Modulation of type I interferon responses potently inhibits SARS-CoV-2 replication and inflammation in rhesus macaques

Abstract

Type I interferons (IFN-I) are critical mediators of innate control of viral infections but also drive the recruitment of inflammatory cells to sites of infection, a key feature of severe coronavirus disease 2019. Here, IFN-I signaling was modulated in rhesus macaques (RMs) before and during acute SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection using a mutated IFN-α2 (IFN-modulator; IFNmod), which has previously been shown to reduce the binding and signaling of endogenous IFN-I. IFNmod treatment in uninfected RMs was observed to induce a modest up-regulation of only antiviral IFN-stimulated genes (ISGs); however, in SARS-CoV-2-infected RMs, IFNmod reduced both antiviral and inflammatory ISGs. IFNmod treatment resulted in a potent reduction in SARS-CoV-2 viral loads both in vitro in Calu-3 cells and in vivo in bronchoalveolar lavage (BAL), upper airways, lung, and hilar lymph nodes of RMs. Furthermore, in SARS-CoV-2-infected RMs, IFNmod treatment potently reduced inflammatory cytokines, chemokines, and CD163⁺ MRC1^- inflammatory macrophages in BAL and expression of Siglec-1 on circulating monocytes. In the lung, IFNmod also reduced pathogenesis and attenuated pathways of inflammasome activation and stress response during acute SARS-CoV-2 infection. Using an intervention targeting both IFN-α and IFN-β pathways, this study shows that, whereas early IFN-I restrains SARS-CoV-2 replication, uncontrolled IFN-I signaling critically contributes to SARS-CoV-2 inflammation and pathogenesis in the moderate disease model of RMs.

Fig. 1.. Administration of IFNmod in uninfected RMs resulted in modest upregulation of ISGs without changes to inflammatory genes or inflammatory cytokines and chemokines.

(a) Study design of IFNmod treatment in uninfected RMs; 1mg IFNmod was administered intramuscularly to n=4 uninfected RMs (7–10 years old, median = 9.5 years) for four consecutive days. Blood and BAL were collected at pre-treatment baseline (3 days before treatment initiation) and once a day from days 1–3 post treatment initiation, with the exception of day 2 post treatment initiation, where only blood was collected. Heatmaps of ISG expression in (b) BAL and (c) PBMC of uninfected RMs before and after IFNmod treatment. Heatmaps of genes associated with IL-6 signaling and inflammation in (d) BAL and (e) PBMC of uninfected RMs before and after IFNmod treatment. (f) Distribution of log2 fold-changes of IRF1 relative to pre-treatment baseline (−3d tx). Filled dots represent the mean, and lighter dots are individual data points. (g) Fold change of cytokines and chemokines in BAL fluid relative to pre-treatment baseline (−3d tx) measured by mesoscale. Statistical analyses for mesoscale analysis were performed using one-tailed Wilcoxon signed-rank tests. Each black, open symbol represents an uninfected RM. Black lines represent median fold change. Gray-shaded boxes indicate that timepoint occurred during IFNmod treatment. The color scale depicted in the top right of panel c indicates log2 expression relative to the mean of all samples and is applicable to panels b-e.

Fig. 2.. Administration of IFNmod in Calu-3 human lung cells modulated type I IFN responses and inhibited SARS-CoV-2 replication to levels comparable to Nirmatrelvir.

(a) Overview of uninfected Calu-3 cell culture setup with IFNmod (0.004, 0.04, and 0.4 µg/ml) +/− IFNα (0.04 µg/ml) treatment. mRNA fold induction of (b) antiviral genes OAS1 and Mx1 and (c) inflammatory gene CXCL10 in Calu-3 cells following IFNmod (0.004, 0.04, or 0.4 µg/ml) +/− IFNα (0.04 µg/ml) treatment from three independent Calu-3 experiments. Statistical analyses were performed using ordinary ANOVA with Dunnett’s multiple comparisons tests comparing IFNmod +/− IFNα treated samples to the respective no IFNmod controls. (d) Overview of Calu-3 SARS-CoV-2 cell culture setups with IFNmod (0.004, 0.04, and 0.4 µg/ml), IFNα (20, 100 and 500 IU/ml), or Nirmatrelvir (0.1, 1, and 10µM) treatment initiated pre-infection. (e) Viral N RNA copies/mL quantified by qRT-PCR and (f) normalized relative to the no treatment condition (N= 3 +/− SEM). Statistical analyses were performed using ordinary ANOVA with Dunnett’s multiple comparisons tests comparing untreated to IFNmod-treated samples. mRNA fold induction of (g) antiviral gene OAS1 and (h) inflammatory gene CXCL10 in SARS-CoV-2-infected Calu-3 cells following IFNmod (0.004, 0.04, or 0.4 µg/ml), IFNα (20, 100 and 500 IU/ml), or Nirmatrelvir (0.1, 1, and 10µM) treatment that was initiated pre-infection relative to untreated, uninfected controls. Statistical analyses were performed using ordinary ANOVA with Dunnett’s multiple comparisons test comparing untreated, infected samples to treated, infected samples. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value <0.0001.

Fig. 3.. IFNmod administration reduced viral loads of SARS-CoV-2 infected RMs.

(a) Study Design; n=18 RMs (6–20 years old, mean = 10 years; 10 males and 8 females) were infected intranasally and intratracheally with SARS-CoV-2. 1 day prior to infection (−1 dpi), n=9 RMs started a 4-dose regimen of IFNmod (1mg/day) that continued up until 2 dpi while the other n=9 RMs remained untreated. RMs were sacrificed at 2 dpi (n=3 RMs per treatment arm), 4 dpi (n=3 RMs per treatment arm), or 7 dpi (n=3 RMs per treatment arm). Levels of SARS-CoV-2 (b-e) gRNA N and (f-i) sgRNA E in BAL, nasopharyngeal swabs, throat swabs, and cranial (upper) lung, caudal (lower) lung, and hilar lymph nodes (LNs) of RMs. For BAL viral loads, n=9 RMs per treatment arm for −7 and 2 dpi, n=6 RMs per treatment arm for 4 dpi, and n=3 RMs per treatment arm for 7 dpi. For nasopharyngeal and throat swab viral loads, n=9 RMs per treatment arm for −7, 1, and 2 dpi, n=6 RMs per treatment arm for 3 and 4 dpi, and n=3 RMs per treatment arm for 5 and 7 dpi. Lung and hilar LN viral loads are from RMs necropsied at 2 dpi (n=3 RMs per treatment arm). Untreated animals are depicted in red and IFNmod-treated animals are depicted in blue. Black lines in h-m represent median viral loads for each treatment group at each timepoint. Gray-shaded boxes indicate that timepoint occurred during IFNmod treatment. Statistical analyses were performed using non-parametric Mann-Whitney tests. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value <0.0001.

Fig. 4.. IFNmod administration resulted in lower levels of lung pathology and inflammatory cytokines and chemokines in SARS-CoV-2-infected RMs.

(a) Individual lung pathology scoring parameters, (b) total lung pathology scores, and (c) average lung pathology scores per lobe of RMs necropsied at 4 and 7 dpi (n=6 RMs per treatment arm). (d-k) Fold change of cytokines and chemokines in BAL fluid relative to –7 dpi measured by mesoscale. One untreated animal was excluded from all mesoscale analysis due to abnormally high baseline levels. n=8 Untreated and 9 IFNmod for 2 dpi, n=5 Untreated and 6 IFNmod for 4 dpi, and n=2 Untreated and 3 IFNmod for 7 dpi. Untreated animals are depicted in red and IFNmod treated animals are depicted in blue. Black lines represent the median viral load, pathology score, or fold change in animals from each respective treatment group. Gray-shaded boxes indicate that timepoint occurred during IFNmod treatment. Statistical analyses were performed using two-sided non-parametric Mann-Whitney tests. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value <0.0001.

Fig. 5.. IFNmod-treated RMs had lower frequencies of CD14+CD16+ monocytes and expression of Siglec-1 compared to untreated RMs.

(a) Representative staining of classical (CD14+CD16-), non-classical (CD14-CD16+), and inflammatory (CD14+CD16+) monocytes in peripheral blood throughout the course of infection with frequency as a percentage of total monocytes. (b-c) Frequency as a percentage of total monocytes and fold change relative to pre-infection baseline (−7 dpi) of inflammatory (CD14+CD16+) monocytes in peripheral blood. (d) Representative staining and (e) frequency as a percentage of total monocytes of Siglec-1+ CD14+ monocytes in peripheral blood. (f) Frequency as a percentage of CD14+ monocytes that were Siglec-1+ in peripheral blood. (g) MFI of Siglec-1 on CD14+ monocytes in peripheral blood. Untreated animals are depicted in red and IFNmod treated animals are depicted in blue. In representative staining plots, frequencies of each quadrant are bolded. Black lines represent the median frequency or fold change in animals from each respective treatment group. Gray-shaded boxes indicate that timepoint occurred during IFNmod treatment. Statistical analyses were performed using two-sided non-parametric Mann-Whitney tests. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value <0.0001.

Fig. 6.. IFNmod treatment suppresses gene expression of ISGs, inflammation, and neutrophil degranulation in the BAL of SARS-CoV-2 infected NHPs.

Bulk RNA-Seq profiles of BAL cell suspensions obtained at −7 dpi (n=9 per treatment arm), 2 dpi (n=9 per treatment arm), 4 dpi (n=6 per treatment arm), and 7 dpi (n=3 per treatment arm). (a) Heatmap of longitudinal gene expression in BAL prior to and following SARS-CoV-2 infection for the ISG gene panel. The color scale indicates log2 expression relative to the mean of all samples. Samples obtained while the animals were receiving IFNmod administration are depicted by a blue bar. (b) Distribution of log2 fold-changes of select ISGs relative to baseline. Filled dots represent the mean, and lighter dots are individual data points. Asterisks indicate statistical significance (p_adj < 0.05) of gene expression relative to baseline within treatment groups; black horizontal bars indicate BH corrected p-values of direct contrasts of the gene expression between groups at time-points (i.e. IFNmod vs Untreated) using the Wald test and the DESeq2 package. (c-e) GSEA enrichment plots depicting pairwise comparison of gene expression of 2 dpi samples vs −7 dpi samples within treatment groups. The untreated group is depicted by red symbols, and data for the IFNmod treated group is shown in blue. The top-scoring (i.e. leading edge) genes are indicated by solid dots. The hash plot under GSEA curves indicate individual genes and their rank in the dataset. Left-leaning curves (i.e. positive enrichment scores) indicate enrichment, or higher expression, of pathways at 2 dpi, right-leaning curves (negative enrichment scores) indicate higher expression at −7 dpi. Sigmoidal curves indicate a lack of enrichment, i.e. equivalent expression between the groups being compared. The normalized enrichment scores and nominal p-values testing the significance of each comparison are indicated. Gene sets were obtained from the MSIGDB (Hallmark and Canonical Pathways) database. (f-h) Heatmaps of longitudinal gene expression after SARS-CoV-2. Genes plotted are the top 10 genes in the leading edge of gene set enrichment analysis calculated in panels c-e for each pathway in the Untreated 2 dpi vs −7 dpi comparison. (i-l) Longitudinal gene expression for selected DEGs in immune signaling pathways. The expression scale is depicted in the top right of panel a and is applicable to panels a, f, g, h, i, j, k, and l.

Fig. 7.. Effect of IFNmod treatment on gene expression of BAL single-cells using 10X.

(n=6 Untreated, n=6 IFNmod except n=5 for Untreated −7dpi, IFNmod 4dpi, and IFNmod 7 dpi) (a) UMAP of BAL samples (62,081 cells) integrated using reciprocal PCA showing cell type annotations. UMAP split by treatment and time points are also shown. (b) Mapping of macrophage/monocyte cells in the BAL of SARS-CoV-2-infected untreated and IFNmod treated RMs to different lung macrophage/monocyte subsets from healthy rhesus macaque (84). (c) Percentage of different macrophage/monocyte subsets out of all the macrophage/monocytes in BAL at −7 dpi and 2 dpi from untreated and IFNmod treated RMs. The black bars represent the median. Statistical analyses between treatment groups were performed using non-parametric Mann-Whitney tests. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value <0.0001. (d) Violin Plots showing the percentage of viral reads in each cell for total macrophages/monocytes and individual macrophages/monocytes subsets at 2 dpi determined using the PercentageFeatureSet in Seurat. (e-h) Dot Plots showing expression of selected (e) ISGs, (f) inflammatory genes, (g) chemokines, and (h) inflammasome genes. The size of the dot indicates the percentage of cells that express a given a gene and the color indicates the level of expression. The numbers of CD16+ monocytes were very low and have been thus omitted.

Fig. 8.. Effect of IFNmod treatment on lung cells.

(a) UMAP based on reciprocal PCA of lung single cells collected at 2 dpi (n = 2 Untreated, 2 IFNmod) and 7 dpi (n = 1 Untreated, 1 IFNmod). The cells were classified into four broad categories – epithelial, lymphoid, myeloid and others (stromal and endothelial). The cells from each category were subset and clustered separately. UMAPs for each category with cell type annotations are also shown. (b) Selected gene sets that were found to be enriched (p-adjusted value < 0.05) in lung cells from untreated RMs at 2 dpi based on over-representation analysis using Hallmark, Reactome, KEGG, and BioCarta gene sets from msigdb. The size of the dots represents the number of genes that were enriched in the gene set and the color indicates the p-adjusted value. The gene set id in order are: M983, M15913, M27255, M27253, M5902, M5890, M5921, M27250, M41804, M5897, M5932, M27698, M27251, M29666, M27436, M27895, M27897, M1014. (c-f) Dot plots showing gene expression in lung cells present at higher frequencies from untreated and IFNmod treated macaques at 2 dpi (c) ISG, (d) genes related to inflammasome, (e) inflammation, and (f) programmed cell death. The size of the dot represents the percent of cells expressing a given gene and the color indicates the average expression.