and Inflammatory Signaling Increase the Expression of the SARS-CoV-2 Receptor ACE2 in the Respiratory Tract

doi: 10.1016/j.devcel.2020.05.012

Figure Lengend Snippet: Patient Characteristics and ACE2 Expression

Article Snippet: Recombinant Anti-ACE2 antibody [ERP4435(2)] , Abcam , ab108252.

Techniques:

Journal: Developmental Cell

Article Title: Cigarette Smoke Exposure and Inflammatory Signaling Increase the Expression of the SARS-CoV-2 Receptor ACE2 in the Respiratory Tract

doi: 10.1016/j.devcel.2020.05.012

Figure Lengend Snippet: ACE2 Is Expressed in Secretory Club and Goblet Cells Along with Alveolar Type 2 Cells in the Mammalian Lung (A) T-SNE clustering of cells from the mouse lung. Cells expressing ACE2 are highlighted in the right panel. (B) Cells in the mouse lung that express various lineage markers (TMEM100 for endothelial cells, EPCAM for epithelial cells, PDGFRA for mesenchymal cells, and PTPRC for immune cells) are highlighted. (C) Cells expressing markers for various epithelial lineages are highlighted: RTKN2 for alveolar type 1 cells, LAMP3 for alveolar type 2 cells, FOXJ1 for ciliated cells, GABRP for both goblet and club cells, MUC5AC for goblet cells, and SCGB1A for club cells. (D) A track plot displaying the expression of ACE2 and several lineage-related genes in different cell populations obtained from Leiden clustering. (E) T-SNE clustering of cells from the human lung. Cells expressing ACE2 are highlighted in the right panel. (F) Cells in the human lung that express various lineage markers (TMEM100 for endothelial cells, EPCAM for epithelial cells, PDGFRA for mesenchymal cells, and PTPRC for immune cells) are highlighted. (G) Cells expressing markers for various epithelial lineages are highlighted: LAMP3 for alveolar type 2 cells, FOXJ1 for ciliated cells, TP63 for basal cells, GABRP for both goblet and club cells, MUC5AC for goblet cells, and SCGB1A1 for club cells. (H) A track plot displaying the expression of ACE2 and several lineage-related genes in different cell populations obtained from Leiden clustering. The gene expression data used in (A)–(D) are from GSE121611 . The gene expression data used in (E)–(H) are from GSE122960 . Additional information on the data sources and sample sizes are included in .

Article Snippet: Recombinant Anti-ACE2 antibody [ERP4435(2)] , Abcam , ab108252.

Techniques: Expressing

Journal: Developmental Cell

Article Title: Cigarette Smoke Exposure and Inflammatory Signaling Increase the Expression of the SARS-CoV-2 Receptor ACE2 in the Respiratory Tract

doi: 10.1016/j.devcel.2020.05.012

Figure Lengend Snippet: Cigarette Smoke Causes the Expansion of ACE2 + Secretory Cells (A) T-SNE clustering of the transcriptomes from single cells derived from the airway epithelia of smokers and never smokers. Cells expressing ACE2 are highlighted in the right panel. (B) Cells in the human airway that express various lineage markers (FOXJ1 for ciliated cells, TP63 for basal cells, MUC5AC for goblet cells, and SCGB1A1 for club cells) are highlighted. (C) GO terms enriched among ACE2-correlated transcripts. (D) Dot plots displaying the expression of the top ten differentially expressed marker genes for various airway lineages and for ACE2+ cells. (E) The fraction of cells expressing the indicated marker genes are displayed. FOXJ1 is a marker for ciliated cells, MUC5AC is a marker for goblet cells, and TP63 is a marker for basal cells. (F) The fractions of cells found in each cell type cluster are displayed. (G) The number of counts per ACE2 + cell are displayed. (H) The fraction of ACE2 + cells co-expressing MUC5AC or that are found within the goblet/club cell cluster are displayed. (I) The 100 top-ranked differentially expressed genes from each cluster in (A) as well as the 100 genes most strongly correlated with ACE2 were used to re-analyze the bulk gene expression data from smokers and non-smokers in Figure 2 F. A volcano plot displays the mean expression change of each cell type signature. (J) The same transcriptional signatures as in Figure 4 I were used to re-analyze the data from current smokers and former smokers in Figure 2 J. A volcano plot displays the mean expression change of each cell type signature. (K) ACE2 expression in mouse tracheal explants undergoing mucociliary differentiation. (L) ACE2 expression in human airway epithelial cells undergoing mucociliary differentiation. (M) ACE2 expression in human airway epithelial cells that underwent mucociliary differentiation in the presence of clean air or cigarette smoke. (N) The same transcriptional signatures as in Figure 4 I were used to re-analyze the data from smoke exposure during differentiation from Figure 4 M. A volcano plot displaying the mean expression change of each cell type signature is displayed. Data analyzed in (A)–(H) were from GSE131391 . Data analyzed in (K) were from GSE75715 . Data analyzed in (L) were from GSE39059 . Data analyzed in (M) were from GSE135188 . Additional information on the data sources and sample sizes are included in . In (E), (F), and (H), a chi-square test is applied. In (G), a Mann-Whitney U test is applied. In (K)–(M), a Student’s t test is applied. ∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.0005.

Article Snippet: Recombinant Anti-ACE2 antibody [ERP4435(2)] , Abcam , ab108252.

Techniques: Derivative Assay, Expressing, Marker, MANN-WHITNEY

Journal: Developmental Cell

Article Title: Cigarette Smoke Exposure and Inflammatory Signaling Increase the Expression of the SARS-CoV-2 Receptor ACE2 in the Respiratory Tract

doi: 10.1016/j.devcel.2020.05.012

Figure Lengend Snippet: ACE2 Is an Interferon-Stimulated Gene that Is Upregulated by Viral Infections (A) ACE2 expression in airway epithelial cells that were infected with influenza. (B) ACE2 expression in airway epithelial cells that were infected with respiratory syncytial virus. (C) ACE2 expression in airway epithelial cells that were infected with SARS. (D) ACE2 expression in airway epithelial cells that were infected with MERS. (E) ACE2 expression in airway epithelial cells that were transfected with the dsRNA mimic poly(I:C). (F) ACE2 expression in human tracheal cells that were cultured in the presence of the indicated cytokine for 24 h. (G) ACE2 expression in human small airway epithelial cells that were cultured in the presence of the indicated cytokine for 24 h. (H) ACE2 expression in airway epithelial cells that were cultured in the presence of IFN-β. Each panel displays log 2 -normalized ACE2 expression relative to a control group. Data analyzed in (A) and (B) were from GSE32138 . Data analyzed in (C) were from GSE47963 . Data analyzed in (D) were from GSE100504 . Data analyzed in (E) were from GSE51392 . Data analyzed in (H) were from GSE19392 . Additional information on the data sources and sample sizes are included in . ∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.0005 (Student’s t test).

Article Snippet: Recombinant Anti-ACE2 antibody [ERP4435(2)] , Abcam , ab108252.

Techniques: Expressing, Infection, Transfection, Cell Culture

Journal: Developmental Cell

Article Title: Cigarette Smoke Exposure and Inflammatory Signaling Increase the Expression of the SARS-CoV-2 Receptor ACE2 in the Respiratory Tract

doi: 10.1016/j.devcel.2020.05.012

Figure Lengend Snippet:

Article Snippet: Recombinant Anti-ACE2 antibody [ERP4435(2)] , Abcam , ab108252.

Techniques: Recombinant, Software

Journal: bioRxiv

Article Title: SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike-ACE2 receptor interaction

doi: 10.1101/2022.01.11.475947

Figure Lengend Snippet: Microglia signature markers, P2RY12 and TMEM119 (in green) and cells nuclei (in blue) assessed by immunofluorescence staining, and representative western blot are presented in panel ( A-B ), respectively. Growth kinetics of SARS-CoV-2 (D614) on MDMi, mouse microglia (mMi), Vero E6 and Caco2 cells in ( C ). Relative expression of ACE2 in MDMi by qPCR compared to Vero E6 and Hek-293T in ( D ). Level of ACE2 receptor in MDMi and mouse microglia compared to Caco2 and Vero E6 cells analysed by western blot shown in panel ( E ). Viral RNA levels from SARS-CoV-2 particles bound on cell surface expressed as N2 copies/well in ( F ) Intracellular luciferase level (LUC) delivered by pseudo-virus (PV) particle for SARS-CoV-2 in MDMi and Vero E6 compared to the non-glycoprotein control (NE) in ( G ). SARS-CoV-2 replication on MDMi (at MOI of 1) and Vero E6 (at MOI of 0.01) using SARS-CoV-2 reporter virus expressing ZsGreen fluorescent protein assessed directly under confocal microscopy at 3dpi are shown in panel ( H ). Data points are means + SEM from at least three different donors. *P < 0.05, **P < 0.01, and ***P < 0.001 and **** P < 0.0001 by two-way ANOVA test with Sidak’s correction.

Article Snippet: The human ACE2 transcript variant 2 was amplified using the OriGene primer set Forward: 5’-TCC ATT GGT CTT CTG TCA CCC G-3’ and Reverse: 5’-AGA CCA TCC ACC TCC ACT TCT C-3’.

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Luciferase, Confocal Microscopy

Journal: bioRxiv

Article Title: SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike-ACE2 receptor interaction

doi: 10.1101/2022.01.11.475947

Figure Lengend Snippet: Relative of infectivity determined by Plaque Reduction Neutralisation Test (PRNT) to verify the neutralizing level of a soluble receptor hACE2-FcM compared to a non-related SARS-CoV-2 receptor NCAM-FcM (top) and the inhibitory concentration (IC50) (bottom) ( A ). Spike–mediated IL-1β secretion (supernatant) in vehicle (untreated) or LPS-primed MDMIs exposed to S-clamp (S; 10-50 μg) in presence or absence of the soluble hACE2-FcM protein. ATP (5 mM) treatment for 1 hour was used as a positive control ( B ). Inhibition of spike-mediated IL-1β secretion by ACE2 inhibition with MLN-4760 (1 or 10 μM) ( C ). Validation of low endotoxin anti-ACE2 (3E8) and anti-Hemagglutinin from influenza A H3 (CO5) proteins by SDS-PAGE and ELISA ( D-E ). Effect of 3E8 in blocking cells activation by spike protein in pre-treatment of LPS-primed MDMi exposed to S-clamp ( F ). Data are means + SEM from at least three different donors. *P < 0.05, **P < 0.01, and ***P < 0.001 and **** P < 0.0001 by one-way analysis of variance (ANOVA) with Tukey’s post hoc test.

Article Snippet: The human ACE2 transcript variant 2 was amplified using the OriGene primer set Forward: 5’-TCC ATT GGT CTT CTG TCA CCC G-3’ and Reverse: 5’-AGA CCA TCC ACC TCC ACT TCT C-3’.

Techniques: Infection, Concentration Assay, Positive Control, Inhibition, SDS Page, Enzyme-linked Immunosorbent Assay, Blocking Assay, Activation Assay

Journal: bioRxiv

Article Title: Functional genomic screens identify human host factors for SARS-CoV-2 and common cold coronaviruses

doi: 10.1101/2020.09.24.312298

Figure Lengend Snippet: (a) Light microscopy images of WT Huh7.5.1 infected with OC43 (7 dpi) and 229E (4 dpi). (b) Quantification of SARS-CoV-2 RNA in WT Huh7.5.1 cells at 24 and 72 hpi by RT-qPCR. Cq values represent mean ± s.e.m. from 3 biological replicates. (c) Light microscopy images of SARS-CoV-2 infected WT Huh7.5.1 cells or Huh7.5.1 cells expressing ACE2-IRES-TMPRSS2 at 3 and 7 dpi. (d) Quantification of ACE2 and TMPRSS2 expression in WT and lentivirally transduced Huh7.5.1 cells by RT-qPCR and Western blot. mRNA levels are displayed as mean ± s.e.m. from two independent harvests and are relative to expression in WT cells. Anti-ACE2 and anti-TMPRSS2 antibodies were used to detect protein levels in WT and overexpression cells. GAPDH was used as loading control. Molecular weight markers are indicated on the left. (e) Quantification of infection with pseudotyped lentivirus bearing SARS-CoV-2 spike and expressing a GFP by flow cytometry. Values are from two biological samples and are displayed as means ± s.d.

Article Snippet: To generate the plenti-CMV-ACE2-IRES-TMPRSS2 construct, ACE2, EMCV IRES (derived from pLenti-DsRed_IRES_EGFP (Addgene, #92194, gift from Huda Zoghbi)), and TMPRSS2 were individually amplified with addition of overlapping sequences and the three fragments were assembled using NEBuilder HiFi DNA Assembly Master Mix.

Techniques: Light Microscopy, Infection, Quantitative RT-PCR, Expressing, Western Blot, Over Expression, Control, Molecular Weight, Flow Cytometry

Journal: bioRxiv

Article Title: Functional genomic screens identify human host factors for SARS-CoV-2 and common cold coronaviruses

doi: 10.1101/2020.09.24.312298

Figure Lengend Snippet: (a) Schematic of CRISPR KO screens for the identification of coronavirus host factors. Huh7.5.1-Cas9 (with bicistronic ACE2-IRES-TMPRSS2 construct for SARS-CoV-2 and without for 229E and OC43 screen) were mutagenized using a genome-wide sgRNA library. Mutant cells were infected with each coronavirus separately and virus-resistant cells were harvested 10–14 days post infection (dpi). The abundance of each sgRNA in the starting and selected population was determined by high-throughput sequencing and a gene enrichment analysis was performed. (b–d) Gene enrichment of CRISPR screens for (b) SARS-CoV-2, (c) 229E and (d) OC43 infection. Enrichment scores were determined by MaGECK analysis and genes were colored by biological function. The SARS-CoV-2 was performed once. The 229E and OC43 screens were performed twice and combined MaGECK scores are displayed.

Article Snippet: To generate the plenti-CMV-ACE2-IRES-TMPRSS2 construct, ACE2, EMCV IRES (derived from pLenti-DsRed_IRES_EGFP (Addgene, #92194, gift from Huda Zoghbi)), and TMPRSS2 were individually amplified with addition of overlapping sequences and the three fragments were assembled using NEBuilder HiFi DNA Assembly Master Mix.

Techniques: CRISPR, Construct, Genome Wide, Mutagenesis, Infection, Virus, Next-Generation Sequencing

Journal: bioRxiv

Article Title: Functional genomic screens identify human host factors for SARS-CoV-2 and common cold coronaviruses

doi: 10.1101/2020.09.24.312298

Figure Lengend Snippet: (a) Indel frequency of RNP-edited polyclonal A549-ACE2 KO cells. Targeted loci were PCR-amplified, Sanger-sequenced and analyzed using Inference of CRISPR Edits (ICE) analysis . (b) Genotyping of clonal Huh7.5.1. Targeted loci were PCR-amplified, Sanger-sequenced and aligned to WT reference sequence. Frameshifts are highlighted in blue. (c) Cell viability measurement of 229E or OC43 infected WT and KO Huh7.5.1 cells. Cells were infected with 229E (moi=0.05) or OC43 (moi=3) and viability was determined 8 dpi using Cell Titer Glo. Values are displayed as means ± s.d. from three (229E) or two (OC43) biological samples.

Article Snippet: To generate the plenti-CMV-ACE2-IRES-TMPRSS2 construct, ACE2, EMCV IRES (derived from pLenti-DsRed_IRES_EGFP (Addgene, #92194, gift from Huda Zoghbi)), and TMPRSS2 were individually amplified with addition of overlapping sequences and the three fragments were assembled using NEBuilder HiFi DNA Assembly Master Mix.

Techniques: Amplification, CRISPR, Sequencing, Infection

Journal: bioRxiv

Article Title: Functional genomic screens identify human host factors for SARS-CoV-2 and common cold coronaviruses

doi: 10.1101/2020.09.24.312298

Figure Lengend Snippet: (a) RT-qPCR quantification of intracellular SARS-CoV-2 levels in RNP edited A549-ACE2 cells. Cells were infected using moi=0.01 and harvested at 72 hours post infection (hpi). (b–d) RT-qPCR quantification of intracellular SARS-CoV-2 levels in WT Huh7.5.1 cells or cells harboring frameshift mutations or frameshift mutant cells complemented with respective cDNAs. Cells were infected using moi=0.01 and harvested at 24 hpi. (e–g) RT-qPCR quantification of intracellular OC43 and 229E RNA levels in WT and KO Huh7.5.1 cells. Cells were infected using moi=0.05 (229E) and moi=3 (OC43) and harvested at 48 hpi. (h) RT-qPCR quantification of intracellular SARS-CoV-2 levels in Huh7.5.1 WT or KO cells by RT-qPCR. Cells were infected using moi=0.01 and harvested at 24 hpi. For SARS-CoV-2 infection, viral transcripts were normalized to cellular RNaseP. For OC43 and 229E experiments, viral RNA was normalized to 18S RNA. For all RT-qPCR experiments, results are displayed relative to infection in WT cells and data represent means ± s.e.m. from 3 biological samples.

Article Snippet: To generate the plenti-CMV-ACE2-IRES-TMPRSS2 construct, ACE2, EMCV IRES (derived from pLenti-DsRed_IRES_EGFP (Addgene, #92194, gift from Huda Zoghbi)), and TMPRSS2 were individually amplified with addition of overlapping sequences and the three fragments were assembled using NEBuilder HiFi DNA Assembly Master Mix.

Techniques: Quantitative RT-PCR, Infection, Mutagenesis