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

  1. Shahanshah Khan
  2. Mahnoush S Shafiei
  3. Christopher Longoria
  4. John W Schoggins
  5. Rashmin C Savani
  6. Hasan Zaki  Is a corresponding author
  1. Department of Pathology, The University of Texas Southwestern Medical Center, United States
  2. Department of Pediatrics, The University of Texas Southwestern Medical Center, United States
  3. Department of Microbiology, The University of Texas Southwestern Medical Center, United States
6 figures, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements
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 with 1 supplement
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 with 2 supplements
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.

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 with 2 supplements
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 with 2 supplements
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.

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent
(Mus musculus)
C57BL/6JJackson
Laboratory
RRID:MGI:3028467JAX:000664
Genetic reagent
(M. musculus)
Myd88−/ C57BL/6JJackson
Laboratory
Genetic reagent
(M. musculus)
Tlr2−/ C57BL/6JJackson
Laboratory
B6.129-Tlr2tm
1kir/JStock No:
004650
Genetic reagent
(M. musculus)
Tlr4−/ C57BL/6JJackson
Laboratory
B6.B10ScN-
Tlr4lps-del/JthJStock No: 007227
Cell line(Homo sapiens)HEK-293TATCCCat# CRL-3216
Cell line(H. sapiens)A549ATCCCat# CCL-185
Cell line(H. sapiens)THP1ATCCCat# CRL-TIB-202Cat# CRL-TIB-202
Cell line(H. sapiens)Calu-3ATCCCat# HTB-55
Cell line(H. sapiens)HEK-Blue-Null2InvivoGenCat# hkb-null2
Cell line(H. sapiens)HEK-Blue-hTLR2InvivoGenCat# hkb-htlr2
Cell line(H. sapiens)HEK-Blue-hTLR2-TLR1InvivoGenCat# hkb-htlr21
Cell line(H. sapiens)HEK-Blue hTLR2-TLR6InvivoGenCat# hkb-htlr26
Cell line(H. sapiens)HEK-Blue-hTLR4InvivoGenCat# hkb-htlr4
Cell line(H. sapiens)Peripheral
blood
mononuclear
cells
StemCell TechnologiesCat# 70025
Cell line(M. musculus)Bone marrow-
derived
macrophage
This paperSee ‘Culture of
mouse bone-
marrow-derived
macrophages’ in
Materials and methods
Cell line(M. musculus)Primary lung
epithelial
cells
This paperSee ‘Culture of
mouse primary
lung epithelial
cells’ in Materials
and methods
Cell line(M. musculus)RAW264.7ATCCCat# TIB-71
Antibody(Rabbit
monoclonal)
Phospho-NF-
kB p65
Cell SignalingCat# 3033WB (1:1000)
Antibody(Mouse monoclonal)
Phospho-IκBα
Cell SignalingCat# 9246WB (1:1000)
Antibody(Rabbit
monoclonal)
IκBα
Cell SignalingCat# 4812WB (1:1000)
Antibody(Rabbit monoclonal)
anti-phospho
p44/42 (ERK1/2)
Cell SignalingCat# 4370WB (1:2000)
Antibody(Rabbit monoclonal)
anti-p44/42 (ERK1/2)
Cell SignalingCat# 4695WB (1:2000)
Antibody(Rabbit monoclonal)
anti-phospho SAPK/JNK
Cell SignalingCat# 4668WB (1:1000)
Antibody(Rabbit monoclonal)
anti-phospho AKT
Cell SignalingCat# 4060WB (1:1000)
Antibody(Rabbit monoclonal)
anti-phospho STAT3
Cell SignalingCat# 9145WB (1:1000)
Antibody(Mouse monoclonal)
anti-SARS-CoV-2 S
GeneTexCat# GTX632604WB (1:1000)FACS (1 μl/
1×106 cells)
Antibody(Monoclonal)
anti-
CD16/CD32
eBioscienceCat# 14-0161-82FACS (1 μg/1×106 cells)
Antibody(Mouse
monoclonal)
anti-β-actin
Sigma-AldrichCat# A2228WB (1:10,000)
Recombinant
DNA reagent
GFP-Flag (plasmid)VectorBuilderCat# VB200507-
2985cmv
Recombinant
DNA reagent
SARS-CoV-2 S-FlagVectorBuilderCat# VB200507-
2984jyv
Recombinant
DNA reagent
TLR1 CRISPR/Cas9Santa CruzCat# sc-423418
Recombinant
DNA reagent
TLR2 CRISPR/Cas9Santa CruzCat# sc-423981
Recombinant
DNA reagent
TLR6 CRISPR/Cas9Santa CruzCat# sc-423420
Sequence-
based reagent
m_Il1b_FThis paperPCR primersGCCTCGTG
CTGTCGG
ACCCATA
Sequence-
based reagent
m_Il1b_RThis paperPCR primersTGCAGGGT
GGGTGTG
CCGTCTT
Sequence-
based reagent
m_Il6_FThis paperPCR primersCAA GAA AGA
CAA AGC
CAG AGT C
Sequence-
based reagent
m_Il6_RThis paperPCR primersGAA ATT GGG
GTA GGA
AGG AC
Sequence-
based reagent
m_Tnfa_FThis paperPCR primersTCCCAGGTTC
TCTTCAAGGGA
Sequence-
based reagent
m_Tnfa_RThis paperPCR primersGGTGAGGAG
CACGTAGTCGG
Sequence-
based reagent
m_Ifng_FThis paperPCR primersGAAAGACAA
TCAGGCCATCA
Sequence-
based reagent
m_Ifng_RThis paperPCR primersTTGCTGTTGC
TGAAGAAGGT
Sequence-
based reagent
m_Ifnb_FThis paperPCR primersGCCTGGATG
GTGGTC
CGAGCA
Sequence-
based reagent
m_Ifnb_RThis paperPCR primersTACCAGTCC
CAGAGTCC
GCCTCT
Sequence-
based reagent
m_Ifna_FThis paperPCR primersTCTGATGCA
GCAGGTGGG
Sequence-
based reagent
m_Ifna_RThis paperPCR primersAGGGCTCT
CCAGACTTC
TGCTCTG
Sequence-
based reagent
m_Cxcl1_FThis paperPCR primersTGAGCTGCG
CTGTCA
GTGCCT
Sequence-
based reagent
m_Cxcl1_RThis paperPCR primersAGAAGCCA
GCGTTCA
CCAGA
Sequence-
based reagent
m_Cxcl2_FThis paperPCR primersCAA GAA CAT
CCA GAG CTT
GAG TGT
Sequence-
based reagent
m_Cxcl2_RThis paperPCR primersGCC CTT GAG
AGT GGC TAT
GAC TT
Sequence-
based reagent
h_IL1B _FThis paperPCR primersAAATACCTG
TGGCCTTGGGC
Sequence-
based reagent
h_IL1B _FThis paperPCR primersTTTGGGATC
TACACTC
TCCAGCT
Sequence-
based reagent
h_IL6 _FThis paperPCR primersGTAGCCGC
CCCACACAGA
Sequence-
based reagent
h_IL6 _RThis paperPCR primersCATGTCTCCT
TTCTCAG
GGCTG
Sequence-
based reagent
h_TNFA_FThis paperPCR primersCCCAGGGA
CCTCTCT
CTAATCA
Sequence-
based reagent
h_TNFA_RThis paperPCR primersGCTTGAGGG
TTTGCTA
CAACATG
Sequence-
based reagent
h_IFNG_FThis paperPCR primersCCAACGCAAA
GCAATACATGA
Sequence-
based reagent
h_IFNG_RThis paperPCR primersCCTTTTTCG
CTTCCCT
GTTTTA
Sequence-
based reagent
h_IFNB_FThis paperPCR primersATTGCCTCAA
GGACAGGATG
Sequence-
based reagent
h_IFNB_RThis paperPCR primersGGCCTTCA
GGTAA
TGCAGAA
Sequence-
based reagent
h_IFNA_FThis paperPCR primersGTGAGGAAAT
ACTTCCAAA
GAATCAC
Sequence-
based
reagent
h_IFNA_RThis paperPCR primersTCTCATGAT
TTCTGCTCT
GACAA
Sequence-
based reagent
h_CXCL1_FThis paperPCR primersAACCGAAGT
CATAGCCACAC
Sequence-
based reagent
h_CXCL1_RThis paperPCR primersCCTCCCTTC
TGGTCAGTT
Sequence-
based reagent
h_CXCL2_FThis paperPCR primersCGCCCAAAC
CGAAGTCAT
Sequence-
based reagent
h_CXCL2_RThis paperPCR primersGATTTGCCATT
TTTCAG
CATCTTT
Sequence-
based reagent
h_CCL2_FThis paperPCR primersAGGTGACTGG
GGCATTGAT
Sequence-
based reagent
h_CCL2_RThis paperPCR primersGCCTCCAGCA
TGAAAGTCTC
Peptide,
recombinant
protein
SARS-CoV-2 S1RayBiotechCat# 230-30161
Peptide,
recombinant
protein
SARS-CoV-2 S1R&DCat# 10569-
CV-100
Peptide,
recombinant
protein
SARS-CoV-2 S2RayBiotechCat# 230-30163
Peptide,
recombinant
protein
SARS-CoV-2 S2R&DCat# 10594-
CV-100
Peptide,
recombinant
protein
SARS-CoV-2 S-trimerR&DCat# 10549-
CV-100
Peptide,
recombinant
protein
SARS-CoV-2 NRayBiotechCat# 230-30164
Peptide,
recombinant
protein
SARS-CoV-2 MMyBioSourceCat# MBS8574735
Peptide,
recombinant
protein
SARS-CoV-2 EMyBioSourceCat# MBS9141944
Commercial
assay or kit
Pierce BCA
Protein Assay
Kit
Thermo Fisher
Scientific
Cat# 23227
Commercial
assay or kit
Mouse IL-6 ELISA KitR&D SystemsCat# DY406-05
Commercial
assay or kit
Mouse IL-1β ELISA KitR&D SystemsCat# DY401-05
Commercial
assay or kit
Mouse TNF-α ELISA KitR&D SystemsCat# DY410-05
Commercial
assay or kit
Human IL-6 ELISA KitR&D SystemsCat# DY206-05
commercial
assay or kit
Human IL-1β ELISA KitR&D SystemsCat#DY201-05
Commercial
assay or kit
Human TNF-α ELISA KitR&D SystemsCat# DY210-05
Commercial
assay or kit
SARS-CoV-2 S ELISA KitRayBiotechCat# ELV-COVID19S2
Chemical
compound,
drug
Phorbol-12-
myristate 13-
acetate (PMA)
InvivoGenCat# tlrl100 ng/ ml
Chemical
compound,
drug
TLR2-IN-C29SelleckchemS6597150 μM/ ml
Chemical
compound,
drug
Pam3CSK4InvivoGentlrl-pms
Chemical
compound,
drug
FSL-1InvivoGentlrl-fsl
Chemical
compound,
drug
ACE2 Inhibitor,
MLN-4760
Sigma-Aldrich530616000110 μM/ ml
Chemical
compound,
drug
Lipofectamine 3000Thermo Fisher ScientificCat# L3000015
Chemical
compound,
drug
Ultrapure
Escherichia coli-
derived LPS
InvivoGenCat# tlrl-smlps
Software,
algorithm
Flowjo v10Treestar, IncRRID:SCR_008520
Software,
algorithm
CytoFLEX-Beckman Coulter
Software,
algorithm
GraphPad PrismGraphpad.comRRID:SCR_00279

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  1. Shahanshah Khan
  2. Mahnoush S Shafiei
  3. Christopher Longoria
  4. John W Schoggins
  5. Rashmin C Savani
  6. Hasan Zaki
(2021)
SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway
eLife 10:e68563.
https://doi.org/10.7554/eLife.68563