Extended data figure 15. Blockade of inflammasome activation leads to reduced cytokine production in vitro (matched to figure 4).
a. Representative flow cytometry plots of CXCL10 and TNF staining in total macrophages and histograms of CXCL10 expression in infected (mNG+) and uninfected (mNG−) macrophages from lungs of SARS-CoV-2-mNG infected MISTRG6-hACE2 mice treated with caspase-1 (Casp1) or NLRP3 inhibitors in vivo. Mice were treated on days 6,8,10,12 post-infection and analyzed at 14dpi. Representative of n=5 biologically independent mice.
b. Mean fluorescent intensity (MFI) of CXCL10 expression in human macrophages isolated from infected MISTRG6-hACE2 mice treated with Casp1 inhibitor or left untreated. N=3 biologically independent mice. Representative of 3 independent experiments. Means with all datapoints and SD. Unpaired, two-tailed t-test.
c. CXCL10 levels in serum of SARS-CoV-2-mNG infected MISTRG6-hACE2 mice (14dpi) treated with Casp1 or NLRP3 inhibitors. n=4 biologically independent mice examined over 2 independent experiments. Means with all datapoints and SD. Unpaired, two-tailed t-test.
d. Frequencies of mNG+ bone marrow-derived macrophages (BMDMs) infected with SARS-CoV-2-mNG in vitro. BMDMs were treated with caspase-1 (Casp1) or NLRP3 inhibitors or left untreated and analyzed at 48hpi. CTRL infected n=22, Casp1 inhibitor n=17, NLRP3 inhibitor n=6 independent datapoints collected over at least 3-experiments. Means with all datapoints and SD. Unpaired, two-tailed t-test.
e. Frequencies of FLICA+ BMDMs infected with SARS-CoV-2 in vitro for 48 hours. BMDMs were treated with Casp1 or NLRP3 inhibitors or left untreated. CTRL infected n=13, Casp1 inhibitor n=12, NLRP3 inhibitor n=6 independent datapoints collected over at least 3 independent experiments. Means with all datapoints. Unpaired, two-tailed t-test. P<0.0001=3.33×10−5.
f. Human IL-18 levels in supernatants of SARS-CoV-2-mNG infected BMDMs treated with caspase-1 (Casp1) inhibitor or left untreated. CTRL infected n=8, Casp1 inhibitor-treated n=6 independent datapoints collected over 2 independent experiments. Means with all datapoints and SD. Unpaired, two-tailed t-test.
g. Representative histograms and quantification of IL-1β in supernatants of BMDMs infected with SARS-CoV-2 in vitro. Cultures were treated with caspase-1 (Casp1) inhibitor. over at least 2 independent experiments. Uninfected n=7; 48hpi CTRL infected n=10, Casp1 inhibitor n=9, NLRP3 inhibitor n=3; 72hpi CTRL infected n=5 Casp1 inhibitor n=3, NLRP3 inhibitor n=3 independent datapoints collected over at least 2 experiments. Means with all datapoints and SD. Unpaired, two-tailed t-test. P<0.0001=4.96×10−7.
h. Human Gasdermin D (GSDMD) levels at 48hpi in supernatants of SARS-CoV-2-mNG infected BMDMs treated with Casp1 inhibitor or left untreated. CTRL infected n=10, Casp1 inhibitor-treated n=6 independent datapoints collected over at least 3 independent experiments. Means with all datapoints. Unpaired, two-tailed t-test.
i. LDH levels measured as absorbance at OD 490nm in supernatants of uninfected or SARS-CoV-2-mNG infected BMDMs treated with Casp1 or NLRP3 inhibitor or left untreated in vitro. Uninfected n=6, CTRL infected (48hpi) n=11, Casp1 inhibitor-treated(48hpi) n=9, NLRP3 inhibitor-treated (48hpi) n=5 independent datapoints collected over 2 independent experiments. Means with all datapoints and SD. Unpaired, two-tailed t-test. P<0.0001=7.38×10−6.
j. Zombie Aqua incorporation in SARS-CoV-2-mNG infected BMDMs treated with Casp1 or NLRP3 inhibitor or left untreated (CTRL infected). Frequencies of Zombie+ cells within Annexin V− population at 48hpi are reported. Uninfected n=4, CTRL infected n=7, Casp1 inhibitor n=4, NLRP3 inhibitor n=3 over 2 experiments. Means with all datapoints. Unpaired, two-tailed t-test.
k. Human CXCL10 levels in supernatants of infected human BMDMs treated with Casp1 inhibitor or NLRP3 inhibitor or left untreated. Supernatants were collected at 48hpi. Uninfected n=5, CTRL infected n=12, Casp1 inhibitor n=5, NLRP3 n=4 independent datapoints over at least 2 independent experiments. Means with all datapoints and SD. Unpaired, two-tailed t-test.
l. Human IL-1RA levels in supernatants of SARS-CoV-2-mNG infected human BMDMs treated with Casp1 inhibitor or not. BMDMs were treated with Casp1 inhibitor. Supernatants were collected at 48hpi. CTRL infected n=7, Casp1 inhibitor-treated n=6, NLRP3 inhibitor-treated n=4 independent datapoints collected over at least 2 independent experiments. Means with all datapoints. Unpaired, two-tailed t-test.
m. Viral titers measured as PFU in lung homogenates of MISTRG6-hACE2 mice infected with SARS-CoV-2 and treated with caspase-1 inhibitor in vivo. Infected MISTRG6-hACE2 mice were treated with Caspase 1 inhibitor on days 6,8,10,12 post-infection and analyzed at 14dpi. Lung homogenates were plaqued using Vero ACE2+TMPRSS2+ cells. of CTRL infected: n=7, Casp1 inhibitor-treated: n=6 biologically independent mice examined over 3 independent experiments. Box and whisker plot (min to max, with all datapoints) 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. Ratio paired, two-tailed t-test.
n. Representative images of plaque assays used to quantify infectious virus in supernatants of BMDMs infected with SARS-CoV-2-mNG and treated with caspase-1 or NLRP3 inhibitors. Supernatants of infected macrophage cultures were collected at 48hpi and plaqued using Vero ACE2+TMPRSS2+ cells. Plaques were resolved at 48hpi. Representative of CTRL infected: n=13, Casp1 inhibitor-treated: n=8, NLRP3 inhibitor-treated: n=5 independent datapoints collected over 3 independent experiments.