Fig. 3.

Inflammation induced by SARS-CoV-2 variants is more pronounced in aged mice, and disease is driven by TNF, TGF-β, and E2F pathways. (A and B) C57BL/6 mice of different ages (young = 6 to 8 wk and aged = 6 to 8 mo) were intranasally inoculated with 10⁴ TCID50 SARS-CoV-2 P2 or P21 and monitored for (A) the proportion of mice that became moribund, reaching humane endpoint and (B) daily percentual weight change over time, relative to the initial weight. (n= 5 to 9 mice per group; results are representative of at least 3 independent experiments) (C) Representative images of hematoxylin and eosin (H&E), SARS-CoV-2 nucleocapsid, MPO (neutrophils), F4/80 (macrophages), or CD3 (T cells) stains of mouse lungs infected with P21 and harvested 3 dpi. Histological images are representative of at least 3 animals. (Scale bars, 50 µm.) (D) Average of sum of 26 cytokines/chemokines expressed in supernatants of lung homogenates of young and aged mice challenged intranasally with 10⁴ TCID50 of P2, P21, or mock at 3 dpi (n = 5 mice per group; mean ± SD of each cytokine is shown; one-way ANOVA with multiple comparisons was performed). (E) Levels of cytokine and chemokines in lung homogenates 3 dpi. Protein levels were measured by ELISA, and corresponding gene expression levels were quantified by bulk RNA-seq. Gene expression data are annotated by protein name rather than the original mouse gene identifier for each of comparison. The Wilcoxon rank-sum test, with Bonferroni adjustment for multiple comparisons, was performed between aged and young mice for each infection group. Boxplots depict the median and interquartile ranges. (F) Pathway enrichment analysis of significantly DE genes identified by directly comparing P21-infected aged and young animals at 3 dpi, using Hallmark gene sets. Negative log₁₀ false discovery rate–adjusted P values associated with each pathway are plotted; dot sizes correspond to the proportion of all genes from that pathway that were found to be significantly DE for a given comparison (Gene Ratio). (G) DE genes attributable to P21 infection in aged and young mice were converted to human homologs and performed a comparative pathway analysis between the two age groups using IPA. Among the top significantly enriched pathways for both age groups was “Role of Hypercytokinemia/Hyperchemokinemia in the Pathogenesis of Influenza” and “Coronavirus Pathogenesis”. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data are representative of 2 to 3 independent experiments (A–C).