Extended Data Fig. 14: Promoting or blocking viral entry or replication in human macrophages in vivo and in vitro impacts inflammatory profile of macrophages (matched to figure 3).
From: Inflammasome activation in infected macrophages drives COVID-19 pathology
a. Human IL-18 levels measured in lung homogenates of infected (4 d.p.i.) MISTRG6-hACE2 mice treated (or not) with CD16 blocking antibody (Abcam). CTRL infected n = 7, anti-CD16 n = 5 biologically independent mice examined over 3 independent experiments. Means with datapoints. Unpaired, two-tailed t-test. b. Human IL-1RA levels measured in lung homogenates of infected (4 d.p.i.) MISTRG6-hACE2 mice treated (or not) with CD16 blocking antibody (Abcam). N = 4 biologically independent mice examined over 2 independent experiments. Means with all datapoints. Unpaired, two-tailed t-test. c. Human CXCL10 levels measured in serum of infected (4 d.p.i.) MISTRG6-hACE2 mice treated (or not) with CD16 blocking antibody (Abcam). CTRL infected n = 7, anti-CD16 n = 4 biologically independent mice examined over at least 2 experiments. Means with individual datapoints. Unpaired, two-tailed t-test. d. Human IL-18 levels measured in serum of infected (14 d.p.i.) MISTRG6-hACE2 mice treated (or not) with CD16 blocking antibody (Abcam). Mice were treated with anti-CD16 blocking antibody at 7 d.p.i. and 11 d.p.i. CTRL infected n = 4, anti-CD16 n = 4 biologically independent mice examined over 2 independent experiments. Means with individual datapoints. Unpaired, two-tailed t-test. e. Human IL-18 levels measured in serum of infected (4 d.p.i.) MISTRG6 mice treated (or not) with anti-ACE2 antibody (Abcam). Mice were treated with anti-ACE2 antibody at 1,2,3 d.p.i. Uninfected n = 4, CTRL infected n = 5, anti-ACE2 n = 4 biologically independent mice examined over 2 independent experiments. Means with individual datapoints. Unpaired, two-tailed t-test. P < 0.0001 = uninfected vs. CTRL-infected = 1.43x10−5, CTRL-infected vs. anti-ACE2 = 6.95x10−5. f. Representative flow cytometry plots of CD14 staining on total human immune cells (hCD45+) as a proxy for myeloid cells in infected MISTRG6-hACE2 mice (4 d.p.i.) treated (or not) with a depleting antibody against CD16 (ThermoFisher, clone 3G8). MISTRG6-hACE2 mice were infected with SARS-CoV-2-mNG. Representative of n = 4 biologically independent mice examined over 2 independent experiments. g. Frequencies of mNG+ cells in infected MISTRG6-hACE2 mice at 4 d.p.i. treated (or not) with a depleting antibody against CD16 (ThermoFisher, clone 3G8). N = 4 biologically independent mice examined over 2 independent experiments. Means with datapoints. Unpaired, two-tailed t-test. h. Human IL-18 levels in serum of infected or uninfected MISTRG6-hACE2 mice that were therapeutically treated with monoclonal antibodies (mAb) at 36 h.p.i. or not. Sera from infected mice were analysed at 4 d.p.i. N = 4 biologically independent mice examined over 2 independent experiments. Means with datapoints. Unpaired, two-tailed t-test. i. Human IL-1RA levels in serum of infected (4 d.p.i.) or uninfected MISTRG6-hACE2 mice that were therapeutically treated with mAb at 36 h.p.i. or not. Uninfected and mAb treated n = 4, CTRL infected n = 3 (matched to mAb treatment) biologically independent mice examined over 2 independent experiments. Means with datapoints. Paired, two-tailed t-test. j. Human IL-18 levels measured in serum of infected (14 d.p.i.) MISTRG6-hACE2 mice treated (or not) with mAb (clone 135+ clone 144) at 7 d.p.i. and analysed at 14 d.p.i. N = 3 biologically independent mice examined over 2 independent experiments. Mean with individual datapoints. Unpaired, two-tailed t-test. k. Frequencies of CXCL10+ macrophages within total human macrophages (hCD45+hCD68+) in lungs of infected MISTRG6-hACE2 mice treated (or not) with mAb (clone 135, clone 144) at 7 d.p.i. and analysed at 14 d.p.i. Mean with individual values. Unpaired, two-tailed t-test, not significant (ns). CTRL-infected n = 5, mAb n = 4 biologically independent mice examined over 2 independent experiments. l. Box and whisker plot (min to max, with all datapoints) of the histopathological scoring of the H&E staining of infected MISTRG6-hACE2 lungs at 4 d.p.i. Mice were either treated with monoclonal antibodies at 35 h.p.i. or anti-CD16 at 2 d.p.i. CTRL infected n = 9, mAb treated n = 6, anti-CD16 treated n = 3 biologically independent mice examined over at least 2 independent experiments. The whiskers go down to the smallest value (minimum) and up to the largest value (maximum). The box extends from the 25th to 75th percentiles. The median is shown as a line in the center of the box. Unpaired t-test not significant. m. Box and whisker plot (min to max, with all datapoints) of the histopathological scoring of the H&E staining of infected MISTRG6-hACE2 lungs at 14 d.p.i. Mice were either treated with monoclonal antibodies at 7 d.p.i. or anti-CD16 at 7 and 11 d.p.i. CTRL infected n = 4, mAb treated n = 4, anti-CD16 treated n = 4 biologically independent mice examined over 2 independent experiments. The whiskers go down to the smallest value (minimum) and up to the largest value (maximum). The box extends from the 25th to 75th percentiles. The median is shown as a line in the center of the box. Unpaired, two-tailed t-test, not significant. n. Human IL-18 levels in supernatants of SARS-CoV-2 infected BMDMs treated with anti-CD16 and anti-ACE2 antibodies to block viral entry or with Remdesivir to block viral replication. CTRL infected n = 8, anti-CD16+anti-ACE2 n = 5, RDV n = 5 independent datapoints over 3 independent experiments. Means with all datapoints. Unpaired, two-tailed t-test. P < 0.0001 = 5.0x10−5. o. Human IL-1β levels in supernatants of SARS-CoV-2 infected BMDMs treated with anti-CD16 and anti-ACE2 antibodies to block viral entry or with Remdesivir to block viral replication. N = 4 independent datapoints over 2 independent experiments. Means with all datapoints. Unpaired, two-tailed, t-test. p. Human IL-1RA in supernatants of SARS-CoV-2 infected BMDMs treated with anti-CD16 and anti-ACE2 antibodies to block viral entry or with Remdesivir to block viral replication. CTRL infected n = 7, anti-CD16+anti-ACE2 n = 3, RDV n = 3 independent datapoints over 2 independent experiments. Means with all datapoints. Unpaired, two-tailed, t-test. q. Human CXCL10 levels in supernatants of BMDMs infected in vitro in presence or absence of anti-CD16 and anti-ACE2 antibodies to block viral entry. CTRL infected N = 12, anti-ACE2+ anti-CD16 treated n = 6 independent datapoints over 2 independent experiments. Means with all datapoints. Unpaired, two-tailed, t-test.
