SARS-CoV-2 Inhibits NRF2-Mediated Antioxidant Responses in Airway Epithelial Cells and in the Lung of a Murine Model of Infection

Abstract

Several viruses have been shown to modulate the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), the master regulator of redox homeostasis. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, also seems to disrupt the balance between oxidants and antioxidants, which likely contributes to lung damage. Using in vitro and in vivo models of infection, we investigated how SARS-CoV-2 modulates the transcription factor NRF2 and its dependent genes, as well as the role of NRF2 during SARS-CoV-2 infection. We found that SARS-CoV-2 infection downregulates NRF2 protein levels and NRF2-dependent gene expression in human airway epithelial cells and in lungs of BALB/c mice. Reductions in cellular levels of NRF2 seem to be independent of proteasomal degradation and the interferon/promyelocytic leukemia (IFN/PML) pathway. Furthermore, lack of the Nrf2 gene in SARS-CoV-2-infected mice exacerbates clinical disease, increases lung inflammation, and is associated with a trend toward increased lung viral titers, indicating that NRF2 has a protective role during this viral infection. In summary, our results suggest that SARS-CoV-2 infection alters the cellular redox balance by downregulating NRF2 and its dependent genes, which exacerbates lung inflammation and disease, therefore, suggesting that the activation of NRF2 could be explored as therapeutic approach during SARS-CoV-2 infection. IMPORTANCE The antioxidant defense system plays a major function in protecting the organism against oxidative damage caused by free radicals. COVID-19 patients often present with biochemical characteristics of uncontrolled pro-oxidative responses in the respiratory tract. We show herein that SARS-CoV-2 variants, including Omicron, are potent inhibitors of cellular and lung nuclear factor erythroid 2-related factor 2 (NRF2), the master transcription factor that controls the expression of antioxidant and cytoprotective enzymes. Moreover, we show that mice lacking the Nrf2 gene show increased clinical signs of disease and lung pathology when infected with a mouse-adapted strain of SARS-CoV-2. Overall, this study provides a mechanistic explanation for the observed unbalanced pro-oxidative response in SARS-CoV-2 infections and suggests that therapeutic strategies for COVID-19 may consider the use of pharmacologic agents that are known to boost the expression levels of cellular NRF2.

Keywords: COVID-19; NRF2; Nrf2-deficient mice; SARS-CoV-2; antioxidant enzymes.

FIG 1

SARS-CoV-2 infection downregulates NRF2 protein levels in epithelial cell lines, including human lung-derived cells. (a) Vero E6 cells were infected with SARS-CoV-2 USA-WA1/2020, (b) Vero-TMPRSS2 cells were infected with the SARS-CoV-2 Omicron (B.1.1.529) variant, and (c) A549-hACE2 cells were infected with SARS-CoV-2 USA-WA1/2020. Cells were harvested at 16 and 24 h postinfection (hpi), and whole-cell lysates were analyzed by Western blotting with anti-NRF2 antibody. The membranes were reprobed for anti-β-actin antibody for a loading control. Western blot images are one representative of two independent experiments. (d) Calu-3 cells cultured on an air-liquid interface (ALI) were infected with SARS-CoV-2 USA-WA1/2020. Cells were harvested at 24 and 48 hpi, and whole-cell lysates were analyzed by Western blotting with anti-NRF2 antibody. The membrane was reprobed for anti-β-actin antibody for a loading control. Data from one experiment performed in triplicate are shown.

FIG 2

SARS-CoV-2 infection downregulates NRF2-dependent gene expression in human lung epithelial cells. A549-hACE2 cells mock infected or infected with SARS-CoV-2 (USA-WA1/2020) were harvested 16 and 24 hpi to prepare total RNA. SOD1, CAT, GPX1, GCLC, NQO1, and HMOX1 gene expression was quantified by RT-qPCR. The graphs show combined data from two independent experiments expressed as mean ± SEM. Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s test (*, P < 0.05).

FIG 3

NRF2 decreased cellular levels are independent of proteasomal degradation and the interferon/promyelocytic leukemia (IFN/PML) pathway. (a) A549-hACE2 cells mock infected or infected with SARS-CoV-2 (USA-WA1/2020) were treated with 10 μM lactacystin or its vehicle (at 6 hpi) and harvested 20 hpi. Whole-cell lysates were analyzed by Western blotting with anti-NRF2 antibody. The membranes were reprobed for anti-β-actin antibody for loading control. The Western blot image is one representative of four independent experiments. The graph shows the densitometric analysis of NRF2 after normalization to β-actin expressed as mean ± SEM (*, P < 0.05 by two-way ANOVA followed by Tukey’s test). (b) Whole-cell lysates from A549-hACE2 cells mock infected or infected with SARS-CoV-2 for 16 and 24 h were analyzed by Western blotting with anti-PML protein antibody. (c) Whole-cell lysates from A549-hACE2 cells untreated and treated with human IFN-β for 16 h and mock infected or infected with RSV for 16 and 24 h were analyzed by Western blotting with anti-PML protein antibody. The membranes were reprobed for anti-β-actin antibody for a loading control. Western blot images are one representative of two independent experiments.

FIG 4

SARS-CoV-2 infection downregulates NRF2-dependent gene expression in lungs of BALB/c mice. (a) Eleven- to 12-week-old female BALB/c mice were inoculated intranasally with 5 × 10⁶ to 10⁷ TCID₅₀s of mouse-adapted SARS-CoV-2 (CMA3p20) or mock inoculated with PBS. The schematic figure was created with BioRender.com. (b) Body weight loss was monitored for 7 days to examine the kinetics of the disease. Data are expressed as mean ± SEM (n = 3 or 4 mice/group; *, P < 0.05 by Student’s t test). (c) A group of mice were euthanized at 2 and 3 days postinfection (dpi), and lungs were harvested to isolate total RNA. Sod1, Cat, Gstm1, Prdx1, Prdx6, and Hmox1 gene expression was quantified by RT-qPCR. Data are expressed as mean ± SEM (n = 9 mice/group; one-way ANOVA followed by Tukey’s test: *, P < 0.05).

FIG 5

Lack of the Nrf2 gene in mice exacerbates clinical disease and peribronchiolitis following SARS-CoV-2 infection. (a) Sixteen- to 20-week-old Nrf2^−/− and wild-type (WT) age-matched BALB/c female mice were infected with 10⁶ TCID₅₀s of mouse-adapted SARS-CoV-2 (CMA3p20) or mock inoculated with PBS. Schematic figure created with BioRender.com. (b) Changes in body weight were monitored for 7 days. Data are expressed as mean ± SEM (mock, n = 3 mice/group; SARS-CoV-2, n = 10 to 12 mice/group; *, P < 0.05 for WT mock infected versus WT SARS-CoV-2, and #, P < 0.05 for WT SARS-CoV-2 versus Nrf2^−/− SARS-CoV-2, by two-way ANOVA followed by Tukey’s test). (c) Lung viral titers at 2 dpi were determined by TCID₅₀ assay. Data are expressed as mean ± SEM (n = 12 mice/group; P = 0.08 by Student’s t test). (d) Peribronchiolitis and (e) perivasculitis scored at 7 dpi in hematoxylin and eosin (H&E) stained sections of lung; (f) percentage of collagen in lung tissue at 7 dpi determined in Masson’s trichrome-stained sections as described in Materials and Methods. Data are expressed as mean ± SEM (n = 3 or 4 mice/group; *, P < 0.05 by two-way ANOVA followed by Tukey’s test). (g) Representative images of H&E-stained sections of lung depicting inflammation surrounding bronchioles and vessels (subgross images at ×1.0 and insets at ×10 magnification).

FIG 6

Cytokines and chemokines in response to SARS-CoV-2 infection in wild-type (WT) versus Nrf2^−/− mice. Sixteen- to 20-week-old BALB/c WT and Nrf2^−/− female mice were infected with 10⁶ TCID₅₀s of mouse-adapted SARS-CoV-2 (CMA3p20) or mock inoculated. Bronchoalveolar lavage fluid (BALF) was collected at 2 dpi, and (a) cytokine and (b) chemokine levels were determined by using a Bio-Plex (23-plex assay). Data are expressed as mean ± SEM (mock, n = 3 mice/group [not shown but included in the statistical analysis]; SARS-CoV-2, n = 12 to 16 mice/group; *, P < 0.05 for WT SARS-CoV-2 versus Nrf2^−/− SARS-CoV-2 by two-way ANOVA followed by Tukey’s test).