key: cord-0872691-gz1qc240
authors: Olajide, Olumayokun A; Iwuanyanwu, Victoria U; Adegbola, Oyinkansola D; Al-Hindawi, Alaa A
title: SARS-CoV-2 spike glycoprotein S1 induces neuroinflammation in BV-2 microglia
date: 2021-08-19
journal: bioRxiv
DOI: 10.1101/2020.12.29.424619
sha: 292e37b868c6e9ac28c3de4f0358f549302f6e8b
doc_id: 872691
cord_uid: gz1qc240

In addition to respiratory complications produced by SARS-CoV-2, accumulating evidence suggests that neurological symptoms are associated with the disease caused by the coronavirus. In this study, we investigated the effects of the SARS-CoV-2 spike protein S1 stimulation on neuroinflammation in BV-2 microglia. Analyses of culture supernatants revealed an increase in the production of TNFα, IL-6, IL-1β and iNOS/NO. S1 also increased protein levels of phospho-p65 and phospho-IκBα, as well as enhancing DNA binding and transcriptional activity of NF-κB. These effects of the protein were blocked in the presence of BAY11-7082 (1 μM). Exposure of S1 to BV-2 microglia also increased the protein levels of NLRP3 inflammasome and enhanced caspase-1 activity. Increased protein levels of p38 MAPK was observed in BV-2 microglia stimulated with the spike protein S1 (100 ng/mL), an action that was reduced in the presence of SKF 86002 (1 μM). Results of immunofluorescence microscopy showed an increase in TLR4 protein expression in S1-stimulated BV-2 microglia. Furthermore, pharmacological inhibition with TAK 242 (1 µM) and transfection with TLR4 siRNA resulted in significant reduction in TNFα and IL-6 production in S1-stimulated BV-2 microglia. These results have provided the first evidence demonstrating S1-induced neuroinflammation in BV-2 microglia. We propose that induction of neuroinflammation by this protein in the microglia is mediated through activation of NF-κB and p38 MAPK, possibly as a result of TLR4 activation. These results contribute to our understanding of some of the mechanisms involved in CNS pathologies of SARS-CoV-2.

The emergence of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus in 2019 has resulted in a global pandemic affecting most countries and territories. To date, there are 207,784,507 confirmed cases and 4,370,424 deaths [1] . The most common complications of illness due to SARS-CoV-2 infection are mainly related to respiratory symptoms [2] [3] [4] . SARS-CoV-2 infection causes significant damage to the alveolus, interstitial and intra-alveolar oedema, infiltration of inflammatory cells (mainly macrophages) [5] .

In addition to respiratory complications of SARS-CoV-2 illness, accumulating evidence suggest that neurological symptoms are associated with COVID-19, the disease caused by the novel coronavirus [6] . These symptoms include headaches, loss of smell, confusion and strokes [7] . Furthermore, a retrospective study in China reported neurologic symptoms such as cerebrovascular diseases, consciousness impairment, and skeletal muscle symptoms [8] . Interestingly, the SARS-CoV-2 subunit S1 has been shown by a study to promote loss of barrier integrity and trigger a pro-inflammatory response in a 3D model of the human blood-brain barrier [9] , suggesting a possible mechanism for the neurological symptoms induced by the coronavirus. Other studies have proposed mechanisms of SARS-CoV-2-mediated CNS dysfunction [10, 11] . Further understanding of the cellular mechanisms involved in neurological consequences of SARS-CoV-2 infection is critical for identifying pharmacological strategies for prevention and treatment.

Microglia are brain-resident macrophages that regulate brain development, maintenance of neuronal networks, and injury repair [12] . In the resting state,

The Griess reagent (Promega) was used to determine the levels of levels of nitric oxide (NO) in supernatants of cultured BV-2 microglia treated with protein S1 (10, 50 and 100 ng/mL) for 24 h. Following treatment with S1, Griess assay (which measures nitrite, a stable breakdown product of NO) was carried out by adding 50 L of sulphanilamide to 50 L of culture supernatants, followed by incubation in the dark at room temperature for 10 min. Thereafter 50 µL of N-1-naphthyl ethylenediamine dihydrochloride (NED) was added and the mixture incubated for a further 10 min. Absorbance was measured at 540nm using a Tecan Infinite M microplate reader. Nitrite concentrations were determined from a nitrite standard reference curve.

The in-cell western is a proven method for the rapid quantification of proteins in cells [28, 29] . BV-2 microglia were seeded into a 96-well plate at 5 × 10 4 cells/mL, and cells treated at 70% confluence. At the end of each experiment, cells were fixed with 8% formaldehyde solution (100 L) for 15 min., followed by washing with PBS. The cells were then incubated with primary antibodies overnight at 4°C. The following antibodies were used: rabbit anti-iNOS (Abcam), rabbit anti-phospho-p65 (Cell Signalling Technology), rabbit anti-phospho-IB (Santa Cruz Biotechnology), rabbit anti-NLRP3 (Abcam), and rabbit anti-phospho-p38 (Cell Signalling Technology) antibodies. Thereafter, cells were washed with PBS and incubated with anti-rabbit HRP secondary antibody for 2 h at room temperature. Then, 100 µL HRP substrate was added to each well and absorbance measured at 450nm with a Tecan Infinite M microplate reader. Readings were normalised with Janus Green normalisation stain (Abcam).

BV-2 cells were seeded in 24-well plate at a density of 4 × 10 4 cells/mL. At 60% confluence, RPMI medium was replaced with Opti-MEM, with a further incubation for 2 h at 37°C. Transfection of BV-2 cells was performed by preparing a Glia-Mag transfection reagent (OZ Biosciences) and Cignal NF-B luciferase reporter (Qiagen) complex at a ratio 3:1 in 50 L Opti-MEM. The complex was added to BV-2 cells, and the plate placed on a magnetic plate (OZ Biosciences) and incubated at 37C for 30 min, followed by a further magnetic plate-free incubation for 2 h.

Thereafter, medium was changed to serum-free RPMI and cells treated with spike S1 (10, 50 and 100 ng/mL) for 6 h. This was followed by a Dual-Glo  reporter assay (Promega). Firefly and renilla luminescence were measured using a FLUOstar OPTIMA microplate reader (BMG Labtech).

The NF-B p65 transcription factor assay is a non-radioactive ELISA-based assay for evaluating DNA binding activity of NF-B in nuclear extracts. BV-2 microglia were seeded in a 6-well plate at a density of 4 × 10 4 cells/mL. The cells were then incubated with 10, 50 and 100 ng/mL of S1 for 60 min. At the end of the incubation, nuclear extracts were prepared from the cells and subjected to NF-B transcription factor binding assay according to the instructions of the manufacturer (Abcam).

The caspase-Glo ® 1 inflammasome assay (Promega) was used to measure the activity of caspase-1 directly in live cells or culture supernatants [30] . BV-2 microglia were seeded out in 24-well plate at a density of 4 × 10 4 cells/mL and stimulated with S1 (10, 50 and 10 ng/mL) for 24 h. After incubation, cell culture supernatants were collected and mixed with equal volume of Caspase-Glo ® 1 reagent or Caspase-Glo ® 1 reagent + YVAD-CHO (1 M) in a 96-well plate. The contents of the wells were mixed using a plate shaker at 400rpm for 30 seconds. The plate was then incubated at room temperature for 60 min, followed by luminescent measurement of caspase-1 activity with a FLUOstar OPTIMA reader (BMG LABTECH).

The effects of the NF-B inhibitor (BAY11-7082), NLRP3 inhibitor (CRID3) and p38 MAPK inhibitor (SKF 86002) on the release of TNF, IL-6 and IL-1 production was investigated by pre-treating BV-2 microglia with BAY11-7082 (1 M), CRID3 (1 M), and SKF 86002 (1 M) 60 min prior to stimulation with S1 (100 ng/mL) for a further 24 h. Culture supernatants were collected and analysed for levels of TNF, IL-6 and IL-1 using mouse ELISA kits (Thermo Scientific).

Immunofluorescence microscopy was carried out by seeding out BV2 microglia in a 24-well cell imaging plate (Eppendorf) at a density of 5 × 10 4 cells/ml. Thereafter, cells were stimulated with S1 (100 ng/ml) for 24 h. After stimulation, cells were washed with PBS and fixed with 500 µL formaldehyde (4%) for 15 min at room temperature. This was followed by PBS wash and blocking with 500 µL of 5% Bovine Serum Albumin for 60 min at room temperature. Cells were then incubated overnight at 4C with either Iba-1 Alexa Fluor ® 488 antibody (Santa Cruz Biotechnology; 1:500) or TLR4 antibody (Santa Cruz Biotechnology; 1:500). In experiments employing unconjugated primary antibody, cells were washed with PBS and further incubated in the dark for 2 h with Alexa Fluor ® 488-conjugated donkey anti-rabbit IgG secondary antibody (Thermo Scientific; 1:500). Then, cells were stained with 50 nM of 4', 6 diamidino-2-phenylindole dihydrochloride (DAPI; Invitrogen) for 3 min and washed.

Images were captured using EVOS ® FLoid® cell imaging station (Invitrogen).

The effects of TLR4 inhibitor TAK 242 were investigated on S1-induced increased production of TNF, IL-6, IL-1β. BV-2 microglia were pre-treated with TAK 242

(1 µM). After 60 min, cells were stimulated with S1 (100 ng/mL) for a further 24 h.

Levels of TNF and IL-6 in culture supernatants were measured using mouse ELISA kits (Thermo Scientific).

In order to further confirm the possible roles of TLR4 in S1-induced increased production of pro-inflammatory mediators, experiments were carried out to knockdown TLR4 in BV-2 microglia. BV-2 cells were seeded out into 6-well plates at concentration 4 × 10 4 cells/ml and cultured until 60% confluence. Thereafter, culture medium in each well was changed to Opti-MEM (1000 µL) followed by incubation for 2 h at 37°C. Complexes containing 1.8 µL of Glial-Mag transfection reagent (OZ Biosciences) and 2 µL of TLR4 or control siRNA (Santa Cruz Biotechnology) were then added to the cells. The culture plate was placed on a magnetic plate (OZ Biosciences) for 30 min, followed by incubation at 37°C for a further 24 h. Following successful transfection, the effects of loss-of-function of TLR4 were evaluated by stimulating the cells with SARS-CoV-2 spike protein S1 (100 ng/mL) for 24 h. Culture supernatants were collected and analysed for levels of TNFα and IL-6 using mouse ELISA kitsStatistical analyses Data are expressed as mean ± SEM for at least three independent experiments (n=3) and analysed using one-way analysis of variance (ANOVA) with post hoc Tukey's test (for multiple comparisons). Statistical analysis were conducted using the GraphPad Prism software (version 9).

Results of pilot experiments to determine effects of stimulation with 10, 50, 100, 500 and 1000 ng/mL of S1 on the production of TNF in BV-2 microglia at 1, 3, 6, 12 and Furthermore, there was a concentration-dependent increase in S1-induced increase in TNF production, which reached a maximum at 100 ng/mL at 3, 6, 12 and 24 h post-stimulation. When compared with effects of 100 ng/mL of S1, there were no further significant increases in TNF production when the concentration of S1 was increased to 500 and 1000 ng/mL Consequently, in subsequent confirmatory experiments 10, 50 and 100 ng/mL of S1 were then incubated with BV-2 cells for 24 h. Results show that with 10 ng/mL of S1, there was an insignificant (p<0.05) elevation in the secretion of TNF. However, on increasing the concentration to 50 ng/mL, significant (p<0.05) increase in the production of TNF was observed ( Figure 1A ). On increasing the concentration of the protein to 100 ng/mL, there was a ~15-fold increase in the levels of TNF in culture supernatants ( Figure 1A ).

Results in Figure 1B show that release of IL-6 was not significantly elevated in BV-2 microglia treated with 10 and 50 ng/mL of S1, in comparison with untreated (control) cells. However, incubation of the cells with 100 ng/mL of S1 resulted in ~10.2-fold and significant (p<0.001) increase in the secretion of IL-6 into culture medium.

Similar observations were made in experiments to determine the effects of S1 sub-unit on IL-1 production; at 10 and 50 ng/mL of S1, there was no significant effect on the release of IL-1. A ~5.5-fold elevation of IL-1 levels was however observed with 100 ng/mL of S1 ( Figure 1C ).

Analyses of culture supernatants obtained from BV-2 microglia incubated with S1 (10 ng/mL) showed insignificant (p<0.05) increase in the levels of NO, when compared with untreated (control) cells. We further showed that incubation with 50 and 100 ng/mL of S1 caused ~5.1 and 8.7 increase in NO production, respectively (p<0.01) (Figure 2A ).

We also used in cell western analyses to further demonstrate that incubation with S1 (50 and 100 ng/mL) resulted in significant (p<0.05) increase in protein levels of iNOS, in comparison with unstimulated cells ( Figure 2B ).

Immunofluorescence microscopy revealed that unstimulated BV-2 microglia express low levels of Iba-1 protein. However, on activation with S1 (10-100 ng/mL), there were marked increases in expression of ( Figure 3 ).

Following our observation that S1 induced a significant increase in the production of pro-inflammatory mediators at 100 ng/mL, we were next interested in evaluating the role of NF-B in its activity at this concentration. Firstly, we used in cell western assays to investigate effects of the protein on the cytoplasmic activation of p65 and IB. Results show that S1 (100 ng/mL) significantly (p<0.01) increased protein levels of both phospho-p65 and phospho-IB following incubation with BV-2 microglia for 15 min (Figures 4A and 4B ). These results also show that protein levels of phospho-p65 and phospho-IB were reduced in the presence of the NF-B inhibitor, BAY11-7082 (1 M).

Results of an NF-B p65 transcription factor assay revealed that following treatment of BV-2 microglia with S1 (100 ng/mL) for 60 min, there was a significant (p<0.001) increase in the binding of NF-B in nuclear extracts to consensus-binding sites in the DNA. Interestingly, pre-treatment of BV-2 cells with BAY11-7082 (1 M) prior to stimulation with S1 produced an inhibition in binding to a double stranded DNA sequence containing the NF-B response element ( Figure 4C ). Luciferase reporter gene assays to evaluate the effects of S1 on transcriptional activity showed that S1 stimulation of BV-2 microglia that were transfected with NF-B plasmid vector resulted in a significant (p<0.01) increase in luciferase activity, in comparison with untreated transfected cells. We further showed that on pre-treating the cells with BAY11-7082 (1 M), S1-induced increase in NF-B luciferase activity was markedly reduced ( Figure 4D ).

In order to further confirm the roles of NF-B in S1-induced neuroinflammation, BV-2 microglia were pre-treated with BAY11-7082 prior to stimulation with the protein for 24 h, followed by measurements of pro-inflammatory cytokines (TNF, IL-6 and IL-1) release in culture supernatants. In the presence of BAY11-7082 (1 M), S1induced increases in TNF, IL-6 and IL-1 production in BV-2 microglia were reduced by ~59.6%, ~40.6% and ~32.8%, respectively ( Figures 5A, 5B and 5C ). Figures 5D and 5E also show that S1-induced increase in NO production and iNOS protein expression were significantly (p<0.01) reduced when the cells were treated with BAY11-7082 (1 M) prior to S1 stimulation.

The NLRP3 inflammasome is known to contribute to the secretion of IL-1 during inflammation. Encouraged by results of experiments showing an increase in the production of this cytokine following BV-2 stimulation with S1, we next investigated its effect on protein levels of NLRP3 in the presence and absence of known NLRP3 inhibitors, CRID3 and BAY11-7082. Cytoblot analyses revealed that stimulation of BV-2 microglia with S1 (100 ng/mL) for 6 h resulted in ~11-fold increase in levels of NLRP3 protein. We also observed that S1-induced increase in NLRP3 protein levels were significantly reduced (p<0.05) in the presence of CRID3 (1 M) and BAY11-7082 (1 M) ( Figure 6A ).

We next determined measured caspase-1 activity in culture media of BV-2 cells that were stimulated with S1 for 6 h. Results in Figure 6B show a ~4.4-fold increase in caspase-1 activity as a result of S1 stimulation. Furthermore, both CRID3 (1 M) and BAY11-7082 (1 M) produced significant (p<0.001) inhibition in S1-induced increased caspase-1 activity.

Experiments to evaluate the effects of CRID3 on S1-induced increased secretion of TNF, IL-6 and IL-1 in BV-2 microglia revealed that pre-treatment with CRID3

(1 M) was ineffective in reducing TNF ( Figure 7A ) and IL-6 ( Figure 7B ) production.

However, pre-treatment of S1-stimulated BV-2 cells with CRID3 significantly (p<0.001) reduced IL-1 production ( Figure 7C ). We also observed that S1-induced NO production was not reduced by pre-treatment with CRID3 ( Figure 7D ).

Incubation of spike protein S1 (100 ng/mL) with BV-2 microglia for 60 min resulted in a significant (p<0.01) and ~11.3-fold increase in protein levels of phospho-p38; an outcome that was prevented by pre-treating the cells with SKF86002 (1 M) for 60 min prior to S1 stimulation ( Figure 8A ).

Based on the results showing that S1 caused an increase in protein levels of phospho-p38 and its inhibition by SKF86002, we were next interested to determine the effects of this inhibitor on cytokine production in S1-stimulated BV-2 microglia. In the presence of SKF86002 (1 M), TNF production was reduced by ~47.9% compared with S1 alone ( Figure 8B ). Similarly, S1-induced increased production of IL-6 was significantly (p<0.05) reduced in the presence of SKF86002 (1 M) ( Figure 8C ). However, there was insignificant reduction in the production of IL -1β by SKF86002 ( Figure 8D ).

Immunofluorescence microscopy revealed that stimulation of BV-2 microglia with 100 ng/mL S1 resulted in marked increase in TLR4 expression ( Figure 9A) . Further experiments showed that pre-treatment with the TLR4 signalling inhibitor, TAK 242

(1 µM) resulted in significant (p<0.05) reduction in the production of TNF and IL-6 ( Figures 9B and 9C ).

Furthermore TLR4 knockdown experiments revealed that TNF production was significantly (p<0.01) reduced in S1-stimulated BV-2 microglia transfected with TLR4 siRNA, in comparison with cells transfected with control siRNA ( Figure 9D) . Similarly, TLR4 knockdown resulted in reduced IL-6 production, when compared with control ( Figure 9E ).

During SARS-CoV-2 infection, viral attachment, fusion and entry into the host's cells are facilitated by the spike proteins which protrude from the surface of mature virions, by binding to the host ACE2 protein [31, 32] . However, accumulating evidence show that in addition to facilitating its fusion to the cell membrane, the location of the spike protein on SARS-CoV-2 also makes it a direct target for host immune responses [31] . With respect to immune responses induced by SARS-CoV-2 infection, it is now established that this may involve an exaggerated inflammation, also known as cytokine storm [33] [34] [35] . Recently, we reported induction of increased release of pro-inflammatory cytokines in human peripheral blood mononuclear cells stimulated with a recombinant SARS-CoV-2 spike protein sub-unit S1, further confirming the role of the spike protein in COVID-19 cytokine storm [36] .

In this study, we showed for the first time that a recombinant SARS-CoV-2 spike protein sub-unit S1 activated BV-2 microglia as demonstrated by an increase in the protein expression of Iba-1, which is mainly expressed by the microglia and increased by the activation of these cells [37, 38] . We further demonstrated that activation of BV-2 microglia by S1 resulted in increased release of TNF, IL-6 and IL-1β, which are hallmarks of neuroinflammation. Activation of neuroinflammatory processes by the spike S1 protein was further confirmed by results showing increased iNOS-mediated production of NO by the protein in microglia. Elevated iNOS/NO has been previously linked to a wide range of CNS disorders including Alzheimer's disease, Parkinson's disease, multiple sclerosis, epilepsy, and migraine

[39].

These are interesting outcomes, considering the roles played by microglia activation and subsequent increased release of pro-inflammatory mediators in neurological disorders. In Parkinson's disease for example, significant increase in the expression of IL-1β was shown in the substantia nigra and frontal cortex, compared to controls [40] . Pro-inflammatory cytokine (IL-1β, TNF-α, IL-6) release has also been linked to depression-like behaviours and cognitive defects in mice [41] . Further studies are therefore needed to elucidate the link between COVID-19 mediated hyperinflammation and the pathogenesis of neurological symptoms that have been reported to be associated with the disease.

In neuroinflammation, the NF-B transcription factor regulates the production of multiple pro-inflammatory genes, including the pro-inflammatory cytokines such as TNF, IL-6 and IL -1, as well as iNOS. We further showed that activation of microglial NF-B signalling mediates the production of pro-inflammatory mediators in the microglia by SARS-CoV-2 spike S1 protein through its ability to promote cytoplasmic phosphorylation of the p65 sub-unit and IB, as well as DNA binding of p65 sub-unit and NF-B transcriptional activity. Interestingly, these effects were blocked by the potent NF-B inhibitor, BAY11-7082. The involvement of NF-B in S1 [46] [47] [48] . In this study, we showed that in addition to increasing IL-1β production, SARS-CoV-2 spike S1 protein activated NLRP3 inflammasome and increased caspase-1 activity in BV-2 microglia. These effects were also shown to be attenuated by CRID3 and BAY11-7082, which are known inhibitors of NLRP3. Furthermore, pre-treatment of BV-2 microglia with CRID3 prevented SARS-CoV-2 spike S1 protein-induced IL-1βproduction, but not TNF, IL-6 or NO, suggesting an involvement of the NLRP3 inflammasome activation in neuroinflammation induced by S1 in BV-2 microglia.

It has been suggested that activation of NF-B could trigger NLRP3 through caspase-1, with subsequent release of mature IL-1β [49] . It is not clear from this study if the activation of NLRP3/caspase-1 by S1 was due to a direct effect on NLRP3 or an indirect action as a result of activating NF-B. The role of NLRP3 inflammasome activation in S1-induced neuroinflammation therefore requires further investigation.

Our results showing activation of p38 MAPK by S1, coupled with inhibition of S1induced increased production of TNF and IL-6 by the p38 inhibitor (SKF 86002) suggest the involvement of the p38 MAPK signalling in its pro-inflammatory effect in BV-2 microglia. One possible explanation for this effect is the activation by S1 of upstream signals which activate both NF-B and MAPK signalling in the microglia.

To explore the potential mechanisms involved in dual activation of NF-B and MAPK by S1 in BV-2 microglia, we conducted experiments that showed an increase in the expression of toll-like receptor 4 (TLR4) by the protein. Interestingly, a known inhibitor of TLR4 signalling, TAK 242 inhibited S1-induced increased production of both TNF and IL-6, which further demonstrates a potential involvement of this pattern recognition receptor (PRR) in S1-induced neuroinflammation. Our findings seem to confirm those reported from a study by Shirato and Kizaki [50] , as they demonstrated involvement of TLR4 in the induction of pro-inflammatory responses in mouse and human macrophages by SARS-CoV-2 spike protein S1 subunit. Similar observations linking TLR4 to induction of inflammation in THP-1 cells have been reported by Zhao et al. [51] . Studies to elucidate detailed modulatory effects of SARS-CoV-2 spike protein S1 subunit on microglia TLR4 signalling are required in order to provide better understanding of the impact of SARS-CoV-2 infection on the brain.

The obtained from these experiments have provided the first evidence demonstrating activation of BV-2 microglia by SARS-CoV-2 spike S1 protein, resulting in the production pro-inflammatory mediators TNF, IL-6, IL-1β and NO. We propose that induction of neuroinflammation by this protein in the microglia is mediated through activation of NF-B and p38 MAPK, possibly as a result of TLR4 activation. These results contribute to our understanding of some of the mechanisms involved in CNS pathologies of SARS-CoV-2. 

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The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. experiments. Data were analysed using One-way ANOVA followed by a post hoc Tukey's multiple comparison test. *p < 0.05, ***p < 0.001, ****p<0.0001, ns (not significant at p<0.05), compared with untreated control.

S1 increased levels of NO (A) and iNOS protein (B) in BV-2 microglia after a 24-h incubation. NO levels in culture supernatants were measured using the Griess assay, while in-cell western (cytoblot) assay was used to detect iNOS protein expression. All values are expressed as mean ± SEM for three independent experiments. Data were analysed using One-way ANOVA followed by a post hoc Tukey's multiple comparison test. *p<0.05, **p < 0.01, ***p < 0.001, ns (not significant at p<0.05), compared with untreated control.

Immunofluorescence microscopy showing increase in Iba-1 protein expression in BV-2 microglia incubated with 10, 50 and 100 ng/mL of S1.

S1 activated NF-B signalling in BV-2 microglia. Nuclear extracts were collected from cells stimulated with S1 (100 ng/mL) in the absence or presence of BAY11-7082 (1 µM) for 60 min and subjected to DNA binding assays (A). Results of luciferase reporter gene assay showing increased NF-B transcriptional activity by S1 and its inhibition by BAY11-7082 (1 µM) (B). In-cell western (cytoblot) analyses showing increased protein expressions of phospho-p65 sub-unit (C) and phospho-IB (D) following stimulation with S1 (100 ng/mL) for 15 min and inhibition by BAY11-7082 (1 µM). Data were analysed using One-way ANOVA followed by a post hoc Tukey's multiple comparison test. *p<0.05, **p < 0.01, ***p < 0.001, ns (not significant at p<0.05), compared with untreated control, or S1 stimulation alone.

Pre-treatment with BAY11-7082 (1 µM) resulted in inhibition of S1-induced increased production of TNF (A), IL-6 (B) and IL-1β (C) in BV-2 microglia. Culture supernatants were analysed using ELISA following stimulation for 24 h. Data were analysed using One-way ANOVA followed by a post hoc Tukey's multiple comparison test. **p < 0.01, ****p<0.0001, compared with untreated control, or S1 stimulation alone.

Pre-treatment with BAY11-7082 (1 µM) resulted in inhibition of S1-induced increased production of NO (D), iNOS protein (E) in BV-2 microglia, following stimulation for 24 h. Culture supernatants were analysed using Griess assay, in-cell western (cytoblot) was used for iNOS detection. Data were analysed using One-way ANOVA followed by a post hoc Tukey's multiple comparison test. *p<0.05, **p < 0.01, ***p<0.001, ****p<0.0001, compared with untreated control, or S1 stimulation alone.

(A) Stimulation of BV-2 microglia with S1 (100 ng/mL) increased protein expression of NLRP3 inflammasome, and was inhibited in the presence of CRID3 (1 µM) and BAY11-7082 (1 µM).

(B) Increased caspase-1 activity by S1 (100 ng/mL) BV-2 microglia was reduced in the presence of CRID3 (1 µM) and BAY11-7082 (1 µM).

Data were analysed using One-way ANOVA followed by a post hoc Tukey's multiple comparison test. *p<0.05, **p < 0.01, ***p<0.001, ****p<0.0001, compared with untreated control, or S1 stimulation alone.

Pre-treatment with CRID3 (1 µM) did not prevent S1-induced increased production of TNF (A), IL-6 (B), NO (D) while IL-1β production was reduced (C) in BV-2 microglia.

Culture supernatants were analysed using ELISA following stimulation for 24 h. Data were analysed using One-way ANOVA followed by a post hoc Tukey's multiple comparison test. **p < 0.01, ***p<0.001, ****p<0.0001, ns (not significant at p<0.05), compared with untreated control, or S1 stimulation alone.

Stimulation of BV-2 microglia with S1 (100 ng/mL) increased protein expression of phospho-p38 MAPK, which was inhibited in the presence of SKF 86002 (1 µM). Data were analysed using One-way ANOVA followed by a post hoc Tukey's multiple comparison test. *p<0.05, **p < 0.01, compared with untreated control, or S1 stimulation alone.

Effects of SKF 86002 (1 µM) on S1 -induced increased production of TNF (B), IL-6 (C) and IL-1β (D) in BV-2 microglia. Culture supernatants were analysed using ELISA following stimulation for 24 h. Data were analysed using One-way ANOVA followed by a post hoc Tukey's multiple comparison test. **p < 0.01, ***p<0.001, ****p<0.0001, ns (not significant at p<0.05), compared with untreated control, or S1 stimulation alone.

Immunofluorescence microscopy showing an increase in TLR4 protein expression following stimulation of BV-2 microglia with S1 (100 ng/mL) (A). Pre-treatment of S1stimulated BV-2 microglia with TAK 242 (1 µM) reduced the production of TNF (B) and IL-6 (C). Data were analysed using One-way ANOVA followed by a post hoc Tukey's multiple comparison test. *p < 0.05, **p<0.01, ***p<0.001, compared with untreated control, or S1 stimulation alone.

Transfection of S1-stimulated BV-2 microglia with mouse TLR4 siRNA reduced TNF (D) and IL-6 production (E). Data were analysed using One-way ANOVA followed by a post hoc Tukey's multiple comparison test. **p<0.01, control siRNA versus TLR4 siRNA.

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Methyl 3,4,5-trimethoxycinnamate suppresses inflammation in RAW264.7 macrophages and blocks macrophage-adipocyte interaction

Induction of neuroinflammation and neurotoxicity by synthetic hemozoin

Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein

The SARS-CoV-2 spike glycoprotein biosynthesis, structure, function, and antigenicity: implications for the design of spike-based vaccine immunogens

Cytokine release syndrome: inhibition of pro-inflammatory cytokines as a solution for reducing COVID-19 mortality

Targeting inflammatory cytokine storm to fight against COVID-19 associated severe complications

Inflammasomes are activated in response to SARS-CoV-2 infection and are associated with COVID-19 severity in patients

Danger-associated molecular patterns in Alzheimer's disease

Mechanisms of NLRP3 inflammasome activation: its role in the treatment of Alzheimer's disease

NLRP3 inflammasome-mediated microglial pyroptosis is critically involved in the development of post-cardiac arrest brain injury

NOD-like receptors and the innate immune system: coping with danger, damage and death

SARS-CoV-2 spike protein S1 subunit induces proinflammatory responses via toll-like receptor 4 signaling in murine and human macrophages

You F (2021) SARS-CoV-2 spike protein interacts with and activates TLR41