SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway

Abstract

The pathogenesis of COVID-19 is associated with a hyperinflammatory response; however, the precise mechanism of SARS-CoV-2-induced inflammation is poorly understood. Here, we investigated direct inflammatory functions of major structural proteins of SARS-CoV-2. We observed that spike (S) protein potently induced inflammatory cytokines and chemokines, including IL-6, IL-1β, TNFα, CXCL1, CXCL2, and CCL2, but not IFNs in human and mouse macrophages. No such inflammatory response was observed in response to membrane (M), envelope (E), and nucleocapsid (N) proteins. When stimulated with extracellular S protein, human and mouse lung epithelial cells also produced inflammatory cytokines and chemokines. Interestingly, epithelial cells expressing S protein intracellularly were non-inflammatory, but elicited an inflammatory response in macrophages when co-cultured. Biochemical studies revealed that S protein triggers inflammation via activation of the NF-κB pathway in a MyD88-dependent manner. Further, such an activation of the NF-κB pathway was abrogated in Tlr2-deficient macrophages. Consistently, administration of S protein-induced IL-6, TNF-α, and IL-1β in wild-type, but not Tlr2-deficient mice. Notably, upon recognition of S protein, TLR2 dimerizes with TLR1 or TLR6 to activate the NF-κB pathway. Taken together, these data reveal a mechanism for the cytokine storm during SARS-CoV-2 infection and suggest that TLR2 could be a potential therapeutic target for COVID-19.

Keywords: COVID-19; SARS-CoV-2; TLR2; cytokine storm; immunology; inflammation; spike protein; viruses.

Figure 1.. SARS-CoV-2 S protein induces cytokines and chemokines in macrophages and monocytes.

(A) Human monocytic cells THP1-derived macrophages were stimulated with recombinant S1, S2, M, N, and E proteins of SARS-CoV-2 at a concentration of 500 ng/ml. Four hours post-stimulation, the expression of IL6, IL1B, TNFA, CXCL1, CXCL2, CCL2, IFNA, IFNB, and IFNG was measured by real-time RT-PCR. (B) THP1 cells were stimulated with S2 protein at various concentrations for 4 hr and measured the indicated cytokines by real-time RT-PCR. (C) THP1 cells were stimulated with S2 protein (500 ng/ml). RNA isolated at 2, 4, and 8 hr post-stimulation was measured for IL6, IL1B, TNFA, CXCL1, and CXCL2 by real-time RT-PCR. (D) Human peripheral blood mononuclear cells (PBMCs) were incubated with S2 (500 ng/ml) protein for 4 hr. The expression of IL6, IL1B, TNFA, CXCL1, and CXCL2 was measured by real-time RT-PCR. (E) THP1 cells were incubated with S1 (500 ng/ml) or S2 (500 ng/ml) in the presence or absence of ACE2 inhibitor MLN-4760 (10 mM). The expression of cytokines was measured at 4 hr by real-time RT-PCR. Data represent mean ± SD (n=3); *p<0.05, **p<0.001, ***p<0.0001, ****p<0.00001 by unpaired Student’s t-test. Experiments described in (A) were repeated three times, and (B–E) were repeated two times. Data of representative experiments are presented.

Figure 1—figure supplement 1.. SARS-CoV-2 S protein induces inflammatory cytokines in macrophages.

(A) THP1 cells were stimulated with S2 protein. Four hours post-stimulation, culture supernatants were analyzed for IL-6, IL-1β, and TNFα by ELISA. (B) THP1 cells were stimulated with S1 and S2 individually or together or in combination with M, N, and E proteins (500 ng/ml each protein). RNA isolated at 4 hr post-stimulation was measured for IL6, IL1B, and TNFA. (C) THP-1 cells were stimulated with PolyI:C (1 μg/ml). The expression of interferons was measured by real-time RT-PCR. (D) THP1 cells were stimulated with native S2 or heat-denatured (HD) S2 (500 ng/ml). The induction of IL6, IL1B, and TNFA was measured by real-time RT-PCR at 4 hr post-stimulation. (E) THP1 cells were stimulated with S1, S2, or S-tri manufactured by R&D for 4 hr. The induction of inflammatory cytokines was measured by real-time RT-PCR. Data represent mean ± SD (n=3); **p<0.001, ***p<0.0001, ****p<0.00001 by unpaired Student’s t-test. Experiments were repeated two times and data of representative experiments are presented.

Figure 1—figure supplement 2.. Mouse macrophages are stimulated by SARS-CoV-2 S protein.

(A) Bone marrow-derived macrophages from WT mice were stimulated with S1 and S2 proteins (500 ng/ml) for 4 hr. The expression of Il6, Il1b, Tnfa, Cxcl1, Cxcl2, Ifna, Ifnb, and Ifng was measured by real-time RT-PCR. (B) RAW264.7 murine macrophage cells were stimulated with S1 or S2 (500 ng/ml) for 4 hr. The expression of Il6, Il1b, and Tnfa was measured by real-time RT-PCR. Data represent mean ± SD (n=3); *p<0.05, **p<0.001, ***p<0.0001 by unpaired Student’s t-test. Experiments were repeated two times and data of representative experiments are presented.

Figure 2.. Lung epithelial cells produce inflammatory molecules in response to SARS-CoV-2 S protein.

(A, B) A549 or Calu3 cells were incubated with SARS-CoV-2 S1 or S2 proteins (500 ng/ml) for 12 and 24 hr. The expression of inflammatory cytokines and chemokines was measured by real-time RT-PCR. (C) Primary mouse lung epithelial cells were stimulated with S2 (500 ng/ml) for 12 and 24 hr. The expression of inflammatory cytokines and chemokines was measured by real-time RT-PCR. (D, E) Calu3 cells or mouse lung primary epithelial cells were stimulated with S2 (500 ng/ml). Culture supernatant collected at 12 and 24 hr were analyzed for IL-6, IL-1β, and TNFα by ELISA. *p<0.05, **p<0.001, ***p<0.0001, ****p<0.00001 by unpaired Student’s t-test. Experiments in (A, B) were repeated three times. Other experiments were repeated two times and data of representative experiments are presented.

Figure 2—figure supplement 1.. Epithelial cells do not respond to SARS-CoV-2 S protein acutely.

A549 cells were incubated with SARS-CoV-2 S1 or S2 (500 ng/ml) proteins for 4 hr. The expression of inflammatory cytokines and chemokines was measured by real-time RT-PCR. Data represent mean ± SD (n=3). Experiments were repeated two times and data of representative experiments are presented.

Figure 3.. Epithelial cells expressing S protein stimulate macrophages during co-culture.

(A–C) SARS-CoV-2 S protein was overexpressed in A549 or Calu3 cells. Forty-eight hours following transfection with S or GFP plasmids, cell culture supernatants were collected and added into THP1 cells in culture at 30% v/v. (B, C) The expression of IL6, IL1B, and TNFA in THP1 cells at 4 hr was measured by real-time RT-PCR. (D) A549 or Calu3 cells expressing S protein were co-cultured with THP1 cells at 1:2 ratio for 16 hr. (E, F) The expression of IL6, IL1B, and TNFA was measured by real-time RT-PCR. (G, H) Protein levels of IL-6, IL-1β, and TNFα in culture supernatant described in (D) were measured by ELISA. Data represent mean ± SD (n=3); **p<0.001, ***p<0.0001 by unpaired Student’s t-test. Experiments were repeated three times and data of representative experiments are presented.

Figure 3—figure supplement 1.. Cytosolic S protein dose not trigger inflammation in epithelial cells.

A549, Calu3, and HEK293T cells were transfected with plasmids containing flag-tagged S or green fluorescent protein (GFP). (A) Forty-eight hours post-transfection, cell lysates were collected and the expression of S was measured by Western blot analysis of S protein. (B–D) Forty-eight hours following transfection with expression plasmids, the mRNA levels of IL6, IL1B, TNFA, CXCL1, and CXCL2 were measured by real-time RT-PCR. Data represent mean ± SD (n=3). Experiments were repeated two times and data of representative experiments are presented.

Figure 3—figure supplement 2.. HEK293T cells expressing S protein activate macrophages.

(A–B) Spike or GFP (control) were overexpressed in HEK293T cells. THP1 cells were stimulated with cell culture supernatant (30% v/v) of HEK293T-S or HEK293T-GFP for 4 hr. The expression of IL6, IL1B, and TNFA was measured by real-time RT-PCR. (B) Spike or GFP (control) proteins were overexpressed in HEK293T or A549 cells. Cell culture supernatants and cell lysates were collected at 48 hr after transfection, and analyzed for S protein by ELISA. (C) The expression of S on the cell surface of HEK293T-S cells was measured by flow cytometry following surface staining of S protein. (D, E) HEK293T cells expressing S protein were co-cultured with THP1 cells at 1:2 ratio for 16 hr. The expression of IL6, IL1B, and TNFA was measured by real-time RT-PCR (D) and ELISA (E). (F) S or GFP (control) proteins were overexpressed in HEK293T cells. Cells were sonicated, and cell lysate supernatants were collected. THP1 cells were incubated with these cell lysate supernatants for 4 hr. (G) The expression of IL6, IL1B, TNFA, CXCL1, and CXCL2 in THP1 cells was measured by real-time RT-PCR. Data represent mean ± SD (n=3); *p<0.05, **p<0.001, ***p<0.0001 by unpaired Student’s t-test. Experiments were repeated two times and data of representative experiments are presented.

Figure 4.. SARS-CoV-2 S protein activates the NF-κB pathway.

(A, B) THP1 and A549 cells were stimulated with S2 (500 ng/ml) for indicated time points. Phosphorylation of P65, IκBα, ERK, JNK, STAT3, and AKT was measured by Western blotting. (C, D) THP1 cells were stimulated by SARS-CoV-2 S2 protein (500 ng/ml) in the presence or absence of IKKβ inhibitor sc514. Phosphorylation of P65 and IκBα was measured by Western blotting (C). The expression of IL6, IL1B, TNFA, CXCL1, and CXCL2 in stimulated THP1 cells was measured by real-time RT-PCR (D). Data represent mean ± SD (n=3); *p<0.05, **p<0.001, ***p<0.0001, ****p<0.00001 by unpaired Student’s t-test. Experiments in (A, B) were repeated three times and (C, D) were repeated two times. Data of representative experiments are presented.

Figure 5.. TLR2 recognizes SARS-CoV-2 S protein and activate the NF-κB pathway.

(A, B) Bone marrow-derived macrophages (BMDMs) from WT and Myd88^−/− mice were stimulated with S2 protein (500 ng/ml). (A) The activation of the NF-κB pathway was measured by Western blot analysis of P-P65 and P-IκBα. (B) The induction of Il6, Il1b, and Tnfa was measured by real-time RT-PCR. (C) BMDMs from WT, Tlr2^−/−, and Tlr4^−/− mice were treated with S2 protein (500 ng/ml). Cell lysates collected at different times were analyzed for the activation of the NF-κB pathway by Western blotting of P-P65 and P-IκBα. (D) BMDMs from WT and Tlr2^−/− mice were treated with S1 protein (500 ng/ml), and the activation of P65 and IκBα was measured by Western blotting. (E, F) WT, Tlr2^−/−, and Tlr4^−/− macrophages were treated with S2 protein (500 ng/ml) or S-tri (500 ng/ml). The expression of cytokines was measured by real-time RT-PCR at 4 hr post-stimulation. Data represent mean ± SD (n=3); ***p<0.0001, ****p<0.00001 by unpaired Student’s t-test. Experiments were repeated two times and data of representative experiments are presented. (G) THP1 cells were stimulated with S2 protein (500 ng/ml), Pam3CSK4 (500 ng/ml), or LPS (100 ng/ml) in the presence or absence of Tlr2 inhibitor C29 (150 mM) for 4 hr. The expression of IL6, IL1B, and TNFA was measured by real-time RT-PCR. (H) WT and Tlr2^−/− mice were administered with S1 and S2 protein (1 μg each/mouse). Blood collected before and 16 hr post S protein administration was measured for IL-6, IL-1β, and TNFα by ELISA. Data represent mean ± SEM (n=5); ***p<0.0001, ****p<0.00001 by unpaired Student’s t-test. Experiments were repeated two times and data of representative experiments are presented.

Figure 5—figure supplement 1.. Macrophages of Tlr2^−/− mice are defective in sensing TLR2 ligand Pam3CSK4.

BMDMs from WT and Tlr2^−/− mice were stimulated with LPS (1 μg/l) or Pam3CSK4 (1 μg/ml) for 4 hr. The expression of inflammatory cytokines was measured by real-time RT-PCR. Data represent mean ± SD (n=3).

Figure 5—figure supplement 2.. Inhibition of TLR2 abrogates S-mediated inflammatory response in Calu-3 cells.

Calu3 cells were stimulated with S2 (500 ng/ml) in the presence or absence of TLR2-inhibitor C29 (150 mM) for 24 hr. The expression of inflammatory cytokines was measured by real-time RT-PCR. Data represent mean ± SD (n=3); ***p<0.0001 by unpaired Student’s t-test. Experiments were repeated two times and data of representative experiments are presented.

Figure 6.. TLR1 and TLR6 are dispensable in S-mediated activation of TLR2/NF-κB pathway.

(A, B) HEK-Blue-Null, HEK-Blue-TLR2, HEK-Blue-TLR1/2, HEK-Blue-TLR2/6, and HEK-Blue-TLR4 were stimulated with S1, S2, or S-tri for 6 hr. FSL1, Pam3CSK4, and LPS were used as ligands for TLR2/1, TLR2/6, and TLR4, respectively. The activation of NF-κB was monitored by the blue color development (A), which was measured at 620 nm (B). (C) HEK-Blue-Null, HEK-Blue-TLR2, HEK-Blue-TLR1/2, HEK-Blue-TLR2/6, and HEK-Blue-TLR4 cells were stimulated with S2 (500 ng/ml) at indicated times. The activation of P-P65 and P-IκBα was measured by Western blot analysis. (D) HEK-Blue-Null, HEK2-Blue-TLR2, HEK-Blue-TLR1/2, HEK-Blue-TLR2/6, and HEK-Blue-TLR4 cells were stimulated with S2 (500 ng/ml) for 6 hr. The induction of IL6 and IL1B was measured by real-time RT-PCR. (E, F) HEK-Blue-TLR2, HEK-Blue-TLR2/1, and HEK-Blue-TLR2/6 cells were stimulated with S1 or S2 in the presence or absence of TLR2 inhibitor C29 (150 mM) for 6 hr. The NF-κB activity was monitored colorimetrically at 620 nm. (G, H) TLR1, TLR2, TLR6, or TLR1/6 were knocked out in Raw264.7 cells with CRISPR/Cas9. Cells were then stimulated with S2 protein (500 ng/ml) for 4 hr. (G) The expression of cytokines was measured by real-time RT-PCR. Data represent mean ± SD (n=5); ***p<0.0001, ****p<0.00001 by unpaired Student’s t-test. All experiments were repeated three times and data of representative experiments are presented.

Figure 6—figure supplement 1.. M, N, and E proteins do not activate TLR2 pathway.

HEK-Blue-TLR2 and HEK-Blue-TLR4 cells were stimulated with S1, S2, M, N, or E proteins (500 ng/ml of each protein). Six hours following stimulation, blue substrate activation was measured by optical density taken at 620 nm. Data represent mean ± SD (n=3). Experiments were repeated three times and data of representative experiments are presented.

Figure 6—figure supplement 2.. Knocking out of TLRs in Raw264.7 cells with CRISPR/Cas9.

Tlr1, Tlr2, Tlr6, or Tlr1/6 were knocked out in Raw264.7 cells with CRISPR/Cas9. (A) The expression of Tlr1, Tlr2, Tlr6, and Tlr4 was measured by real-time RT-PCR. (B) Knockout cells were stimulated with Pam3CSK4 (500 ng/ml), FSL1 (100 ng/ml), or LPS (100 ng/ml) for 4 hr. The expression of Il6 was measured by real-time RT-PCR. Data represent mean ± SD (n=3). Experiments were repeated two times and data of representative experiments are presented.