IFN signaling and neutrophil degranulation transcriptional signatures are induced during SARS-CoV-2 infection

Abstract

SARS-CoV-2 virus has infected more than 92 million people worldwide resulting in the Coronavirus disease 2019 (COVID-19). Using a rhesus macaque model of SARS-CoV-2 infection, we have characterized the transcriptional signatures induced in the lungs of juvenile and old macaques following infection. Genes associated with Interferon (IFN) signaling, neutrophil degranulation and innate immune pathways are significantly induced in macaque infected lungs, while pathways associated with collagen formation are downregulated, as also seen in lungs of macaques with tuberculosis. In COVID-19, increasing age is a significant risk factor for poor prognosis and increased mortality. Type I IFN and Notch signaling pathways are significantly upregulated in lungs of juvenile infected macaques when compared with old infected macaques. These results are corroborated with increased peripheral neutrophil counts and neutrophil lymphocyte ratio in older individuals with COVID-19 disease. Together, our transcriptomic studies have delineated disease pathways that improve our understanding of the immunopathogenesis of COVID-19.

Fig. 1. Gene expression profiles for lung tissue samples from macaques infected with SARS-CoV-2 are distinct from naive uninfected samples.

a PCA plot showing the clustering of samples based on overall transcriptomic profiles. b Differential gene expression plot showing the relative normalized gene expression levels (FPKM) for each gene, with genes significantly differentially regulated by COVID-19 are indicated.

Fig. 2. Genes differentially regulated in SARS-CoV-2-infected macaques represent pathways characteristic of neutrophil degranulation, IFN signaling, collagen degradation and TFG-β signaling.

The top 20 most significantly a upregulated genes and b downregulated genes in SARS-CoV-2-infected macaque lungs. Expression values are visualized by Z scores of normalized expression data (FPKM) per sample, and Log₂ Fold Change and -Log P values are from the DESeq2 output. Genes are sorted by P value. c Gene expression plot showing the relative normalized gene expression levels (FPKM) for each gene, with “neutrophil degranulation” (R-HSA-6798695) pathway genes highlighted, including those that were significantly upregulated during COVID-19 (blue) and those that were not (orange). d Multilabel confocal immunofluorescence microscopy of FFPE lung sections from SARS-CoV-2-infected rhesus macaques with SARS-CoV-2 Spike specific antibody (green), neutrophil marker CD66abce (red), and DAPI (blue) at 63X magnification.

Fig. 3. 86 genes were significantly upregulated and 96 genes were significantly downregulated with COVID-19 only in juvenile macaques.

Heatmaps visualizing the significance values of COVID-19 a upregulated and b downregulated genes, in juvenile and old macaques. Green shaded areas contain genes significant only in juveniles, and red shaded areas contain genes significant only in old macaques.

Fig. 4. Genes related to Type I interferon signaling are upregulated in juvenile macaques compared to old macaques during COVID-19-infection.

The top 20 most significantly a upregulated genes and b downregulated genes in SARS-CoV-2 infected juvenile macaque lungs but not in old macaques. Expression values are visualized by Z scores of normalized expression data (FPKM) per sample, and Log₂ Fold Change and -Log P values are from the DESeq2 output. Genes are sorted by P value. c The relative gene expression of ACE2 and ADAM17 among naive, juvenile and old SARS-CoV-2-infected macaques.

Fig. 5. Interferon alpha signaling genes are significantly upregulated in juvenile SARS-CoV-2-infected macaques but not old SARS-CoV-2-infected macaques.

a Differential expression significance values for genes from the “Interferon-alpha signaling” (R-HSA-909733) pathway, in both the juvenile vs naive and old vs naive comparisons. Included are significance values for all genes (gray), pathway genes significantly upregulated in juvenile but not old (orange) and other pathway genes (green). P value cutoffs (0.01 and 0.1) are indicated with dashed lines. b The relative expression levels (FPKM) for the five “interferon-alpha signaling” genes belonging to this gene set are shown. P values represent FDR-corrected significance values from DESeq2.

Fig. 6. Genes related to VEGF signaling are downregulated in old macaques compared to juvenile macaques during COVID-19.

The top 20 most significantly a upregulated genes and b downregulated genes in infected old macaque lungs but not in juvenile macaques. Expression values are visualized by Z scores of normalized expression data (FPKM) per sample, and Log₂ fold change and -Log P values are from the DESeq2 output. Genes are sorted by P value.

Fig. 7. VEGF pathway genes are significantly downregulated in old SARS-CoV-2- infected macaques but not juvenile SARS-CoV-2-infected macaques.

a Differential expression significance values for genes from the “Signaling by VEGF” (R-HSA-194138) pathway, in both the juvenile vs naive and old vs naive comparisons. Included are significance values for all genes (gray), pathway genes significantly downregulated in old but not juvenile (orange) and other pathway genes (green). P value cutoffs (0.01 and 0.1) are indicated with dashed lines. b The relative expression levels (FPKM) for the seven “Signaling by VEGF” genes belonging to this gene set are shown. P values represent FDR-corrected significance values from DESeq2.

Fig. 8. VEGF and peripheral neutrophil counts are higher in old COVID-19 patients.

Peripheral blood samples were obtained from a cohort of patients with laboratory-confirmed SARS-CoV-2 infection at hospital admission. Levels of different immune markers were determined by Luminex assay in plasma samples from COVID-19 and healthy volunteer controls. COVID-19 patients were stratified by age as younger than or older than 60 years. a Levels of IFN-γ and b levels of VEGF proteins were measured in plasma of COVID-19 and healthy controls. c Peripheral neutrophil counts and d neutrophil to lymphocyte ratio (NLR) values were retrieved from the medical records of COVID-19 patients and compared between age groups. Each point in the figure corresponds to an independent biological sample for each group. Mean ± SD for each group is shown in the figure. Statistical significance was verified by one-way anova: P = *(<0.05), **(<0.01) with Tukey’s correction (a and b) or by Student’s t-test: P = *(<0.05), **(<0.01) (c and d).

Fig. 9. Genes higher in expression during both COVID-19 and TB share common pathways.

a The top 20 (of 97) most significantly upregulated genes in COVID-19 infected and TB infected macaques. Expression values are visualized by Z scores of normalized expression data (FPKM) per sample, and Log₂ fold change and -Log P values are from the DESeq2 output. Genes are sorted by P value. b Significant Reactome pathway enrichment among the 97 genes.

Fig. 10. Genes higher in expression during COVID-19 than TB are related to blood morphogenesis pathways.

a The top 20 (of 76) most significantly upregulated genes in SARS-CoV-2-infected compared to Mtb-infected macaques. Expression values are visualized by Z scores of normalized expression data (FPKM) per sample, and Log₂ fold change and -Log P values are from the DESeq2 output. Genes are sorted by P value. b Significant Gene Ontology pathway enrichment among the 76 genes.