Lethal COVID-19 associates with RAAS-induced inflammation for multiple organ damage including mediastinal lymph nodes

Abstract

Lethal COVID-19 outcomes are attributed to classic cytokine storm. We revisit this using RNA sequencing of nasopharyngeal and 40 autopsy samples from patients dying of SARS-CoV-2. Subsets of the 100 top-upregulated genes in nasal swabs are upregulated in the heart, lung, kidney, and liver, but not mediastinal lymph nodes. Twenty-two of these are "noncanonical" immune genes, which we link to components of the renin-angiotensin-activation-system that manifest as increased fibrin deposition, leaky vessels, thrombotic tendency, PANoptosis, and mitochondrial dysfunction. Immunohistochemistry of mediastinal lymph nodes reveals altered architecture, excess collagen deposition, and pathogenic fibroblast infiltration. Many of the above findings are paralleled in animal models of SARS-CoV-2 infection and human peripheral blood mononuclear and whole blood samples from individuals with early and later SARS-CoV-2 variants. We then redefine cytokine storm in lethal COVID-19 as driven by upstream immune gene and mitochondrial signaling producing downstream RAAS (renin-angiotensin-aldosterone system) overactivation and organ damage, including compromised mediastinal lymph node function.

Keywords: COVID-19; fibrosis; renin angiotensin aldosterone system.

Fig. 1.

Early-stage innate immune gene expression in nasopharyngeal samples from COVID-19 patients. (A) Box and whisker plot of hallmark interferon alpha response gene sets determined by fGSEA for nasopharyngeal samples ranked by NES (nominal enrichment score). (B) Volcano plots of nasopharyngeal samples with high, SARS-CoV-2 RNA copy levels at collection. (C) Lollipop plots for custom gene sets determined by fGSEA (FDR < 0.25), ranked by NES. (D and E) Heatmaps of t score statistics when comparing viral load versus negative patient samples for specific (D) innate immune genes and (E) downregulated genes of interest, IL-1 family and antimicrobial-associated genes.

Fig. 2.

Mid- and late-stage and innate immune gene expression transcript changes in infected rodent samples and human autopsy tissues from COVID-19 patients. (A) Circular heatmap displaying the t-score statistics for specific innate immune genes, comparing SARS-CoV-2 infected hamster tissues, viral load versus negative patient nasopharyngeal and autopsy tissue samples, and log₂-foldchange for SARS-CoV-2 infected C57BL/6 and BALB/c mouse lungs. (B) Lollipop plots for custom gene sets determined by fGSEA (FDR < 0.25), ranked by NES.

Fig. 3.

SARS-CoV-2 infection alters extracellular-mediated immunity-associated gene expression. (A) Circular heatmap displaying the t-score statistics for extracellular-mediated immunity-associated genes comparing SARS-CoV-2 infected hamster tissues, viral load versus negative patient nasopharyngeal and autopsy tissue samples, and log₂-foldchange for SARS-CoV-2 infected C57BL/6 and BALB/c mouse lungs. (B) Lollipop plots for custom extracellular-mediated immunity-associated gene sets determined by fGSEA (FDR< 0.25) ranked by NES.

Fig. 4.

SARS-CoV-2-infection induces increased transcription of genes involved in RAAS-overaction in human lung, heart, and lymph nodes. (A) Schematic summary of RAAS pathway. (B) Linear heatmap displaying the t score statistics for RAAS target genes comparing SARS-CoV-2 infected hamster tissues, viral load versus negative patient nasopharyngeal and autopsy tissue samples, and log₂-foldchange for SARS-CoV-2 infected C57BL/6 and BALB/c mouse lungs.

Fig. 5.

Severe COVID-19 disrupts organ architecture with an associated accumulation of pathogenic fibroblasts. Mediastinal lymph nodes were obtained from patients without lung disease or from patients who died from COVID-19. 5 regions (at 10× magnification were evaluated from each patient sample and evaluated for (A) CD3 (cells/cm²), (B) FSP (cells/cm²), (c) representative staining of FSP staining, (D) reticulin staining and the percentage of area with reticulin staining, (E) reticulin fiber intensity, (F) representative staining of reticulin staining, (G) percentage of fibrosis staining (based on Trichrome staining), (H) collagen fiber length, and (I) representative staining of Trichrome staining.

Fig. 6.

SARS-CoV-2 infection upregulates mtDNA/mtdsRNA-activated genes despite the absence of viral transcripts. (A) Schematic summary of mtDNA/mtdsRNA-activated pathways. (B) Graphical representation of findings from Guarnieri et al. (80). (C) Lollipop plots for statistically significant changes determined by fGSEA, ranked by NES, and (D) Linear heatmaps displaying the t-score statistics for mtDNA/mtdsRNA-activated genes SARS-CoV-2 infected hamster tissues, viral load versus negative patient nasopharyngeal and autopsy tissue samples, miR-2392-expressing 3D-HUVEC-MT cells, and log₂-foldchange (FC) for SARS-CoV-2 infected C57BL/6 and BALB/c mouse lungs.

Fig. 7.

Transcript changes in PBMCs (Alpha and Omicron) and whole blood (Pre-Delta and Delta) display comparable immune responses. (A) Lollipop plots for statistically significant changes in custom gene sets determined by fGSEA for Whole blood and PBMCs ranked by NES. (B) Linear heatmap displaying the t-score statistics for conserved upregulated genes comparing PBMCs collected from SARS-CoV-2-Alpha and SARS-CoV-2-Omicron-infected patients versus non-infected patients. (C) Schematic summary figure of mtDNA/mtdsRNA-activated immune response with associated induction of EIF2AK2/PKR and ISR pathways driving dysfunctional RAAS pathway state.