Inflammasome activation in infected macrophages drives COVID-19 pathology

Abstract

Severe COVID-19 is characterized by persistent lung inflammation, inflammatory cytokine production, viral RNA and a sustained interferon (IFN) response, all of which are recapitulated and required for pathology in the SARS-CoV-2-infected MISTRG6-hACE2 humanized mouse model of COVID-19, which has a human immune system^1-20. Blocking either viral replication with remdesivir^21-23 or the downstream IFN-stimulated cascade with anti-IFNAR2 antibodies in vivo in the chronic stages of disease attenuates the overactive immune inflammatory response, especially inflammatory macrophages. Here we show that SARS-CoV-2 infection and replication in lung-resident human macrophages is a critical driver of disease. In response to infection mediated by CD16 and ACE2 receptors, human macrophages activate inflammasomes, release interleukin 1 (IL-1) and IL-18, and undergo pyroptosis, thereby contributing to the hyperinflammatory state of the lungs. Inflammasome activation and the accompanying inflammatory response are necessary for lung inflammation, as inhibition of the NLRP3 inflammasome pathway reverses chronic lung pathology. Notably, this blockade of inflammasome activation leads to the release of infectious virus by the infected macrophages. Thus, inflammasomes oppose host infection by SARS-CoV-2 through the production of inflammatory cytokines and suicide by pyroptosis to prevent a productive viral cycle.

Extended data figure 1.. Targeting viral replication and the downstream interferon response attenuates the hyperactive immune/inflammatory response (matched to figure 1).

a. Representative gating strategy of human immune cells in the lungs of SARS-CoV-2 infected MISTRG6-hACE2 mice. Cells isolated from lungs or bronchioalveolar lavage (BAL) were stained with antibodies against human CD45, HLA-DR, CD68, CD16, CD14, CD206, CD86, CD11B, CD11C, CD123, CD3, and mouse CD45. Cell numbers were calculated using counting beads. b. Human immune cells (numbers) in BAL (14dpi) or lungs (14 and 28dpi) of SARS-Cov-2 infected MISTRG6-hACE2 mice treated with dexamethasone (Dex), Remdesivir (RDV), anti-IFNAR2 or a combined therapy of Remdesivir (RDV) and anti-IFNAR2. 14dpi, BAL: CTRL-infected n=5, Dex; RDV anti-IFNAR2 n=3; anti-IFNAR2+RDV n=4 biologically independent mice examined over 2 independent experiments. 14dpi, Lung: CTRL-infected n=7; Dex, RDV, anti-IFNAR2, anti-IFNAR2+RDV n=4 biologically independent mice examined over 3 independent experiments. 28dpi, lung: CTRL infected n=5, Dex n=4, RDV n=3, anti-IFNAR2 n=3, anti-IFNAR2+RDV n=6 biologically independent mice examined over 3 independent experiments. Means with individual datapoints plotted. Unpaired, two-tailed t-test. P<0.0001=8.19×10⁻⁵. c. Representative flow cytometry plots and frequencies of alveolar macrophages (AMs) (middle: hCD206^hihCD86⁺hCD68⁺) or inflammatory macrophages (bottom: hCD206^lo/−hCD86⁺hCD68⁺) in 14dpi or 28dpi lungs of treated or untreated MISTRG6-hACE2 mice. 14dpi: CTRL infected n=5, Dex n=4, RDV n=4, anti-IFNAR2 n=4, anti-IFNAR2+RDV n=4 biologically independent mice examined over at least 2 independent experiments. 28dpi: CTRL infected n=5, Dex n=4, RDV n=3, anti-IFNAR2 n=3, anti-IFNAR2+RDV n=6 biologically independent mice examined over 3 independent experiments. Means with individual datapoints. Unpaired, two-tailed t-test. P<0.0001=4.67×10⁻⁵. d. Frequencies (left) and numbers (right) of lung pDCs at 14dpi. CTRL-infected n=6, Dex n=4, RDV, anti-IFNAR2 n=3, anti-IFNAR2+RDV n=6 mice examined over at least 2 experiments. Means with datapoints. Unpaired, two-tailed t-test. P<0.0001=7.29×10⁻⁵. e. IFNA transcript levels measured by qPCR in treated or control untreated MISTRG6-hACE2 mice infected with SARS-CoV-2: Uninfected n=5; CTRL infected: 4dpi n=8, 14dpi n =9, 28dpi n=: 6; Dex 14dpi n=4, Dex 28dpi= 4; RDV 14 and 28dpi n=3, anti-IFNAR2 14 and 28dpi n=3, anti-IFNAR2+ Remdesivir 14 and 28dpi n=4 biologically independent mice examined over at least 2 independent experiments. Normalized to HPRT1. Violin plots with individual datapoints. Unpaired, two-tailed t test. f. Representative histograms and frequencies of HLA-DR⁺ activated T cells in treated or control mice. 14dpi: CTRL-infected n=5, Dex, RDV, anti-IFNAR2, anti-IFNAR2+RDV n=4; 28dpi: CTRL infected, Dex, anti-IFNAR2+RDV n=4, RDV, anti-IFNAR2 n=3 biologically independent mice examined over 3 independent experiments. Means with datapoints. Unpaired, two-tailed t-test. Some of the data associated with dexamethasone therapy used here as a control have been reported.

Extended data figure 2.. Anti-IFNAR2 and Remdesivir therapy reverses infection induced transcriptional changes (matched to figure 1).

a. Similarity comparison of uninfected, infected, and therapeutically manipulated lungs based on dexamethasone suppressed genes. Pearson correlation. Duplicates analyzed for each condition. b. Genes suppressed by both dexamethasone and combined therapy of Remdesivir and anti-IFNAR2 (Log2, Foldchange <−1, P adj<0.05). P adj: For the adjusted P values the Bonferroni correction was used. Duplicates analyzed for each condition. Dexamethasone suppressed genes significantly overlapped with genes significantly suppressed by combined anti-IFNAR2 and Remdesivir therapy (64% overlap). See Table S1 for a full list of genes and their normalized expression. c. Network analysis (STRING v11.0) of genes suppressed by both dexamethasone and combined therapy of Remdesivir and anti-IFNAR2 (as shown in Extended data figure 2b). Duplicates analyzed for each condition. K- means clustering (n=4). d. Pathway (Ingenuity) analysis of genes suppressed by both dexamethasone and combined therapy of Remdesivir and anti-IFNAR2 (as shown in Extended data Fig. 2b). Duplicates analyzed for each condition. Fisher’s Exact Test was used to determine statistical significance in the overlap between the dataset genes and the genes suppressed by therapy. e. Transcriptional landscape of human immune cells at single cell level in uninfected or infected (28dpi). MISTRG6-hACE2 mice. Cluster identifying genes comparing human immune cells from infected (28dpi) or uninfected lungs for 17 clusters shown in Fig 1e. Marker genes for each cluster of cells were identified using the Wilcoxon test with Seurat. Pooled duplicates analyzed for each condition.

Extended data figure 3.. Deeper characterization of monocyte/macrophage clusters at early (4dpi) or late (14 and 28dpi) SARS-CoV-2 infection(matched to figure 1).

a. Heatmap visualizing cluster identifying genes comparing human monocytes and macrophages from infected (4, 14 or 28dpi) or uninfected lungs (as shown in Fig 1g). Pooled duplicates. Uninfected: 438 cells, 4dpi: 336 cells, 14dpi: 793 cells, 28dpi: 1368 cells were analyzed. This analysis allowed step by step characterization of the inflammatory macrophage response. Marker genes for each cluster of cells were identified using the Wilcoxon test (two-tailed) with Seurat. b. Temporal distribution of transcriptional changes associated with monocytes and macrophages in infected (4, 14 or 28dpi) or uninfected lungs (as shown in Fig 1g). Pooled duplicates analyzed. Uninfected: 438 cells, 4dpi: 336 cells, 14dpi: 793 cells, 28dpi: 1368 cells included in analysis. c. Top: Heatmap of representative genes that are differentially regulated (DEGs) in human macrophages from 4, 14, 28dpi lungs compared with uninfected lungs. Uninfected: 438 cells, 4dpi: 336 cells, 14dpi: 793 cells, 28dpi: 1368 cells included in analysis. Bottom: Distribution of interferon stimulated genes within these DEGs. Pooled duplicates analyzed.

Extended data figure 4.. Therapeutics reduced expression of a representative list of interferon stimulated genes- ISGs (DDX58, IFIH1, IFITM3, IRF7) or inflammatory markers (IL6) (matched to figure 1).

Relative expression of interferon inducible or inflammatory genes in treated or untreated MISTRG6- hACE2 mice infected with SARS-CoV-2 mice at 14dpi or 28dpi. Uninfected baseline expression values are presented as reference. The distribution of cells that preferentially express these genes is overlayed on the tSNE plots showing 14dpi and 28dpi human immune cells. IFITM3 and IL6 were particularly enriched in macrophage/monocyte clusters, while IRF7, DDX58 and IFIH1 were enriched in multiple immune cells such as T cells, B cells, and myeloid cells. Normalized to HPRT1. DDX58: uninfected n=3; CTRL-infected: 14dpi n=8, 28dpi n=: 5; Dex 14dpi n=3, 28dpi n= 6; RDV 14 and 28dpi n=3; anti-IFNAR2 14 and 28 dpi n=3, anti-IFNAR2+ Remdesivir 14 and 28dpi n=5 biologically independent mice examined over at least 2 independent experiments. IFIH1: uninfected n=5; CTRL-infected: 14dpi n =11, 28dpi n=: 3; Dex 14 and 28dpi n=4; RDV 14 and 28dpi n=3; anti-IFNAR2 14 and 28 dpi n=3, anti-IFNAR2+ Remdesivir 14 dpi n=4 and 28dpi n=5 biologically independent mice examined over at least 2 independent experiments. IFITM3: uninfected n=4; CTRL-infected: 14dpi n =7, 28dpi n=: 4; Dex 14 and 28dpi n=4; RDV 14 and 28dpi n=3; anti-IFNAR2 14 and 28 dpi n=3, anti-IFNAR2+ Remdesivir 14 and 28dpi n=4 biologically independent mice examined over at least 2 independent experiments. IRF7: uninfected n=4; CTRL-infected: 14dpi n =7, 28dpi n=: 5; Dex 14 and 28dpi n=4; RDV 14 and 28dpi n=3; anti-IFNAR2 14 and 28 dpi n=3, anti-IFNAR2+ Remdesivir 14dpi n=4, 28dpi n=5 biologically independent mice examined over at least 2 independent experiments. IL6: uninfected n=5; CTRL-infected: 14dpi n =9, 28dpi n=: 4; Dex 14dpi n=3, 28dpi n=4; RDV 14 and 28dpi n=3; anti-IFNAR2 14 and 28 dpi n=3, anti- IFNAR2+ Remdesivir 14dpi n=4, 28dpi n=5 biologically independent mice examined over at least 2 independent experiments. Unpaired, two-tailed t-test.

Extended data figure 5.. Anti-IFNAR2 and Remdesivir combined therapy reverses fibrotic transcriptional signature and prevents the transition to fibrosis seen in the infected mice (matched to figure 1).

a. Relative expression of IFNG in treated or untreated MISTRG6-hACE2 mice infected with SARS-CoV-2 mice at 14dpi or 28dpi. Uninfected baseline expression values are presented as reference. Normalized to HPRT1. Uninfected n=7; CTRL infected: 14dpi n =7, 28dpi n=: 6; Dex 14dpi=3, 28dpi= 5; anti-IFNAR2+ Remdesivir 14dpi n=4, 28dpi n=6. over at least 2 independent experiments. Unpaired, two-tailed t-test. b. Heatmap of AT2 cell self-renewal and AT1 differentiation and pre-alveolar type 1 transitional cell state (PATS) associated genes at in uninfected or infected (14dpi) lungs in response to therapeutics. AT2 cell self-renewal and AT1 differentiation gene signature was inhibited while PATS gene signature was enriched in autopsy lungs of patients with severe COVID-19. Top differentially expressed genes in epithelial cluster 7 of autopsy lungs were used in the analysis. Duplicates were analyzed for each condition. Normalized counts of duplicates visualized as min-max transformed values, calculated by subtracting row mean and diving by SD for each gene. Rows (genes) clustered by hierarchical clustering (one-minus Pearson). c. Representative images of Trichrome staining and box and whisker plot (min to max, with all datapoints) of the trichrome scoring of MISTRG6-hACE2 mice treated with a combined therapy of Remdesivir and anti-IFNAR2 or not (CTRL infected). 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. N=4 biologically independent mice examined over 2 independent experiments. Unpaired, two-tailed t-test.

Extended data figure 6.. Cellular source of persistent SARS-CoV-2 viral RNA and sustained viral replication in lungs (matched to figure 2).

a. Quantification of genomic (gRNA) and subgenomic (sgRNA) viral RNA (E-gene) in whole homogenized lung tissue at 4, 14 and 28dpi. 4dpi: n=7, 14dpi n=5, 28dpi n=4 biologically independent mice examined over 3 independent experiments. Means with all datapoints and SD. b. Quantification of genomic (gRNA) and subgenomic (sgRNA) viral RNA (E-gene) in whole homogenized lung tissue at 14dpi in mice treated with combined therapy of Remdesivir and anti-IFNAR2. CTRL: n=4, anti-IFNAR2+RDV: n=4 biologically independent mice examined over 2 independent experiments. N.D.=not detected. c. Quantification of genomic (gRNA) and subgenomic (sgRNA) viral RNA (E-gene) in whole homogenized lung tissue at 28dpi in mice treated with Remdesivir, anti-IFNAR2 or combined therapy of Remdesivir and anti-IFNAR2. N=3 biologically independent mice representative of 2 independent experiments. N.D.=not detected. d. Representative gating strategy for sorting human immune cells (human CD45+) or epithelial cells (mouse EPCAM+) from lungs of mice infected with SARS-CoV-2 and quantification of viral RNA (E and N genes) in these sorted cells. N gene: 4dpi n=3, 14dpi n=6(epithelial), n=5 (immune), 28dpi n=4 (epithelial) n=3 (immune) biologically independent mice analyzed over 3 independent experiments. E gene: 4dpi n=3, 14dpi n=7 (epithelial), n=6 (immune), 28dpi n=4 (epithelial) n=3 (immune) biologically independent mice analyzed over 3 independent experiments. e. mNG signal in epithelial (EPCAM+) cells from lungs and BAL of mice infected with reporter SARS-CoV-2-mNG or control wild type SARS-CoV-2/WA1. mNG is expressed in infected cells following viral replication. Representative of n=4 biologically independent mice examined over 2 independent experiments. f. Representative histograms of mNG expression in human or mouse lung macrophages isolated from BAL of infected MISTRG6-hACE2 mice at 4dpi. Representative of n=3 biologically independent mice examined over 2 independent experiments. g. Frequencies of mNG+ cells within human lung immune cells (hCD45⁺) of SARS-CoV-2-mNG infected MISTRG6-ACE2 mice at 4dpi and 14dpi. 4dpi n=4, 14dpi n=6 biologically independent mice examined over at least 2 experiments. Unpaired, two-tailed t-test. P value=0.066. h. Viral titers measured as PFU using Vero ACE2+ TMPRSS2+ cells that over express ACE2 from lung homogenates of MISTRG6 mice transduced with AAV-hACE2 (+AAV) or not (−AAV) and infected with SARS-CoV-2. MISTRG6-hACE2 (+AAV): 2dpi n=2, 4dpi n=5, 7dpi n=2, 14dpi n=6 MISTRG6(−AAV): 2dpi n=4, 4dpi n=10 and 7, 14 dpi n=2, biologically independent mice representative of at least 2 independent experiments. Viral titers using standard Vero E6 cells do not have any detectable titers (previously reported) in MISTRG6 mice without AAV. Some of the MISTRG6-hACE2 data presented here have been previously reported as part of the characterization of the model. i. Frequencies of mNG+ cells within human macrophages (human CD68+) isolated from lungs of infected MISTRG6 mice transduced with AAV-hACE2 (AAV+) or not (AAV−). MISTRG6 mice with and without AAV-hACE2 were reconstituted with human progenitor cells from the same donor. AAV+ n=6, AAV− n=5 biologically independent mice examined over 3 independent experiments. j. Representative gating strategy for sorting mNG+ and mNG− human immune cells, mNG+ and mNG− mouse epithelial cells and mouse immune cells. Lung cells from SARS-CoV2-mNG infected MISTRG6-hACE2 mice were stained with antibodies against human CD45, mouse CD45, and mouse EPCAM. Sorted cells were used for viral quantification (Fig. 2) and characterization of the inflammasome pathway (Fig. 3).

Extended figure 7.. Viral replication products are detected in human lung macrophages of infected MISTRG6-hACE2 mice (matched to figure 2).

a. Representative fluorescent microscopy images showing colocalization of double stranded RNA (clone rJ2) staining, mNG signal and DAPI staining in fixed lung tissue at 4dpi. Representative of n=4 biologically independent mice examined over 2 independent experiments. b. Representative fluorescent microscopy images of RNA dependent RNA polymerase (RdRp), anti-human CD68 and DAPI staining in fixed lung tissue from SARS-CoV-2 infected or control MISTRG6-hACE2 mice (Non-SARS-CoV-2 pneumonia). Representative of n=7 biologically independent SARS-CoV-2 infected mice examined over 3 independent experiments. Yellow arrows mark RdRp+ human macrophages. Blue arrows mark RdRp− human macrophages. Isotype controls (bottom panels) and non- COVID pneumonia lungs (bacterial infection, top panels) n=3 biologically independent mice are presented as controls. Pseudo-colors were assigned for visualization.

Extended figure 8.. Viral RNA dependent RNA polymerase (RdRp) and Spike in human lung macrophages of MISTRG6-hACE2 mice infected with SARS-CoV-2 (matched to figure 2).

a. Representative fluorescent microscopy images of Spike (S), human CD68, and DAPI staining in fixed lungs of SARS-CoV-2- infected MISTRG6-hACE2 mice. Yellow rectangle provides a higher magnification view of the selected area. Pseudo-colors were assigned for visualization. Representative of n=5 biologically independent mice examined over 3 independent experiments. b. Quantification of viral replication products or machinery in human lung macrophages from SARS-CoV-2 infected MISTRG6-hACE2 mice measured by immunofluorescent staining. Quantification was performed based on representative high-power images (40×) in areas showing diffuse alveolar damage. Frequencies of dsRNA, mNG, RdRp, and Spike positive human macrophages out of hCD68+DAPI+ cells are plotted. dsRNA: 20, 81, 133, 135, 52 human macrophages were counted. mNG: 30, 103, 110 human macrophages were counted. RdRp monoclonal: 187, 59, 85, 106, 142, 63, 59 human macrophages were counted. RdRp polyclonal: 134, 21, 22, 218, 44 human macrophages were counted. Spike (antibody 1): 21, 22, 218, 44 total human macrophages were counted. Spike (antibody 2): 63, 83, 163, 101, 57 human macrophages were counted. N=5 (dsRNA+), N=3 (mNG), N=7 (RdRp+ monoclonal), N=5 (RdRp+ polyclonal), N=4 (Spike-1), N=5 (Spike-2) biologically independent mice representative of at least 2 independent experiment. Means with all datapoints are shown. See supplementary methods for details of antibodies used. c. Representative fluorescent microscopy images and quantification of colocalization of Spike (S), RNA dependent RNA polymerase (RdRp), human CD68, and DAPI staining in fixed lungs of SARS-CoV-2-infected MISTRG6-hACE2 mice. Top panel: isotype control staining of SARS-CoV-2- infected lungs. Middle panel: control lungs with Non-SARS-CoV-2, bacterial pneumonia. Bottom panels: SARS-CoV-2-infected MISTRG6-hACE2 mice. Yellow rectangle provides a higher magnification view of the selected area. Pseudo-colors are assigned for visualization. Representative of n=5 biologically independent mice over 3 independent experiments.

Extended data figure 9.. Viral RNA dependent RNA polymerase (RdRp) and Spike in human macrophages of human autopsy lungs with SARS-CoV-2 pneumonia (matched to figure 2).

Representative fluorescent microscopy images and quantification of colocalization of Spike (S), RNA dependent RNA polymerase (RdRp), human CD68 and DAPI staining in fixed human autopsy lungs with SARS-CoV-2 pneumonia or non-SARS-CoV-2 pneumonia. Quantification was performed based on representative high-power images (40×) in areas showing diffuse alveolar damage. Top panels: Representative of RdRp staining with human CD68; middle panels: Representative of Spike staining with CD68; bottom panels: RdRp and Spike staining in SARS-CoV-2- infected autopsy lungs. Yellow rectangle provides a higher magnification view of the selected area. Pseudo-colors are assigned for visualization. SARS-CoV-2 pneumonia n=4, non-SARS-CoV-2 pneumonia n=3 biologically independent specimens.

Extended data figure 10.. Human lung macrophage infection was enhanced by antibodies and reduced by CD16, ACE2, or RdRp blockade in vivo and in vitro (matched to figure 2).

a. Viral titers in lung homogenates of Remdesivir (RDV) treated or control untreated MISTRG6-hACE2 mice infected with SARS-CoV-2-mNG. CTRL infected n=6, RDV treated n=6 biologically independent mice examined over 3 independent experiments. b. Representative histograms and mean florescent intensity (MFI) for ACE2 expression in mNG+ or mNG− epithelial cells from MISTRG6-hACE2 mice or total epithelial cells from MISTRG6 (AAV−) mice infected with SARS-CoV-2-mNG. AAV+ N=10, AAV− N=6 biologically independent mice examined over at least 3 independent experiments. Paired, two-tailed t-test. c. Representative histograms for ACE2 expression in mNG+ or mNG− human macrophages, human B cells (CD19+) or mouse immune cells isolated from MISTRG6-hACE2 mice infected with SARS-CoV-2-mNG. Representative of N=10 for epithelial cells, N=7 for human macrophages biologically independent mice examined over at least 3 independent experiments. d. MFI of ACE2 expression in mNG+ or mNG− human macrophages or mouse epithelial cells isolated from SARS-CoV-2-mNG infected MISTRG6-hACE2 mice. Epithelial cells n=10, human macrophages n=7 biologically independent mice examined over at least 3 independent experiments. Paired, two-tailed t-test. e. MFI of ACE2 expression in mNG+ or mNG− human macrophages isolated from MISTRG6 (AAV-) mice infected with SARS-CoV-2-mNG. Epithelial cells are virtually not infected with SARS-CoV-2-mNG in MISTRG6 mice without transduced hACE2. N=8 biologically independent mice examined over at least 3 independent experiments. Paired, two-tailed t-test. f. Representative fluorescent microscopy images showing colocalization of human ACE2 and human CD68 cells in SARS-CoV-2 infected MISTRG6-hACE2 mice. Representative of 3 independent mice over 2 independent experiments. g. Anti-Spike (RBD) IgG levels measured by ELISA in serum or lung homogenates of SARS-CoV-2 infected (4 and 14dpi) or uninfected MISTRG6-hACE2 mice treated therapeutically with mAbs (treated at 35hpi or 7dpi) or not. Lung homogenates: Uninfected n=5, 4dpi n=8, 14dpi n=8, 4dpi+mAB n=2, 14dpi+mAB n=2 biologically independent mice representative of at least 2 experiments. Serum: Uninfected n=3, 4dpi n=3, 14dpi n=3, 4dpi+mAB n=3, 14dpi+mAB n=2 biologically independent mice representative of at least 2 experiments. Unpaired, two-tailed t-test. h. Frequencies of mNG signal in human immune cells in infected mice (14dpi) treated therapeutically with monoclonal antibodies^, (mAb) at 7dpi. CTRL infected n=5, mAb treated n=4 biologically independent mice examined over 2 independent experiments. Means with datapoints and SD. Paired, two-tailed t-test. i. Two-way plot showing anti-Spike (RBD) IgG levels and corresponding mNG+ human immune cell proportions in lungs of infected MISTRG6-hACE2 mice at 4dpi. Pearson’s correlation value =0.70. N=8 biologically independent mice examined over 4 independent experiments. j. Frequencies of mNG+ human macrophages in human immune cells in SARS-CoV-2-mNG infected MISTRG6-hACE2 mice treated with anti-CD16 antibody (Abcam-clone SP175) at 7dpi and 11 dpi and analyzed at 14dpi. n=6 biologically independent mice examined over 3 independent experiments. Unpaired, two-tailed t-test. k. Representative histograms and frequencies of mNG+ cells in BMDMs cultured (or not) with SARS-CoV-2-mNG for 48 hours. Cells were treated with pooled plasma from healthy controls (prior to COVID-19 pandemic) or convalescent COVID-19 patients. Uninfected n=3, infected+ healthy plasma n=7, infected+ COVID plasma n=10 independent samples cultured and analyzed over at least 3 experiments. Means with datapoints. Unpaired t-test. P<0.0001=1.57×10⁻⁵. l. Quantification of genomic (gRNA) and subgenomic (sgRNA) viral RNA (E gene) in infected BMDMs at 48hpi. Cells were treated with plasma from healthy controls or convalescent COVID-19 patients. Healthy plasma: n=4, COVID plasma n=6, RDV: n=6, anti-CD16+anti-ACE2 n=4 independent samples analyzed over at least 2 independent experiments. Means with datapoints. Mann-Whitney, two-tailed, t-test. m. Representative histograms and frequencies of mNG+ cells in BMDMs and lung macrophages cultured with SARS-CoV-2 in presence of plasma of convalescent COVID-19 patients. mNG+ macrophages were pre-gated on live (live-dead stain negative) cells at 48hpi. BMDMs N=6, Lung macrophages N=4 independent samples analyzed over 2 independent experiments. Unpaired, two-tailed t-test. n. Frequencies of mNG+ cells in BMDMs cultured with SARS-CoV-2 or not in presence of healthy patient plasma, COVID plasma, monoclonal antibodies (clones 135 and 144) or no antibodies. COVID plasma n=5, mAb n=4, healthy plasma n=4, no Ab n=5 independent samples analyzed over 2 independent experiments. Means with datapoints and SD. The same monoclonal antibody cocktail used was used in vivo (Fig. 3). Unpaired, two-tailed t-test. o. Representative histograms and frequencies of mNG+ cells in BMDMs cultured with SARS-CoV-2-mNG (or not) in presence or absence of COVID plasma. Cultures were treated with Remdesivir, anti-human CD16 antibody and/or anti-human ACE2 antibody. Healthy plasma n=5, COVID plasma n=10, RDV n=5, anti-CD16 n=6, anti-ACE2 n=4, anti-CD16+ACE2 n=4 independent samples analyzed over at least 2 independent experiments. Means with datapoints. Unpaired two-tailed t-test. P values<0.0001: anti-CD16 vs COVID plasma= 1.98×10⁻⁵, RDV vs. COVID plasma= 5.24×10⁻⁶. p. Viral titers and representative plaque images from supernatants of human or mouse BMDMs infected with SARS-CoV-2 mNG in vitro (without COVID plasma). Infectious virus from supernatants of infected macrophage cultures collected at 24hpi, 48hpi and 72 hpi was plaqued using Vero ACE2+TMPRSS2+ cells. Supernatant collected from Vero E6 cell cultures were provided as reference. Human: 24hpi n=9, 48 hpi n=13, 72hpi n=4. Mouse: 24hpi n=6 independent samples analyzed over at least 2 independent experiments. q. Viral titers from supernatants of BMDMs infected with SARS-CoV-2 mNG in vitro and treated with Remdesivir (RDV) or a combination of anti-CD16 and anti-ACE2 antibodies. Cultures were not supplemented with COVID plasma. Infectious virus from supernatants of infected macrophage cultures collected at 24hpi was plaqued using Vero ACE2+TMPRSS2+ cells. CTRL n=9, RDV n=4, anti-CD16 and anti-ACE2 n=4 independent samples representative of 2 independent experiments. Means with datapoints. r. Viral titers measured as PFUs using supernatants containing concentrations of Remdesivir (1μm) or anti-ACE2 (1μg/ml) and anti-CD16 antibodies diluted to (1:10) allow quantification of PFUs at 24hpi from macrophage cultures. Supernatants were applied on Vero ACE2+ TMPRSS2+ cells which were then infected with a matched inoculum of SARS-CoV-2 mNG (10³ PFU quantified in Vero-E6 cells) to test carry over effect in plaque quantification. Untreated N=9, RDV N=6, anti-ACE2+anti-CD16 n=4 independent datapoints collected over 3 independent experiments. Means with datapoints. Unpaired, two-tailed, t-test.

Extended data figure 11.. SARS-CoV-2 infected human macrophages have a unique transcriptional signature (matched to figure 3).

a. Representative gating strategy of CXCL10 or TNF producing human macrophages in MISTRG6-hACE2 mice infected with SARS-CoV-2-mNG. b. Representative flow cytometry plots of CXCL10 and TNF staining in mice therapeutically treated with mAb or control untreated mice. Representative of n=4 biologically independent mice. C. CXCL10 production measured by ELISA in supernatants of BMDMs infected with SARS- CoV-2 in vitro. Infected BMDM cultures were supplemented with pooled plasma from COVID-19 and were treated with Remdesivir or not. Uninfected n=5, CTRL infected n=12, RDV n=4 over 3 independent experiments. Means with individual values are plotted. Unpaired, two-tailed t-test. c. Spearman correlation values of each gene based on its correlation with CXCL10 or TNF or TLR7. d. Expression and distribution of CXCL10, TNF and TLR7 in human immune cells from infected (4, 14 and 28dpi) MISTRG6-hACE2 mice.

Extended data figure 12.. CXCL10-associated genes(matched to figure 3).

a. Network (STRING v11.0) analysis of top CXCL10-associated genes (top 200 genes). K-means clustering. Clusters and their corresponding pathway analysis are available as source files. Top genes that correlate with CXCL10 (4dpi, Pearson and Spearmen correlation combined) are enriched for distinct inflammatory molecules. b. Network (STRING) analysis of genes that are preferentially associated with CXCL10 but not with TLR7 or TNF. Disconnected nodes in the network are not displayed. K-means clustering. Clusters and their corresponding pathway analysis are presented as source files. c. Proportions of TNF or CXCL10 producing macrophages among alveolar (CD206^hiCD68⁺) macrophages. Unpaired, two-tailed t-test. N=6 biologically independent mice examined over 3 independent experiments. MISTRG6-hACE2 mice were infected with SARS-CoV-2-mNG and lungs were analyzed at 4dpi. d. Distribution of CXCL10 or TNF associated genes at 4, 14, 28 dpi in lungs infected with SARS-CoV-2 or not. Analysis performed on macrophages of 4dpi lungs in Fig 3d was extended to more timepoints. Pearson (right) and Spearman (left) correlation values were calculated for each gene for its correlation with CXCL10 or TNF in human monocytes and macrophages isolated from uninfected and infected (4, 14 and 28 dpi) lungs. K-means clustering analysis.

Extended data figure 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).

a. Representative images of single stained cells for ASC specks, NLRP3, 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 cell (mCD45⁺) and mouse epithelial cell (EPCAM⁺). Representative of n=5 independent mouse examined over 3 independent experiments. b. Visualization of ASC specks as a measure of inflammasome activation in mNG+ (SARS-Cov2+) or mNG− (SARS-Cov2−) human immune cells at 4dpi. 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 4dpi and 14dpi. 4dpi: n=3 biologically independent mice examined over 2 independent experiments, 14dpi n=5 biologically independent mice examined over 3 independent experiments. Lung cells were incubated with FLICA-Casp1 substrate for 30 minutes. Means with individual datapoints plotted. Paired, two-tailed t-test. P<0.0001=4.29×10⁻⁹. d. Quantification of Casp1-FLICA staining as a measure of active caspase-1 in infected (mNG+) human or total mouse CD11b+ cells at 4dpi. 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=1.79×10⁻⁹. 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 4dpi. 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 4dpi. 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 4dpi and 14dpi. Fresh serum was assayed for LDH. Uninfected n=3, 4dpi n=4, 14dpi 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 hours. 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. Coloring 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: 5.85×10⁻⁶. j. Representative histograms and quantification of IL-1β in supernatants of BMDMss infected with (or not) SARS-CoV-2 in vitro for 48 or 72 hours. Uninfected n=7, 48hpi n=10, 72hpi 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 72hpi= 4.96×10⁻⁷, 48hpi vs 72hpi=1.17 ×10⁻⁸. k. Human IL-18 levels at 48hpi in supernatants of BMDMss infected or not with SARS-CoV-2 in vitro. BMDMss cultures were supplemented with COVID plasma for the duration of the infection. Uninfected n=4, 48hpi 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 48hpi in supernatants of BMDMss infected with SARS-CoV-2 in vitro or not. Uninfected n=3, infected 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. m. Gasdermin D (GSDMD) levels in supernatants of BMDMss infected with (or not) SARS-CoV-2 in vitro for 48 hours. 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 490nm in supernatants of infected or uninfected. Uninfected n=6, Infected n=11 independent datapoints collected over 3 independent experiments. Means with individual datapoints. Unpaired, two-tailed t-test. P-value= 7.38×10⁻⁶. o. Zombie Aqua incorporation in uninfected or SARS-CoV-2-mNG infected BMDMs. Frequencies of Zombie+ cells within Annexin V− population at 48hpi are presented. Uninfected n=4, infected n=7 independent datapoints collected over 3 independent experiments. Means with individual datapoints. Unpaired, two-tailed t-test.

Extended data figure 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).

a. Human IL-18 levels measured in lung homogenates of infected (4dpi) MISTRG6-hACE2 mice treated (or not) with CD16 blocking antibody (Abcam). CTRL-infected N=7, ant-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 (4dpi) 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 (4dpi) 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. Mean with individual values. Unpaired, two-tailed t-test. d. Human IL-18 levels measured in serum of infected (14dpi) MISTRG6-hACE2 mice treated (or not) with CD16 blocking antibody (Abcam). Mice were treated with anti-CD16 blocking antibody at 7dpi and 11dpi. 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 (4dpi) MISTRG6 mice treated (or not) with anti-ACE2 antibody (Abcam). Mice were treated with anti-ACE2 antibody at 1,2,3 dpi. 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=1.43×10⁻⁵, CTRL-infected vs anti-ACE2=6.95×10⁻⁵. 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 (4dpi) 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 4dpi 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 36hpi or not. Sera from infected mice were analyzed at 4dpi. 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 (4dpi) or uninfected MISTRG6-hACE2 mice that were therapeutically treated with mAb at 36hpi 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 (14dpi) MISTRG6-hACE2 mice treated (or not) with mAb (clone 135+ clone 144) at 7dpi and analyzed at 14dpi. 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 7dpi and analyzed at 14dpi. Mean with individual values. Unpaired, two-tailed t-test. 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 4dpi. Mice were either treated with monoclonal antibodies at 35hpi or anti-CD16 at 2dpi. 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 14dpi. Mice were either treated with monoclonal antibodies at 7dpi or anti-CD16 at 7 and 11dpi. 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= 5.0×10⁻⁵. 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.

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.

Figure 1.. Targeting viral replication and downstream interferon signaling ameliorates chronic COVID-19.

a. Therapy Schematic: SARS-CoV-2 infected MISTRG6-hACE2 mice were treated with Dexamethasone(dex) and Remdesivir(RDV) at 7,8,9 dpi, with anti-IFNAR2 at 7, 11dpi and analyzed at 14 or 28dpi. b. Post-infection weight changes. 28dpi: CTRL-infected n=5, Dex, anti-IFNAR2+RDV n=4, RDV, anti-IFNAR2 n=3 mice examined over at least 2 experiments. Means with SD. Unpaired, two-tailed t-test. c. Human macrophages in lungs. Uninfected: n=10; 14dpi: CTRL-infected n=7 Dex, RDV, anti-IFNAR, anti-IFNAR+RDV n=4; 28dpi: CTRL-infected, Dex n=4, RDV, anti-IFNAR n=3, anti-IFNAR+RDV n=6 mice examined over 3 experiments. Means with datapoints. Unpaired, two-tailed t-test. d. Heatmap of genes suppressed by therapy in lungs (Log2, Foldchange >1; P-adj with the Bonferroni correction <0.05). Differential expression by DESeq2. Statistics by Wald-test. Transformed (min-max), normalized counts of duplicates. Hierarchical clustering (one-minus Pearson). e. t-distributed stochastic neighbor embedding (t-SNE) plot of human immune cells from uninfected or infected lungs (28dpi). Pooled duplicates. Cluster marker genes identified with Wilcoxon-test (Extended data Fig. 2e). Uninfected=3,655, 28dpi=3,776 cells analyzed. f. t-SNE plots highlighting differentially abundant (DA) human immune cell populations identified by DA-seq. Top: Distribution/enrichment of DA-populations. Bottom: DA-clusters. g. t-SNE plots of human monocyte/macrophage clusters from 4dpi, 14dpi and 28dpi and uninfected lungs. Left: dpi, right: clusters. Different conditions integrated as described in methods. Marker genes identified with Wilcoxon-test (Extended data Fig.3a,b). P-adj with the Bonferroni correction. Uninfected=438, 4dpi=336, 14dpi=793, 28dpi=1368 cells analyzed. h. Heatmap visualizing response to the combined therapy based on DEGs associated with monocytes and macrophages. Transformed (min-max), normalized expression of duplicates. Hierarchical clustering (one-minus Pearson). i. Representative H&E staining and box plot of histopathological scores. Uninfected n=6, CTRL-infected n=7, anti-IFNAR2+RDV n=4 mice examined over 3 experiments. Whiskers: smallest/minimum to the largest/maximum value. Box: 25^th-75^th percentiles. Center line: median. Unpaired, two-tailed, t-test. Data associated with dexamethasone used here as a control have been reported.

Figure 2.. SARS-CoV-2 replicates in human macrophages.

a. Representative flow cytometry plots and frequencies of mNG+ human (CD68+) or mouse (F4/80+) lung macrophages in SARS-CoV-2-mNG infected MISTRG6-hACE2 mice. Human n=7, mouse n=6 mice over at least 3 experiments. Unpaired, two-tailed t-test. b. Quantification of gRNA and sgRNA (E-gene)^, in sorted mNG+ or mNG− epithelial cells or human immune cells. N=3 mice over 2 experiments. Means with datapoints. c. Representative fluorescent microscopy images of dsRNA (rJ2), CD68 and DAPI staining in fixed lung tissues from SARS-CoV-2 infected MISTRG6-hACE2 mice. Representative of n=5 mice examined over 3 experiments. Yellow rectangle: higher magnification view of the selected area. Yellow arrow: colocalization of CD68 with dsRNA. Pseudo-colors were assigned. d. Representative fluorescent microscopy images of RdRp, CD68 and DAPI staining in fixed lung tissues from SARS-CoV-2 infected MISTRG6-hACE2 mice. Representative of n=5 mice examined over 3 experiments. Yellow arrows: colocalization of human CD68 with dsRNA. Yellow rectangle: higher magnification view of the selected area. Pseudo-colors were assigned. e. Frequencies of mNG+ human immune cells in Remdesivir treated (1–3dpi) or control MISTRG6-hACE2 mice infected with SARS-CoV-2-mNG. N=6 mice examined over 3 experiments. Means with datapoints. Paired t-test, two-tailed. f. Frequencies of mNG+ human immune cells upon ACE2 blockade (1–3dpi) in MISTRG6 (no AAV) mice infected with SARS-CoV-2-mNG. CTRL-infected n=5, anti-ACE2 treated n=6 mice examined over 2 experiments. Means with datapoints. Paired t-test, two-tailed. g. Representative flow cytometry plots and frequencies of mNG+ macrophages in infected MISTRG6-hACE2 mice treated with monoclonal antibodies (mAb)^,, at 35hpi. CTRL-infected n=7, treated n=4 mice examined over 2 experiments. Means with datapoints. Unpaired, two-tailed t-test. h. Frequencies of mNG+ human immune cells in MISTRG6-hACE2 mice after CD16 blockade (Abcam, 2dpi). N=6 mice examined over 3 experiments. Means with datapoints shown. Paired t-test, two-tailed.

Figure 3.. SARS-CoV-2 infection of human macrophages activates inflammasomes and pyroptosis.

a. CXCL10+ or TNF+ human macrophages. Representative of n=6 mice over 3 experiments. b. CXCL10+ lung macrophage frequencies upon mAb or Remdesivir therapy. CTRL-infected n=9, mAb n=4, RDV n=6 mice over 2 experiments. Means with datapoints. Unpaired, two-tailed-t-test. c. Serum CXCL10 levels upon mAb or Remdesivir therapy. Means with datapoints. Uninfected, mAb n=4; CTRL-infected n=7, RDV n=3 mice examined over 2 experiments. Unpaired, two-tailed-t-test. d. Correlation (Pearson and Spearman) of each gene with CXCL10, TNF or TLR7 in human lung monocytes and macrophages. K-means clustering. P-values: T-distribution with length(x)-2 degrees of freedom or algorithm AS 89 with exact = TRUE. Two-tailed. e. Representative plots and AM frequencies within mNG+ or mNG-macrophages. N=8 mice examined over 4 experiments. f. ASC speck visualization/quantification and colocalization with active caspase-1 (FLICA) in mNG+ or mNG-human immune cells from MISTRG6-hACE2 mouse lung. Cells sorted based on Extended data Fig. 6j. 1000-cells analyzed/per condition. ASC+specks: 4dpi n=3(A), 5(B-D); 14dpi n=3 mice, FLICA: n=3 mice examined over at least 2 experiments. Means with datapoints, SD. Unpaired, two-tailed-t-test. g. ASC speck visualization/quantification and colocalization with NLRP3 oligomerization in sorted mNG+ or mNG-human lung immune cells. 1000-cells analyzed/per condition. N=3 mice over 2 experiments. Means with datapoints, SD. Unpaired, two-tailed-t-test. h. Serum IL-18, IL-1RA and GSDMD levels. IL-18: n =4 mice examined over 2 experiments. IL-1RA: Uninfected n=5, 4dpi n=7, 14dpi n=4 mice examined over 3 experiments. GSDMD: uninfected, 4dpi n=4, 14dpi n=5 mice over 3 experiments. Means with datapoints. Unpaired, two-tailed t-test. P-value<0.0001=3.32×10⁻⁷. i. Serum IL-18 and IL-1RA levels in mice treated with CD16-blocking (Abcam) or depleting (ThermoFisher), antibodies or Remdesivir. IL-18: uninfected, CD16-blocking, CD16-depletion n=4, CTRL-infected n=7, RDV n=3; IL-1RA: uninfected n=5, CTRL-infected n=7, RDV n=6, CD16-blocking, CD16-depletion n=4 mice examined over at least 2 experiments. Means with datapoints. Unpaired, two-tailed t-test. P-values<0.0001:uninfected=3.28×10⁻⁵, CD16-depl=7.92×10⁻⁷.

Figure 4.. Inflammasome inhibition ameliorates inflammation and disease in infected MISTRG6-hACE2 mice.

a. Schematic of inflammasome inhibition in vivo. SARS-CoV-2 infected MISTRG6-hACE2 mice treated with caspase-1 or NLRP3 inhibitors, 6–12dpi. b. Frequencies of mNG+ human immune cells upon inflammasome inhibition. CTRL-infected n=5, caspase-1 inhibitor n=6, NLRP3 inhibitor n=4 mice examined over at least 2 experiments. Means with datapoints. Paired, two-tailed t-test. c. Frequencies of CXCL10+ or TNF+ human lung macrophages upon inflammasome inhibition. CTRL-infected n=5, caspase-1 inhibitor n=5, NLRP3 inhibitor n=4 mice examined over at least 2 experiments. Means with datapoints. Unpaired, two-tailed t-test. d. Quantification of active caspase-1 in mNG+ human macrophages upon inflammasome inhibition. CTRL-infected n=5, Casp1-inhibitor n=5, NLRP3-inhibitor n=4 mice examined over at least 2 experiments. Means with datapoints. Unpaired, two-tailed t-test. e. Serum human IL-18 levels upon inflammasome inhibition. CTRL, NLRP3: n=4, Casp1 n=5 mice examined over 2 experiments. Means with datapoints. Unpaired, two-tailed t-test. P<0.0001=1.00114×10⁻⁵. f. Human IL-1RA levels in lung homogenates upon inflammasome inhibition. N=4 mice examined over 2 experiments. Means with datapoints. Unpaired, two-tailed t-test. g. Serum GSDMD levels upon inflammasome inhibition. CTRL-infected n=5, Casp1, NLRP3 inhibitors n=4 mice examined over 2 experiments. Means with datapoints. Unpaired, two-tailed t-test. h. Box and whisker plot of histopathological scores upon inflammasome inhibition. CTRL-infected n=4, Casp1-inhibitor n=6, NLRP3-inhibitor n=4 independent mice over at least 2 experiments. Whiskers: smallest (minimum) to the largest value (maximum). Box: 25^th-75^th percentiles. Center line: median. Unpaired, two-tailed t-test. i. Viral titers from supernatants of BMDMs infected with SARS-CoV-2-mNG in vitro and treated with Casp1 or NLRP3 inhibitors. CTRL-infected: n=13, Casp1-inhibitor-: n=8, NLRP3 inhibitor: n=5 independent datapoints collected over 3 experiments. Means with datapoints. Unpaired two-tailed t-test.