SARS-CoV-2 nucleocapsid protein triggers hyperinflammation via protein-protein interaction-mediated intracellular Cl- accumulation in respiratory epithelium

Abstract

SARS-CoV-2, the culprit pathogen of COVID-19, elicits prominent immune responses and cytokine storms. Intracellular Cl^- is a crucial regulator of host defense, whereas the role of Cl^- signaling pathway in modulating pulmonary inflammation associated with SARS-CoV-2 infection remains unclear. By using human respiratory epithelial cell lines, primary cultured human airway epithelial cells, and murine models of viral structural protein stimulation and SARS-CoV-2 direct challenge, we demonstrated that SARS-CoV-2 nucleocapsid (N) protein could interact with Smad3, which downregulated cystic fibrosis transmembrane conductance regulator (CFTR) expression via microRNA-145. The intracellular Cl^- concentration ([Cl^-]_i) was raised, resulting in phosphorylation of serum glucocorticoid regulated kinase 1 (SGK1) and robust inflammatory responses. Inhibition or knockout of SGK1 abrogated the N protein-elicited airway inflammation. Moreover, N protein promoted a sustained elevation of [Cl^-]_i by depleting intracellular cAMP via upregulation of phosphodiesterase 4 (PDE4). Rolipram, a selective PDE4 inhibitor, countered airway inflammation by reducing [Cl^-]_i. Our findings suggested that Cl^- acted as the crucial pathological second messenger mediating the inflammatory responses after SARS-CoV-2 infection. Targeting the Cl^- signaling pathway might be a novel therapeutic strategy for COVID-19.

Fig. 1

SARS-CoV-2 N protein-elicited respiratory epithelial inflammation. a The mRNA expression levels of pro-inflammatory cytokines in N protein (50 μg/ml)-stimulated BEAS-2B cells (n = 3). b Fluorescence labeling of β-tubulin (green) and N protein (red) in BEAS-2B cells with DAPI-labeled nuclei (blue) after stimulation with N protein (50 μg/ml) for 24 h. Scale bars, 10 μm. c The phosphorylation level of IκB and NF-κB p65 subunit after N protein (50 μg/ml) stimulation in BEAS-2B cells, with the gray value analysis (n = 3). d Volcano plot visualization of the global RNA-seq DEGs in N protein (50 μg/ml)-stimulated BEAS-2B cells. e Dot plot of the enriched GO terms of the mRNAs exhibiting the enriched genes of BEAS-2B cells which were stimulated with N protein (50 μg/ml). f A heatmap showing the mRNA expression levels of the DEGs upregulated by N protein (50 μg/ml) stimulation compared with the control group, with the genes belonging to the GO annotations for cytokine activity and chemokine activity (GO: 0005125 and GO: 0008009). g The mRNA expression levels of cytokines in mice stimulated with N protein (0.25 mg/kg) (n = 3). h The phosphorylation level of IκB and p65 after N protein (0.25 mg/kg) stimulation in mice, with the gray value analysis (n = 3). i H&E staining of lung slices from N protein (0.25 mg/kg)-stimulated mice. Scale bars, 50 and 25 μm. j The mRNA expression levels of cytokines in SARS-CoV-2 (1 × 10⁵ PFU)-infected hACE2-transduced mice (n = 3). Data were shown as mean ± SD, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with the control group or indicated by lines

Fig. 2

SARS-CoV-2 N protein triggered the downregulation of CFTR and increased [Cl⁻]_i in RECs. a Representative traces showing the effect of N protein (50 μg/ml) on I_SC responses induced by forskolin (FSK, 10 μM) in 16HBE14o- cells, with the statistical analysis (n = 3). b The mRNA expression levels of CFTR in N protein (50 μg/ml)-stimulated BEAS-2B cells (n = 3). c The expression of CFTR after N protein (50 μg/ml) stimulation in BEAS-2B cells, with the gray value analysis (n = 3). d Fluorescence labeling of CFTR (green) and N protein (red) in BEAS-2B cells with DAPI-labeled nuclei (blue) after stimulation with N protein (50 μg/ml) for 24 h. Scale bars, 10 μm. e The changes in [Cl⁻]_i of BEAS-2B cells after N protein (50 μg/ml) stimulation (n = 12 cells). f The mRNA expression level of Cftr in the lungs of N protein (0.25 mg/kg)-stimulated mice (n = 3). g The expression of CFTR after N protein (0.25 mg/kg) stimulation in the lungs of mice, with the gray value analysis (n = 3). h Fluorescence labeling of CFTR (green) and N protein (red) in mouse lung slices with DAPI-labeled nuclei (blue) after N protein (0.25 mg/kg) stimulation. Scale bars, 10 μm. i The changes in [Cl⁻]_i of mPAECs from mice after N protein (0.25 mg/kg) stimulation (n = 20 cells). j The mRNA expression level of Cftr in the lungs of hACE2-transduced mice infected with SARS-CoV-2 (1 × 10⁵ PFU) (n = 3). k Fluorescence labeling of CFTR (green) and N protein (red) in lung slices of hACE2-transduced mice infected with SARS-CoV-2 (1 × 10⁵ PFU). Scale bars, 50 μm. Data were shown as mean ± SD, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with the control group or indicated by lines

Fig. 3

SARS-CoV-2 N protein interacted with Smad3 to induce miR-145-mediated downregulation of CFTR and increased [Cl⁻]_i in RECs. a Whole-cell lysates (WCL) from BEAS-2B cells stimulated by N protein (50 μg/ml) for 24 h were subject to immunoprecipitation with anti-Smad3 beads and immunoblotted with an anti-N protein antibody. b GST pull-down assays using GST-Smad3 and N protein. N proteins binding to GST-Smad3 were detected by using an anti-N protein antibody. c Fluorescence labeling of Smad3 (red) and N protein (green) in BEAS-2B cells with DAPI-labeled nuclei (blue) after stimulation with N protein (50 μg/ml) for 24 h. Scale bars, 2 μm. d The effect of Inh miR-145 (100 nM) on the mRNA expression levels of CFTR in BEAS-2B cells stimulated with N protein (50 μg/ml) for 24 h (n = 3). e The effect of Inh miR-145 (100 nM) on the expression of CFTR in BEAS-2B cells stimulated by N protein (50 μg/ml) for 24 h, with the gray value analysis (n = 3). f The effect of Inh miR-145 (100 nM) on [Cl⁻]_i of BEAS-2B cells stimulated by N protein (50 μg/ml) for 24 h (n = 20 cells). Data are shown as mean ± SD, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, n.s. P > 0.05

Fig. 4

SARS-CoV-2 N protein-triggered inflammation via the activation of SGK1 in RECs. a The phosphorylation level of SGK1 after N protein (50 μg/ml) stimulation in BEAS-2B cells, with the gray value analysis (n = 3). b The effect of EMD638683 (50 μM) on the mRNA expressions of pro-inflammatory cytokines in N protein (50 μg/ml)-stimulated BEAS-2B cells (n = 3). c The mRNA expressions of cytokines after N protein (50 μg/ml) stimulation in the empty vector control or SGK1 KO BEAS-2B cells (n = 3). d The phosphorylation level of SGK1 in lungs of N protein (0.25 mg/kg)-stimulated mice, with the gray value analysis (n = 3). e Fluorescence labeling of phosphorylated SGK1 (green) and N protein (red) in lung slices of hACE2-transduced mice infected with SARS-CoV-2 (1 × 10⁵ PFU). Scale bars, 10 μm. f The effect of EMD638683 (10 mg/kg) on the mRNA expressions of cytokines in N protein (0.25 mg/kg)-stimulated mice (n = 3). g The effect of EMD638683 (10 mg/kg) on the phosphorylation level of IκB and NF-κB p65 subunit in N protein (0.25 mg/kg)-stimulated mice, with the gray value analysis (n = 3). h H&E staining of lung slices from N protein (0.25 mg/kg)-stimulated mice with or without treatment with EMD638683 (10 mg/kg). Scale bars, 50 μm and 25 μm. Data were shown as mean ± SD, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with the control group or indicated by lines, n.s. P > 0.05

Fig. 5

SARS-CoV-2 N protein contributed to the sustained elevation in [Cl⁻]_i via NF-κB-PDE4-cAMP signaling pathway in RECs. a The mRNA expressions of PDE4C and PDE4D in the N protein (50 μg/ml)-stimulated BEAS-2B cells (n = 3). b The mRNA expressions of Pde4 in the lung tissues of N protein (0.25 mg/kg)-stimulated mice (n = 3). c The mRNA expressions of Pde4 in the lung tissues of SARS-CoV-2 (1 × 10⁵ PFU)-infected hACE2-transduced mice (n = 3). d The effect of PDTC (100 μM) on PDE4 mRNA expression in BEAS-2B cells stimulated with N protein (50 μg/ml) (n = 3). e The effect of PDTC (100 μM) on intracellular cAMP levels in BEAS-2B cells stimulated with N protein (50 μg/ml) (n = 3). f The effect of PDTC (100 μM) on [Cl⁻]_i of BEAS-2B cells stimulated with the N protein (50 μg/ml) (n = 12 cells). g The effect of PDTC (100 mg/kg) on Pde4 mRNA expression in the lung tissues of N protein (0.25 mg/kg)-stimulated mice (n = 3). h The effect of PDTC (100 mg/kg) on [Cl⁻]_i of mPAECs from mice after N protein (0.25 mg/kg) stimulation (n = 20 cells). Data were shown as mean ± SD, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with the control group or indicated by lines, n.s. P > 0.05

Fig. 6

Inhibition of PDE4 suppressed the SARS-CoV-2 N protein-induced ongoing inflammation via downregulation of the [Cl⁻]_i. a The effect of rolipram (20 μM) on the intracellular cAMP content after the N protein (50 μg/ml) stimulation in BEAS-2B cells (n = 3). b The effect of rolipram (20 μM) on [Cl⁻]_i in BEAS-2B cells stimulated with the N protein (50 μg/ml) (n = 12 cells). c The effect of rolipram (20 μM) on phosphorylation level of SGK1, IκB, and NF-κB p65 subunit after N protein (50 μg/ml) stimulation in BEAS-2B cells. d The effect of rolipram (20 μM) on the mRNA expression levels of pro-inflammatory cytokines stimulated with the N protein (50 μg/ml) in BEAS-2B cells (n = 3). e The effect of rolipram (10 mg/kg) on [Cl⁻]_i in mPAECs from mice after N protein (0.25 mg/kg) stimulation (n = 20 cells). f The effect of rolipram (10 mg/kg) on the expression levels of cytokines stimulated with the N protein (0.25 mg/kg) in mice (n = 3). g The effect of rolipram (10 mg/kg) on the phosphorylation level of IκB and p65 after N protein (0.25 mg/kg) stimulation in mice, with the gray value analysis (n = 3). h H&E staining of lung slices from N protein (0.25 mg/kg)-stimulated mice with or without treatment with rolipram (10 mg/kg). Scale bars, 50 μm and 25 μm. Data were shown as mean ± SD, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, n.s. P > 0.05

Fig. 7

Involvement of Cl⁻ signaling mediated by SARS-CoV-2 N protein-Smad3 interactions in N protein-triggered inflammatory responses in hPAECs. a The mRNA expression levels of IL1B in N protein (50 μg/ml)-stimulated hPAECs (n = 3). b The mRNA expression levels of CFTR in N protein (50 μg/ml)-stimulated hPAECs (n = 3). c The changes in [Cl⁻]_i of hPAECs after N protein (50 μg/ml) stimulation (n = 39 cells from 3 individuals). d Fluorescence labeling of Smad3 (red) and N protein (green) in hPAECs with DAPI-labeled nuclei (blue) after stimulation with N protein (50 μg/ml) for 24 h. Scale bars, 2 μm. e The effect of Inh miR-145 (100 nM) on the mRNA expression levels of CFTR in hPAECs stimulated with N protein (50 μg/ml) for 24 h (n = 3). f The effect of Inh miR-145 (100 nM) on the expression of CFTR in hPAECs stimulated by N protein (50 μg/ml) for 24 h, with the gray value analysis (n = 3). g The effect of Inh miR-145 (100 nM) on the [Cl⁻]_i of hPAECs stimulated with N protein (50 μg/ml) for 24 h (n = 20 cells from three individuals). h The effect of EMD638683 (50 μM) on the mRNA expressions of IL1B in N protein (50 μg/ml)-stimulated hPAECs (n = 3). i The mRNA expressions of PDE4 in the N protein (50 μg/ml)-stimulated hPAECs (n = 3). j The effect of PDTC (100 μM) on the [Cl⁻]_i of the N protein (50 μg/ml)-stimulated hPAECs (n = 20 cells from three individuals). k The effect of rolipram (20 μM) on [Cl⁻]_i of the N protein (50 μg/ml)-stimulated hPAECs (n = 3 cells from 3 individuals). l The effect of rolipram (20 μM) on the mRNA expression levels of IL1B in the N protein (50 μg/ml)-stimulated hPAECs (n = 3). Data were shown as mean ± SD, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with the control group or indicated by lines, n.s. P > 0.05

Fig. 8

Schematic diagram depicting the role of intracellular Cl⁻ accumulation in eliciting an ongoing inflammatory response after SARS-CoV-2 N protein stimulation in RECs. In RECs, the N protein interacted with Smad3, which triggered the downregulation of the CFTR via miR-145. Thereafter, the [Cl⁻]_i was elevated and elicited inflammatory responses through activating the Cl⁻-sensing SGK1. Moreover, the expression of PDE4 was upregulated owing to the activation of NF-κB, which resulted in cAMP degradation and dysfunction of CFTR, contributing to the sustained high [Cl⁻]_i and ongoing airway inflammation. [Created with BioRender.com (Canada)]