Fig 2: scRNAseq reveals cell type‐specific expression of SARS‐CoV‐2 receptors and processing enzymes in COPD and control lungs. (A) Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) representation of cell clusters in control and COPD lungs. Dots represent single cells. ATI, alveolar type I cells; ATII, alveolar type II cells; B_Plasma, plasma cells; DC, dendritic cells; ILC, innate lymphoid cells; Lymphatic, lymphatic endothelium; Mast, mast cells; NK, natural killer cells; pDC, plasmacytoid dendritic cells; PNEC, pulmonary neuroendocrine cell; SMC, smooth muscle cell. (B) Expression of ACE2, TMPRSS2, BSG (CD147), FURIN, and EZR in single cells of control and COPD lungs. Cell clusters are shown as indicated in (A). (C) Dot plot representation of ACE2, TMPRSS2, BSG (CD147), FURIN, and EZR expression in specific cell populations within control and COPD lungs. Dot sizes represent the percent of cells within each cluster with positive gene expression and color intensity indicates the average expression of within one cluster.

Fig 3: Local and systemic levels of SARS‐CoV‐2 receptors and processing enzymes in patients and controls. (A) Quantitative real‐time PCR analysis of ACE2, TMPRSS2, Furin (FURIN), Ezrin (EZR), and CD147/Basigin (BSG) expression levels in lung homogenates of donors and transplant patients with COPD, IPAH, and PF. (B) sACE2 levels measured by ELISA in plasma samples of healthy controls (Ctrl), COPD, IPAH, and PF patients. Boxplots with single data points are shown. Statistical analysis was performed using a nonparametric Kruskal–Wallis test with post hoc comparison of multiple groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 4: Ezrin preferentially interacts with L-selectin in non-transmigrated uropods, whereas moesin interacts with L-selectin in transmigrated subendothelial pseudopods. (A–F) THP-1 cells co-expressing either ezrin–RFP (Ezr-RFP) or moesin–RFP (Moe-RFP) (in ezrin- or moesin-knockdown cells, respectively), and WT or non-cleavable (ΔM-N) L-selectin–GFP, were resuspended into neat RPMI and seeded onto poly-L-lysine (PLL)-coated coverslips for 10 min at 37°C and subsequently fixed for FRET measurements by FLIM (see Materials and Methods). Representative images of each cell line, showing individual fluorescence channels and lifetime profiles, are provided. The lifetime of fluorescence is expressed in a pseudocolour scale from red (low lifetime with a very high probability of interaction) to blue (high lifetime with a very low probability of interaction). (G–L) THP-1 cells expressing WT L-selectin–GFP and ezrin–RFP or moesin–RFP (in ezrin- or moesin-knockdown cells, respectively) were perfused over TNF-activated HUVECs and fixed in 4% PFA at 6 min or 25 min to represent early and late phases of TEM, respectively (see ‘timed perfusions’ in the Materials and Methods). FLIM measurements were taken from two optical sections: above (‘Top’) and below (‘Base’) the endothelium. FRET efficiencies (FRET Eff) were derived from measurements of at least 45 cells from three independent experiments. Individual fluorescence channels and lifetime image are provided for each cell line. Note that RFP channel was taken in wide field and so could not be used to represent ‘Top’ and ‘Base’ images in this channel. Error bars represent s.e.m. **P<0.01, ***P<0.001 (two-tailed unpaired Student's t-test).

Fig 5: Pharmacological or genetic blockade of ezrin function impacts on early phases of leukocyte recruitment. (A) Primary human CD14+ monocytes were isolated from healthy donors on the day of the assay, pretreated for 10 min with either 10 µM of the ezrin inhibitor (NSC668394) or an equal volume of carrier (DMSO), and then perfused over TNF-activated endothelial monolayers at 1.5 dynes cm2 for a period of 20 min. Percentage recruitment was scored as the proportion of CD14+ cells stably interacting with the underlying endothelium, which included transmigrated cells. Rolling cells were excluded from the scoring, but made up the total percentage of recruited cells to 100%. (B) THP-1 cell lines stably expressing L-selectin–GFP (L-sel), but lacking endogenous ezrin or moesin expression by virtue of stable expression of shRNA clones for ezrin (Ezr KD, clone 5 or 6) or moesin (Moe KD, clone 2 or 3). Cells were perfused at 1.5 dynes/cm2 and analysed by time-lapse microscopy for a 25 min period. Cells were continually perfused over monolayers at a density of 0.5×106 cells per ml. THP-1 cells expressing non-targeting shRNA (NT) along with L-selectin–GFP were used in control experiments. Data is expressed as the percentage of cells actively engaged in TEM over time. Significance is expressed. Error bars represent s.e.m. **P<0.01, ****P<0.0001 (compared with moesin-depleted cells in B; one-way ANOVA followed by Bonferroni's post-test).