The S1 spike protein of SARS-CoV-2 upregulates the ERK/MAPK signaling pathway in DC-SIGN-expressing THP-1 cells

Abstract

Dendritic cells, macrophages, neutrophils, and other antigen-presenting cells express various C-type lectin receptors that function to recognize the glycans associated with pathogens. The dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) binds various pathogens such as HIV glycoprotein 120, the Ebola glycoprotein, hemagglutinin, and the dengue virus glycoprotein in addition to the SARS-CoV-2 spike protein, and also triggers antigen-presenting cell endocytosis and immune escape from systemic infections. Many studies on the binding of SARS-CoV-2 spike protein with glycans have been published, but the underlying mechanism by which intracellular signaling occurs remains unclear. In this study, we report that the S1 spike protein of SARS-CoV-2 induces the phosphorylation of extracellular signal-regulated kinases (ERKs) in THP-1 cells, a DC-SIGN-expressing human monocytic leukemic cell line. On the other hand, the phosphorylation level of NF-κB remained unchanged under the same conditions. These data suggest that the major cell signaling pathway regulated by the S1 spike protein is the ERK pathway, which is superior to the NF-κB pathway in these DC-SIGN-expressing THP-1 cells and may contribute to immune hyperactivation in SARS-CoV-2 infections. Additionally, several glycans such as mannans, mannosylated bovine serum albumin, the serum amyloid beta protein, and intracellular adhesion molecule 3 suppressed ERK phosphorylation, suggesting that these molecules are target molecules for SARS-CoV-2 infection by suppressing immune hyperactivation that occurs in the ERK signaling pathway.

Keywords: DC-SIGN; Dendritic cell; ERK; NF-κB; SARS-CoV-2 S1.

Fig. 1

The expression of DC-SIGN in THP-1 cells. (a) DC-SIGN expression in THP-1 cells was examined by Western blotting. After incubation with only PMA, both PMA and IL-4, or with GM-CSF and IL-4 for 3 days or 5 days, THP-1 cells were lysed and analyzed. GAPDH was used as a loading control. (b) The expression of DC-SIGN was examined by immunocytochemistry. After incubation with PMA and IL-4, the THP-1 cells were stained with an anti-DC-SIGN antibody and an anti-rabbit IgG antibody labeled with Alexa488. Counterstaining with Hoechst 333342 was additionally performed. Bars indicate 50 µm. Abbreviations used: DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL-4, interleukin-4; PMA, phorbol 12-myristate 13-acetate.

Fig. 2

The phosphorylation level of ERKs in THP-1 cells after incubation with DC-SIGN-targeting molecules. (a) Representative results of Western blotting indicating the phosphorylation level of ERKs. After incubation with PMA and IL-4, THP-1 cells were subsequently incubated with recombinant ICAM3, SARS-CoV-2 S1, SAP, mannan, or mannosylated BSA for 1 and 2 h. Then, the cells were lysed, and the 20 µg of total proteins were analyzed. The band intensities of phospho-ERKs relative to total-ERKs were measured, and they were normalized to the control results. GAPDH is a loading control. (b) The phosphorylation levels of ERKs in three independent experiments are summarized. The band images of two additional times of Western blotting are shown in Supplementary Figure S1. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations used: BSA, bovine serum albumin; DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICAM3, intracellular adhesion molecule 3; IL-4, interleukin-4; ns, not significant; PMA, phorbol 12-myristate 13-acetate; SAP, serum amyloid P.

Fig. 3

The phosphorylation level of NF-κB p65 in THP-1 cells after incubation with DC-SIGN-targeting molecules. (a) Representative results of Western blotting indicating the phosphorylation level of NF-κB p65. The band intensities of phospho-p65 relative to total-p65 were measured, and were normalized to the control results. GAPDH is a loading control. (b) The phosphorylation levels of NF-κB p65 in three independent experiments are summarized. The band images of two additional times of Western blotting are shown in Supplementary Figure S2. Abbreviations used: BSA, bovine serum albumin; DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICAM3, intracellular adhesion molecule 3; ns, not significant; SAP, serum amyloid P.

Fig. 4

The S1 spike protein induces ERK pathway, which is superior to the NF-κB pathway in the DC-SIGN-expressing THP-1 cells. Abbreviations used: BSA, bovine serum albumin; DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin; ERK, extracellular signal-regulated kinase; ICAM3, intracellular adhesion molecule 3; SAP, serum amyloid P.