SARS-CoV-2 Switches 'on' MAPK and NFκB Signaling via the Reduction of Nuclear DUSP1 and DUSP5 Expression

Abstract

Mitogen-activated protein kinases (MAPK) and NF-kappaB (NF-κB) pathway regulate many cellular processes and are essential for immune cells function. Their activity is controlled by dual-specificity phosphatases (DUSPs). A comprehensive analysis of publicly available gene expression data sets of human airway epithelial cells (AECs) infected with SARS-CoV-2 identified DUSP1 and DUSP5 among the lowest induced transcripts within these pathways. These proteins are known to downregulate MAPK and NF-κB pathways; and their lower expression was associated with increased activity of MAPK and NF-κB signaling and enhanced expression of proinflammatory cytokines such as TNF-α. Infection with other coronaviruses did not have a similar effect on these genes. Interestingly, treatment with chloroquine and/or non-steroidal anti-inflammatory drugs counteracted the SARS-CoV-2 induced reduction of DUSP1 and DUSP5 genes expression. Therapeutically, impeding this evasion mechanism of SARS-CoV-2 may help control the exaggerated activation of these immune regulatory pathways during a COVID-19 infection.

Keywords: COVID-19; DUSP1; DUSP5; MAPK; NFkB; SARS-CoV-2; chroloquine.

FIGURE 1

SARS-CoV-2 activates MAPK and NF-κB pathways via the reduced expression of DUSP1 and DUSP5 expression compared to other coronaviruses (A) Enrichment of MAPK pathway in SARS-CoV-2 infected human AECs along with the expression levels of enriched MAPK pathway related genes in SARS-CoV-2 infected AECs (ES = 0.437; NES = 1.57) (B) Enrichment of MAPK pathway in SARS-CoV-1 infected human AECs (ES = 0.489; NES = 1.51) (C) Enrichment of MAPK pathway in MERS-CoV infected human AECs (ES = 0.432; NES = 1.37) (D) Enrichment of NF-κB pathway in SARS-CoV-2 infected human AECs along with the expression levels of enriched NF-κB pathway related genes in SARS-CoV-2 infected AECs (ES = 0.663; NES = 1.24) (E) Enrichment of NF-κB pathway in SARS-CoV-1 infected human AECs (ES = 0.643; NES = 1.6) (F) Enrichment of NF-κB pathway in MERS-CoV infected human AECs (ES = 0.704; NES = 1.13). Enrichment Score (ES), Normalized enrichment Score (NES) (G,H) Gene expression of DUSP1 and DUSP5 in SARS-CoV-2, SARS-CoV-1, or MERS-CoV infected human AECs, 24 h post-infection. Representative data shows lower expression level of DUSP1 and DUSP5 following SARS-CoV-2 infection as compared to other coronaviruses. Two-way comparison was done using t-test or Mann-Whitney U test, depending on the skewness of the data. ***p < 0.001, ****p < 0.0001.

FIGURE 2

Lower gene expression levels of DUSP1 and DUSP5 in nasopharyngeal swabs from COVID-19 patients compared to healthy controls (A,B) Gene expression level of DUSP1 and DUSP5 in COVID-19 (n = 430) and in healthy (n = 54) nasopharyngeal swabs (Dataset GSE152075). Expression level of DUSP1 and DUSP5 is significantly lower in nasopharyngeal swabs from COVID-19 patients as compared to healthy controls (C,D) Confirmation of gene expression level of DUSP1 and DUSP5 as measured by RT-PCR, in nasopharyngeal swabs of severe COVID-19 patients (n=16) and healthy controls (n=6). Expression level of DUSP1 and DUSP5 is lower in nasopharyngeal swabs from COVID-19 patients as compared to healthy controls. Two-way comparison was done using t-test or Mann-Whitney U test, depending on the skewness of the data. *p < 0.05, ****p < 0.0001.

FIGURE 3

Increased gene expression of MAPK associated pro-inflammatory cytokines in nasopharyngeal swabs of COVID-19 patients (A–C) Gene expression level of TNF-α, IL-1β, and IL-1A in nasopharyngeal swabs fromm COVID-19 (n = 430) and healthy controls (n = 54) (Dataset GSE152075). Expression level of TNF-α, IL-1β, and IL-1A is higher in nasopharyngeal swabs of COVID-19 patients as compared to healthy controls (D–F) Confirmation of gene expression level of TNF-α, IL-1β, and IL-1A as measured by RT-PCR, in nasopharyngeal swabs from COVID-19 pateints (n = 16) and healthy controls (n = 6). Expression level of TNF-α, IL-1β, and IL-1A is higher in nasopharyngeal swabs from COVID-19 patients as compared to healthy controls (G–I) Gene expression level of TNF-α, IL-1β, and IL-1A in spleen tissue of DUSP1 ^+/+ mice (n = 3) and DUSP1^−/− mice (n = 3). Expression level of TNF-α, IL-1β, and IL-1A is higher in spleen tissue of DUSP1^−/− as compared to DUSP1 ^+/+ mice. Two-way comparison was done using t-test or Mann-Whitney U test, depending on the skewness of the data. *p < 0.05, **p < 0.01, ****p < 0.0001.

FIGURE 4

Increased gene expression of NF-κB associated pro-inflammatory cytokines in nasopharyngeal swabs of COVID-19 patients (A–C) Gene expression level of IL-6, IL-8 and IL-23 in nasopharyngeal swabs from COVID-19 patients (n = 430) and healthy controls (n = 54) (Dataset GSE152075). Expression level of IL-6, IL-8 and IL-23 is higher in nasopharyngeal swabs from COVID-19 patients as compared to healthy controls (D–F) Confirmation of gene expression level of IL-6, IL-8 and IL-23 as measured by RT-PCR, in nasopharyngeal swabs from COVID-19 patients (n = 16) and healthy control (n = 6). Expression level of IL-6, IL-8 and IL-23 is higher in nasopharyngeal swabs from COVID-19 patients as compared to healthy controls (G–L) Gene expression level of TNF-α, IL-1β, IL-1A, IL-6, IL-23, and IL-8 in bone marrow derived eosinophils from DUSP5^+/+ (n = 3) and DUSP5^−/− mice (n = 3). Expression level of TNF-α, IL-1β, IL-1A, IL-6, IL-23, and IL-8 was higher in DUSP5 deficient eosinophils as compared to the WT eosinophils. Two-way comparison was done using t-test or Mann-Whitney U test, depending on the skewness of the data. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 5

Effect of common medications on the expression levels of DUSP1 and DUSP5 in human primary hepatocytes (A,B) Medications with a significant regulatory effect on the gene expression levels of DUSP1 (A) and DUSP5 (B). Statistical test: Limma adjusted p < 0.05 (C) Baseline expression of DUSP1 and DUSP5 in healthy liver and lung tissue extracted from the Genotype-Tissue Expression (GTEx) project. The data shows that the gene expression levels of DUSP1 and DUSP5 in healthy liver tissues, on which the medication has been tested, is comparable with their lung expression levels.