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. 2024 May 15;27(6):109975.
doi: 10.1016/j.isci.2024.109975. eCollection 2024 Jun 21.

SARS-CoV-2 envelope protein regulates innate immune tolerance

Eric S Geanes  1 , Rebecca McLennan  1 , Stephen H Pierce  2 , Heather L Menden  3 , Oishi Paul  1 , Venkatesh Sampath  3   4 , Todd Bradley  1   2   4   5
Affiliations

SARS-CoV-2 envelope protein regulates innate immune tolerance

Eric S Geanes et al. iScience. .

Abstract

Severe COVID-19 often leads to secondary infections and sepsis that contribute to long hospital stays and mortality. However, our understanding of the precise immune mechanisms driving severe complications after SARS-CoV-2 infection remains incompletely understood. Here, we provide evidence that the SARS-CoV-2 envelope (E) protein initiates innate immune inflammation, via toll-like receptor 2 signaling, and establishes a sustained state of innate immune tolerance following initial activation. Monocytes in this tolerant state exhibit reduced responsiveness to secondary stimuli, releasing lower levels of cytokines and chemokines. Mice exposed to E protein before secondary lipopolysaccharide challenge show diminished pro-inflammatory cytokine expression in the lung, indicating that E protein drives this tolerant state in vivo. These findings highlight the potential of the SARS-CoV-2 E protein to induce innate immune tolerance, contributing to long-term immune dysfunction that could lead to susceptibility to subsequent infections, and uncovers therapeutic targets aimed at restoring immune function following SARS-CoV-2 infection.

Keywords: components of the immune system; immunity; molecular biology; transcriptomics; virology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
SARS-CoV-2 envelope protein stimulates human monocyte cytokine secretion (A) Heatmap of secreted cytokine levels represented as z-scores after human monocyte stimulation with mock, envelope (E), spike (S), and nucleocapsid (N) SARS-CoV-2 viral proteins after 24 hours (n = 5 individuals). Hierarchical clustering of analyte rows using 1-Pearson metric. (B) Bar graph showing log2 fold-change of each condition compared to control (mock activated) conditions for individual analytes. Analytes ordered by descending Log2FC. (C) HEK-Blue cell lines that express reporters for TLR2 (solid line) or TLR4 (dashed line) were stimulated with either the spike (blue) or E (red) proteins from SARS-CoV-2 at various concentrations (x-axis). Activation measured by optical density of SEAP levels in the supernatant. (D) THP1-Dual (black line) or THP1-Dual-TLR2-KO (red line) cells stimulated with SARS-CoV-2 E protein at various concentrations (x-axis). Activation measured by optical density of SEAP levels in the supernatant.
Figure 2
Figure 2
SARS-CoV-2 Envelope protein tolerized monocytes to secondary stimulation (A) Timeline of primary and secondary stimulation of human monocytes. (B) Heatmap of secreted cytokine levels represented as z-scores after human monocyte primary stimulation with mock (n = 13), envelope (E; n = 13), spike (S; n = 5), and nucleocapsid (N; n = 5) SARS-CoV-2 viral proteins followed by secondary stimulation with LPS. Hierarchical clustering of analyte rows using 1-Pearson metric. Control samples were not given any primary stimulation on day 1 but did receive the secondary LPS stimulation on day 6. (C) Bar graph showing log 2-fold change of each condition compared to control (mock activated) conditions for select individual analytes measured in (B).∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, Paired Wilcoxon Test. Analytes ordered by descending Log2FC of E protein treated samples. (D) Heatmap of secreted cytokine levels presented as z-scores after human monocyte primary stimulation with envelope protein followed by secondary stimulation with envelop protein (autologous; n = 5). Baseline samples received no stimulations during the experiment. (E) Bar graph showing log 2-fold change of each condition compared to control (mock activated) conditions for select individual analytes measured in (D).∗p ≤ 0.05, ∗∗p ≤ 0.01, Paired Wilcoxon Test.
Figure 3
Figure 3
SARS-CoV-2 E protein priming of monocytes is dose dependent (A) Heatmap of secreted cytokine levels represented as z-scores after human monocyte primary stimulation with mock (n = 4) or E protein stimulation at 1 μg/mL, 10 ng/mL, 1 ng/mL or 10 pg/mL dose (n = 4 at each dose) followed by secondary stimulation with LPS. Hierarchical clustering of analyte rows using 1-Pearson metric. (B) Bar graph showing log 2-fold change of each condition compared to control (mock activated) conditions for individual analytes.∗p ≤ 0.05, Paired Wilcoxon Test. Error bars are standard error of the mean. Analytes ordered by descending Log2FC of highest-dose-treated samples.
Figure 4
Figure 4
SARS-CoV-2 E protein changes transcriptome of monocytes (A) Volcano plot of differentially expressed genes determined by RNA-seq at day 6 after either E protein (n = 3) or control (n = 3) stimulation of human monocytes. Genes upregulated by ≥ 1.5 or downregulated by ≤ −1.5 log2 Fold-Change in E protein stimulated compared to control are colored red or blue, respectively. Statistical significance threshold was adjusted p value ≤ 0.01. (B) Gene set enrichment analysis (GSEA) comparing the changed genes measured by RNA-seq enriched in the E protein stimulated monocytes compared to control. Top 5 enriched pathways and GSEA output criteria shown in the table, with the enrichment plots for the top two GSEA identified pathways enriched in the E protein stimulated monocytes.
Figure 5
Figure 5
Neonatal pups exposed to E protein priming have suppressed lung cytokine expression and TLR pathway activation upon challenge with systemic LPS (A) 5-day-old mice were injected with 2 mg/kg E protein or vehicle i.p. Subsequently, on day of life 12, mice were injected with 2 mg/kg i.p. LPS or vehicle, followed by collection of whole lung tissues on day of life 14. Whole lung homogenates were used for assays. (B) mRNA expression of cytokine genes after various treatments. N = 4/group. Error bars represent standard deviations, ∗p < 0.01. (C and D) Protein expression of p-P65, p-P38, TLR2, and TLR4 after different treatments with densitometry quantification shown. N = 4/group. Error bars represent standard deviations, ∗p < 0.05, one-way ANOVA with Bonferroni correction.
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