SARS coronavirus spike protein-induced innate immune response occurs via activation of the NF-kappaB pathway in human monocyte macrophages in vitro

Abstract

A purified recombinant spike (S) protein was studied for its effect on stimulating human peripheral blood monocyte macrophages (PBMC). We examined inflammatory gene mRNA abundances found in S protein-treated PBMC using gene arrays. We identified differential mRNA abundances of genes with functional properties associated with antiviral (CXCL10) and inflammatory (IL-6 and IL-8) responses. We confirmed cytokine mRNA increases by real-time quantitative(q) RT-PCR or ELISA. We further analyzed the sensitivity and specificity of the prominent IL-8 response. By real-time qRT-PCR, S protein was shown to stimulate IL-8 mRNA accumulation in a dose dependent manner while treatment with E protein did not. Also, titration of S protein-specific production and secretion of IL-8 by ELISA showed that the dose of 5.6nM of S produced a significant increase in IL-8 (p=0.003) compared to mock-treated controls. The increase in IL-8 stimulated by a concentration of 5.6nM of S was comparable to concentrations seen for S protein binding to ACE2 or to neutralizing monoclonal antibody suggesting a physiological relevance. An NF-kappaB inhibitor, TPCK (N-Tosyl-L-Phenylalanine Chloromethyl Ketone) could suppress IL-8 production and secretion in response to S protein in PBMC and THP-1 cells and in HCoV-229E virus-infected PBMC. Activation and translocation of NF-kappaB was shown to occur rapidly following exposure of PBMC or THP-1 cells to S protein using a highly sensitive assay for active nuclear NF-kappaB p65 transcription factor. The results further suggested that released or secreted S protein could activate blood monocytes through recognition by toll-like receptor (TLR)2 ligand.

Fig. 1

Differential mRNA abundances determined by a microarray focused on immunologically relevant genes after treatment of PBMC (2 × 10⁵ cells) with 11.2 nM S protein for 24 h. Statistically significant (p value <0.05) differences in mRNA abundance were identified using the student's t-test by comparing treated with mock-treated culture from three or more independent experiments. Sixteen differences in mRNA abundance were identified. ¹CXCL10 was further analyzed by qRT-PCR.

Fig. 2

S protein-treated PBMC synthesized and secreted significantly (p < 0.05) more of IL-8, MIP-1β, IL-6, IL-1β inflammatory cytokines than the control. PBMC (2 × 10⁵ cells) were cultured with and without 11.2 nM S protein for 24 h and supernatants were assayed for cytokines by ELISA. Mean ± SE of three or more independent experiments is shown. p values were determined by student's t-test comparing S protein-treated and mock-treated control culture. TNFα p = ns.

Fig. 3

Production and secretion of IL-8 was significantly increased in PBMC after treatment with S protein. The kinetics and dose response effect of S protein on in vitro PBMC 3, 6 and 9 h after treatment with S protein (0.28, 2.8 and 5.6 nM) were evaluated. Secreted IL-8 is expressed as pg/ml (mean ± SE of three separate experiments). Statistical significance was determined using the student's t-test based on comparison of S protein-treated with the mock-treated control for each time point. Our results indicated that S protein treatment increased IL-8 production in a dose dependent manner and that 5.6 nM gave a statistically significant response compared to control.

Fig. 4

S protein but not E protein treatment increased IL-8 mRNA abundance and IL-8 production and secretion in PBMC. In vitro PBMC (2 × 10⁵ cells) were treated with S protein (0.28, 2.8, 5.6 and 28 nM) or E protein (0.5, 2.5 and 25 nM) for 24 h. (A) RNA was extracted, reverse transcribed, cDNA amplified and IL-8 gene expression was determined by real-time quantitative PCR. Relative expression of mRNA species was calculated using the comparative C_T method. Statistical significance was determined using the student's t-test based on comparison with the mock-treated control for each dose. Significant differences in IL-8 mRNA abundance were observed when the PBMC were treated with S protein ≥5.6 nM compared to control (p = 0.047, data of three separate experiments) while E protein did not increase IL-8 mRNA levels. (B) Secreted IL-8 concentrations at 24 h following S protein and E protein treatment of PBMC were determined by ELISA. IL-8 concentration increased in S protein-treated PBMC in proportion to mRNA abundance whereas secreted IL-8 did not increase in E protein-treated PBMC, compared to control.

Fig. 5

Effect of treatment with TPCK on the IL-8 response to S protein in PBMC and THP-1 cells and in HCoV-229E-infected PBMC. PBMC or THP-1 cells were pretreated with 25 μM TPCK for 20 min prior to addition of 11.2 nM S protein. Secretion of IL-8 into culture supernatants after 24 h of incubation was measured using ELISA: (A) In PBMC, in the presence of TPCK the response to S protein was reduced by 95% (p = 0.024) (B) In THP-1 cells, in the presence of TPCK the response to S protein was reduced by 95% (p = 0.022). (C) In HCoV-229E-infected PBMC, in the presence of TPCK the secretion of IL-8 was reduced by 51% at 6 h post-infection and 34% at 8 h post-infection (p = 0.018). The data are from three independent experiments.

Fig. 6

Quantitation of the effect of S protein on the activation of NF-κB. Nuclear proteins were extracted from PBMC or THP-1 cells treated with S protein (11.2 nM) or mock-treated (control). To assay for activated NF-κB, 1 μg of the nuclear extract was added per well coated with NF-κB consensus DNA sequence and the amount of active NF-κB p65 bound after 1 h incubation was measured as luminescence units using EZ Detect NF-κB p65 Transcription Factor Assay. Each bar represents luminescence activity per μg nuclear extract (mean ± S.E from three independent experiments). The results show that in (A) PBMC and (B) THP-1 cells, treatment with S protein increased NF-κB activity compared to controls. In THP-1 cells, TPCK pretreatment reduced NF-κB binding activity in response to S protein by 62% (p = 0.04) at 1 h post-treatment and by 77% (p = 0.008) at 4 h post-treatment with S protein.

Fig. 7

S protein is a ligand for human TLR2. Cultures of (A) hTLR2-293, (B) 293 and (C) hACE2-293 cells (1 × 10⁶ cells) were stimulated with S (spike) protein (11.2 μM), SARS pseudovirus (3 × 10⁵ RLU), supernatant from pSARS pHIV cotransfected 293 cells (3 × 10⁶ RLU), or infected with HCoV-229E (MOI = 1). Culture media were collected at 18 h and secreted IL-8 was measured by ELISA. Data are from two or more experiments. p = 0.03 for S protein compared with control in hTLR2-293 cells by the paired one-tailed t-test.