Immune Cells Are Differentially Affected by SARS-CoV-2 Viral Loads in K18-hACE2 Mice

Abstract

Viral load and the duration of viral shedding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are important determinants of the transmission of coronavirus disease 2019. In this study, we examined the effects of viral doses on the lung and spleen of K18-hACE2 transgenic mice by temporal histological and transcriptional analyses. Approximately, 1×10⁵ plaque-forming units (PFU) of SARS-CoV-2 induced strong host responses in the lungs from 2 days post inoculation (dpi) which did not recover until the mice died, whereas responses to the virus were obvious at 5 days, recovering to the basal state by 14 dpi at 1×10² PFU. Further, flow cytometry showed that number of CD8+ T cells continuously increased in 1×10² PFU-virus-infected lungs from 2 dpi, but not in 1×10⁵ PFU-virus-infected lungs. In spleens, responses to the virus were prominent from 2 dpi, and number of B cells was significantly decreased at 1×10⁵ PFU; however, 1×10² PFU of virus induced very weak responses from 2 dpi which recovered by 10 dpi. Although the defense responses returned to normal and the mice survived, lung histology showed evidence of fibrosis, suggesting sequelae of SARS-CoV-2 infection. Our findings indicate that specific effectors of the immune response in the lung and spleen were either increased or depleted in response to doses of SARS-CoV-2. This study demonstrated that the response of local and systemic immune effectors to a viral infection varies with viral dose, which either exacerbates the severity of the infection or accelerates its elimination.

Keywords: Dose-response relationship, immunologic; Immune response; K18-hACE2 mice; SARS-CoV-2; Transcriptome profiling.

Figure 1. SARS-CoV-2 viral loads influence mortalities.

(A, B) Body temperature (A) and body weight (B) of PBS-infected control K18-hACE2 mice (n=6), 1×10² PFU-infected mice (n=8), and 1×10⁵ PFU-infected mice (n=8). (C) Survival rate after infection with SARS-CoV-2. (D) Virus titer was measured at 2 and 7 dpi. Error bars are SEM and significance was calculated using 2-way ANOVA with Bonferroni’s multiple comparison test. ^*p<0.05; ^****p<0.0001.

Figure 2. A lower dose of SARS-CoV-2 infection induces pulmonary fibrosis.

(A, B) H&E staining of lung sections in control, 1×10² PFU-, and 1×10⁵ PFU-infected mice. Lungs infected with SARS-CoV-2 exhibit inflammation (white +), vascular edema (arrow), capillary dilatation (arrowhead), and pulmonary fibrosis (open circled). The letters ‘B’ and ‘V’ represent the bronchiole and vessel, respectively (scale bars, 100 μm). (C, D) Pathological score. Error bars are SEM and significance was calculated using 2-way ANOVA with Bonferroni’s multiple comparison test. ^***p<0.001; ^****p<0.0001.

Figure 3. Viral loads of SARS-CoV-2 induce temporally distinct but equivalent transcriptional changes in the lungs, while only 1×10⁵ PFU SARS-CoV-2 induced obvious immune response in spleens.

(A) Heatmap for 108 DEGs with 1×10⁵ PFU-infected lungs (red: 0 dpi, green: 1 dpi, blue: 2 dpi, yellow: 5 dpi, purple: 7 dpi). (B) Heatmap for 153 DEGs with 1×10² PFU-infected lungs (red: 0 dpi, green: 1 dpi, blue: 2 dpi, yellow: 5 dpi, purple: 7 dpi, cyan: 10 dpi, dark red: 14 dpi). (C) Heatmap for 41 DEGs with 1×10⁵ PFU-infected spleens (red: 0 dpi, green: 1 dpi, blue: 2 dpi, yellow: 5 dpi, purple: 7 dpi). (D) Heatmap for 13 DEGs with 1×10² PFU-infected spleens (red: 0 dpi, green: 1 dpi, blue: 2 dpi, yellow: 5 dpi, purple: 7 dpi, cyan: 10 dpi, dark red: 14 dpi). The enriched GO terms of the biological process are displayed on the right side of heatmaps.

Figure 4. SARS-CoV-2 dosage determines gene expression levels and temporal changes in immune response-related genes.

(A) Heatmaps of the GSVA results for response to virus (GO: 0009615) and immunoglobulin production (GO: 0002377) in lungs infected with 1×10⁵ PFU or 1×10² PFU. (B, D) Graphs illustrating the temporal changes in expression levels caused by SARS-CoV-2 infection for representative genes (orange: 1×10⁵ PFU, blue: 1×10² PFU). (C) Heatmaps for GSVA results for response to virus (GO: 0009615), response to IL-1 (GO: 0070555), and lymphocyte mediated immunity (GO: 0002449) in spleens.

Figure 5. Changes in CD8+ T cells and B cells in the lungs and spleens of SARS-CoV-2 infected mice.

(A) Immunohistochemistry for CD8+ T cells during the progression of infection. The graph depicts the number of CD8+ cells relative to total number of cells at 40× high power field (scale bars, 50 µm). (B, C) Flow cytometric analysis of total T cells (B) and CD8+ T cells (C) in SARS-CoV-2-infected lungs at 2, 5, 7, or 14 dpi with SARS-CoV-2 (left: 1×10² PFU, right: 1×10⁵ PFU). A 0 dpi indicates naïve mice. Bars represent the relative proportion of T cells in each dpi relative to naïve mice (at least 2 independent experiments per group). (D) Volcano plots comparing total genes from lung samples collected at 10 dpi relative to 0 dpi at 1×10² PFU. Red indicates 28 genes that were related to T cell identification and profiling in the previous studies (2122). The 6 most significant genes are labeled. (E) Gene set enrichment analysis for genes related to cytolytic CD8+ effector T cells (22). (F) Immune deconvolution results identifying B cell composition in the spleens. Bars represent mean ± SEM. (G) Flow cytometric analysis of B cells in SARS-CoV-2-infected spleens at 2, 5, 7, or 14 dpi with SARS-CoV-2 (left: 1×10² PFU, right: 1×10⁵ PFU). A 0 dpi indicates naïve mice. Bars represent the relative proportion of B cells in each dpi relative to naïve mice (at least 2 independent experiments per group). (H) Volcano plots comparing total genes from spleen samples collected at 7 dpi relative to 0 dpi at 1×10⁵ PFU. Red represents the 32 B cell markers utilized in the immune deconvolution analysis. The 6 most significant markers are labeled. (I) Gene set enrichment analysis for genes related to adaptive B2 lymphocytes (23).

Figure 6. Genes associated with pulmonary fibrosis are upregulated by SARS-CoV-2 infection.

(A) Heatmaps for genes related to pulmonary fibrosis in the lungs (top: 1×10⁵ PFU, bottom: 1×10² PFU). (B, C) Gene set enrichment analysis for genes associated with lung fibrosis using C2 gene sets from molecular signatures database (MsigDB) at 1×10⁵ PFU (B) and at 1×10² PFU (C). (D) Gene set enrichment analysis of for genes associated with apoptosis using C2 gene sets from MsigDB (left: 7 dpi versus 0 dpi 1×10⁵ PFU, right: 7 dpi versus 0 dpi 1×10² PFU).