Recombinant SARS-CoV-2 Spike Protein and Its Receptor Binding Domain Stimulate Release of Different Pro-Inflammatory Mediators via Activation of Distinct Receptors on Human Microglia Cells

SARS-CoV-2 infects cells via its spike (S) protein binding to its surface receptor angiotensin converting enzyme 2 (ACE2) on target cells and results in acute symptoms involving especially the lungs known as COVID-19. However, increasing evidence indicates that SARS-CoV-2 infection produces neuroinflammation associated with neurological, neuropsychiatric, and cognitive symptoms persists well past the resolution of the infection, known as post-COVID-19 sequalae or long-COVID. The neuroimmune mechanism(s) involved in long-COVID have not been adequately characterized. In this study, we show that recombinant SARS-CoV-2 full-length S protein stimulates release of pro-inflammatory IL-1b, CXCL8, IL-6, and MMP-9 from cultured human microglia via TLR4 receptor activation. Instead, recombinant receptor-binding domain (RBD) stimulates release of TNF-α, IL-18, and S100B via ACE2 signaling. These results provide evidence that SARS-CoV-2 spike protein contributes to neuroinflammation through different mechanisms that may be involved in CNS pathologies associated with long-COVID.

Here, we show that SARS-CoV-2 spike protein contributes to neuro-inflammation through different mechanisms that may be involved in CNS pathologies associated with long-COVID.

Human Microglia Cell Culture
The immortalized human microglia-SV40 cell line derived from primary human microglia was purchased from Applied Biological Materials Inc. (ABM Inc.; Richmond, BC, Canada) and cultured in Prigrow III medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in type I collagen-coated T25-flasks (ABM Inc.).Microglia-SV40 maintain their phenotype and proliferation rates for over 10 passages, during which all experiments were performed using multiple microglia thaws and sub-cultured cells.Experiments were carried out in type I collagen-coated plates (BD PureCoat™ ECM Mimetic Cultureware Collagen I peptide plates, Becton Dickinson, Bedford, MA).Cell viability was determined by trypan blue (0.4%) exclusion.

Statistical Analysis
All in vitro conditions were performed in triplicate, and all experiments were repeated at least three times (n = 3).Results are presented as mean ± standard error of the mean (SEM).Differences between two groups were assessed using the Student's t-test.Comparisons among at least three groups were tested by one-way analysis of variance (ANOVA), and then post hoc comparisons to determine significant differences between several experimental groups and the control group and between two groups were performed using Dunnett's test and Bonferroni test, respectively.Differences with P-values less than 0.05 were considered statistically significant.All analyses were performed using Graph Pad Prism 5.

SARS-CoV-2 Spike Protein Stimulates Secretion of Pro-Inflammatory Mediators from Human Microglia in a Dose-Dependent Manner
We analyzed the effect of full-length S protein on secretion of pro-inflammatory mediators, including IL-1β and CXCL8, in human cultured microglia.Upon stimulation with different concentrations (1, 5, or 10 ng/mL) of recombinant SARS-CoV-2 full-length S protein for 24 h, pro-inflammatory protein levels in the cell culture supernatants were significantly elevated, compared with those in controls in a dosedependent manner (Fig. 1A, B).The data were confirmed by two different sources (Abcam and GeneTex) of recombinant SARS-CoV-2 full length S (Supplemental Fig. 1).

SARS-CoV-2 Spike Protein and RBD Stimulate Secretion of Different Pro-Inflammatory Mediators from Human Microglia
We examined the effects of recombinant SARS-CoV-2 fulllength S and RBD on secretion of the pro-inflammatory mediators IL-1β, CXCL8, IL-6, TNF-α, IL-18, MMP9, and S100B from human cultured microglia.Stimulation with full-length S, but not RBD for 24 h, stimulated significant release of IL-1β, CXCL8, IL-6 and MMP9 compared to controls (Fig. 2A-D).Interestingly, both full-length S and RBD were able to stimulate secretion of TNF-α and S100B levels (Fig. 2E and F).However, stimulation with RBD alone for 24 h was able to stimulate significant secretion of IL-18 compared to controls (Fig. 2G).The data were confirmed by two different sources (Abcam and GeneTex) of SARS-CoV-2 S and RBD (Supplemental Fig. 2).

Discussion
The SARS-CoV-2 S protein attaches to the surface receptor ACE2 via the S1 subunit containing the RBD, while the S2 subunit containing a transmembrane anchor that is needed to fuse the viral envelope with the host's cell surface membrane [1].
Our results showing the release of IL-18 and S100B are novel.IL-18 is longer acting than other proinflammatory cytokines [95] and its expression was increased in the amygdala of children with ASD [96][97][98].The mechanism of action of IL-18 is different than that of IL-1β because unlike the former, IL-18, the latter activates MAP kinases and not NF-kB [99].
In conclusion, the SARS-CoV-2 spike protein can stimulate secretion of different pro-inflammatory molecules via activation of distinct receptors on cultured human microglia leading to neuro-inflammation [64][65][66] that could damage brain cells [64,69,70].Preventing or minimizing the detrimental effects of the spike protein could lead to novel targeted treatment approaches [126,127].