Extended Data Fig. 13: SARS-CoV-2 infection of human macrophages activates inflammasomes and leads to death by pyroptosis in vivo and in vitro (matched to figure 3).
From: Inflammasome activation in infected macrophages drives COVID-19 pathology
a. Representative images of single stained cells for ASC specks, NLRP3, human CD14, human CD45, mouse CD45 and mouse EPCAM. Cells from SARS-CoV-2 infected humanized mice were sorted based on (Extended Data Fig. 6j): human immune cells (hCD45+); mouse immune cells (mCD45+) or epithelial mouse cells (EPCAM+). Sorted cells were stained with single antibodies against ASC, CD14 or NLRP3. Left panel shows human immune cells and right panel shows mouse immune cells (mCD45+) and mouse epithelial cells (EPCAM+). Representative of n = 5 independent mice examined over 3 independent experiments. b. Visualization of ASC specks as a measure of inflammasome activation in mNG+ (SARS-CoV-2+) or mNG− (SARS-CoV-2−) human immune cells at 4 d.p.i. Human immune cells were sorted from SARS-CoV-2 infected humanized mice were sorted based on expression of human CD45 and mNG and lack of mouse CD45 and EPCAM expression (Extended Data Fig. 6j). Representative of n = 5 biologically independent mice examined over 3 independent experiments. c. Left: Representative flow cytometry plot displaying SARS-CoV-2-mNG and Casp1-FLICA staining of CD11b+ human immune cells. Right: quantification of FLICA+ cells (%) as a measure of active caspase-1 in infected (mNG+) and uninfected (mNG−) human lung macrophages (CD11b+hCD45+) at 4 d.p.i. and 14 d.p.i. 4 d.p.i.: n = 3 biologically independent mice examined over 2 independent experiments, 14 d.p.i. n = 5 biologically independent mice examined over 3 independent experiments. Lung cells were incubated with FLICA-Casp1 substrate for 30 min. Means with individual datapoints plotted. Paired, two-tailed t-test. P < 0.0001 = 4.29x10−9. d. Quantification of Casp1-FLICA staining as a measure of active caspase-1 in infected (mNG+) human or total mouse CD11b+ cells at 4 d.p.i. Mouse cells: mCD45+CD11b+hCD45−. Human cells: mCD45−CD11b+hCD45+mNG+. N = 6 biologically independent mice examined over 3 independent experiments. Means with individual datapoints plotted. Paired, two-tailed t-test. P < 0.0001 = 1.79x10−9. e. Human IL-18 (measured by ELISA) in lungs and serum and corresponding mNG levels (measured as percent within human immune cells by flow cytometry) in lungs of infected MISTRG6-hCE2 mice at 4 d.p.i. Lung: Pearson’s correlation value = 0.69. N = 8 biologically independent mice examined over 3 independent experiments. Serum: Pearson’s correlation value = 0.75. n = 7 biologically independent mice examined over 3 independent experiments. Unpaired, two-tailed t-test. f. Human IL-1RA (measured by ELISA) in lungs and serum and corresponding mNG levels (measured as percent within human immune cells by flow cytometry) in lungs of infected MISTRG6-hCE2 mice at 4 d.p.i. Lung: Pearson’s correlation value = 0.82 n = 8 biologically independent mice examined over 3 independent experiments. Serum: Pearson’s correlation value = 0.46 n = 8 biologically independent mice examined over 3 independent experiments. Unpaired t-test, two-tailed. g. LHD levels measured as absorbance at OD 490nm in serum of uninfected or infected MISTRG6-hACE2 mice at 4 d.p.i. and 14 d.p.i. Fresh serum was assayed for LDH. Uninfected n = 3, 4 d.p.i. n = 4, 14 d.p.i. n = 4 biologically independent mice examined over 2 independent experiments. Means with individual datapoints. Unpaired, two-tailed t-test. h. Zombie Aqua incorporation in infected (mNG+) or uninfected (mNG−) CD16+CD11b+ or CD16−CD11b+ human myeloid cells. Frequencies of Zombie+ cells were measured in Annexin V− cells. N = 5 biologically independent mice examined over 2 independent experiments. Means with individual datapoints. Paired, two-tailed t-test. i. Representative histograms and quantification of Casp1-FLICA staining as a measure of active caspase-1 in bone marrow derived macrophages (BMDMs) infected with SARS-CoV-2 in vitro or not for 48 h. BMDM cultures were either supplemented with healthy or COVID plasma or monoclonal antibodies for the duration of the infection. Cultures were treated with Remdesivir, anti-ACE2 and anti-CD16 to block viral replication or viral entry. Colouring on the histograms matches the bar graph legend. Uninfected n = 4; healthy plasma CTRL infected n = 9, anti-ACE2 n = 4, RDV n = 4; mAb n = 4; COVID plasma CTRL infected n = 12, anti-ACE2+anti-CD16 n = 6, RDV n = 5 independent datapoints collected over at least 2 independent experiments. Means with all datapoints. Unpaired, two-tailed t-test. P- values < 0.0001: COVID plasma vs. RDV: 5.85x10−6. j.Representative histograms and quantification of IL-1β in supernatants of BMDMs infected with (or not) SARS-CoV-2 in vitro for 48 or 72 h. Uninfected n = 7, 48 h.p.i. n = 10, 72 h.p.i. n = 5 independent datapoints collected over 3 independent experiments. Means with SD and individual datapoints. Unpaired, two-tailed t-test. P- values < 0.0001: uninfected vs. 72 h.p.i. = 4.96x10−7, 48 h.p.i. vs. 72 h.p.i. = 1.17 x10−8. k. Human IL-18 levels at 48 h.p.i. in supernatants of BMDMs infected or not with SARS-CoV-2 in vitro. BMDM cultures were supplemented with COVID plasma for the duration of the infection. Uninfected n = 4, 48 h.p.i. n = 7 independent datapoints collected over at least 2 independent experiments. Unpaired, two-tailed t-test. Means with individual datapoints. Unpaired, two-tailed t-test. l. Human IL-1RA levels at 48 h.p.i in supernatants of BMDMs infected with SARS-CoV-2 in vitro or not. Uninfected n = 3, infected n = 7 independent datapoints collected over at least 2 independent experiments. Means with individual datapoints. Unpaired, two-tailed t-test. m. Gasdermin D (GSDMD) levels in supernatants of BMDMs infected with (or not) SARS-CoV-2 in vitro for 48 h. BMDM cultures were supplemented with COVID plasma for the duration of the infection. Cultures were treated with Remdesivir to block viral replication. Uninfected N = 3, CTRL infected n = 10, RDV n = 3 independent datapoints collected over at least 2 independent experiments. Means with individual datapoints. Unpaired, two-tailed t-test. n. LHD levels measured by absorbance at OD 490 nm in supernatants of infected or uninfected BMDMs. Uninfected n = 6, Infected n = 11 independent datapoints collected over 3 independent experiments. Means with individual datapoints. Unpaired, two-tailed t-test. P-value = 7.38x10−6. o. Zombie Aqua incorporation in uninfected or SARS-CoV-2-mNG infected BMDMs. Frequencies of Zombie+ cells within Annexin V− population at 48 h.p.i. are presented. Uninfected n = 4, infected n = 7 independent datapoints collected over 3 independent experiments. Means with individual datapoints. Unpaired, two-tailed t-test.
