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SARS-CoV-2 spike protein and its RBD stimulate human microglia to secrete various proinflammatory mediators

Sep 30, 2023 | About the Virus

Coronavirus disease 2019 (COVID-19) is a clinical syndrome caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is a systemic multiple-organ disease characterized by a broad spectrum of clinical manifestations. SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA virus. Its genome encodes four structural proteins, namely the spike (S), envelope (E), nucleocapsid (N), and membrane (M) protein. In this in vitro study, researchers from the United States investigated the effects of the full-length recombinant SARS-CoV-2 S protein and its receptor-binding domain (RBD) on the release of proinflammatory mediators in human microglia. 

The S protein appears to be a major pathogenic factor that contributes to the unique pathogenesis of SARS-CoV-2. The S protein is a glycosylated homotrimer with each monomer composed of subunits S1 and S2, separated by host cell proteases. The S1 domain comprises the N-terminal domain (NTD), the RBD with a receptor binding motif (RBM), and two C-terminal domains. The RBD is a short immunogenic fragment that facilitates the binding of the S protein to the host cell angiotensin-converting enzyme 2 (ACE2) receptor.

Microglia, the resident mononuclear phagocytes of the central nervous system (CNS), act as the first line of defense in the brain. These cells are highly heterogeneous in the healthy CNS and account for between 5 and 10 percent of total brain cells. Activated microglia have important functions in the CNS, especially in neuroinflammation and neurodegenerative diseases. The postmortem examination of brains from deceased individuals infected with SARS-CoV-2 revealed extensive microglial activation and neuroinflammation associated with brain pathology. In addition, the alterations in neurometabolites, which are indicators of glial dysfunction, can be detected in the brains of patients diagnosed with long COVID syndrome.

About the study

The authors conducted an in vitro analysis to evaluate the influence of the full-length recombinant SARS-CoV-2  S protein and its RBD on the secretion of proinflammatory mediators such as interleukin (IL)-1β, IL-6, IL-18, the chemokine CXCL8, tumor necrosis factor-alpha  (TNF-α), matrix metallopeptidase 9 (MMP-9), and S100B protein in the immortalized human microglia-SV40 cell line, derived from primary human microglia.


The SARS-CoV-2 S protein stimulated the release of proinflammatory mediators from human microglia in a dose-dependent manner. After 24 hours of stimulation with various concentrations (1, 5, or 10 ng/ml) of the full-length recombinant S protein, the levels of proinflammatory proteins in cell culture supernatants were significantly increased in a dose-dependent manner compared to those observed in controls.

After 24 hours of stimulation, the full-length S protein significantly increased IL-1β, TNF-α, S100B, CXCL8, IL-6, and MMP9 levels compared with controls. RBD alone significantly increased TNF-α, IL-18, and S100B levels compared with controls. These data were confirmed with the S protein and RBD from two different sources.

The authors then investigated whether the proinflammatory responses triggered by the full-length recombinant S protein in human microglia were mediated by TLR2 or TLR4 signaling. They preincubated human microglia with anti-TLR2, anti-TLR4, and anti-ACE2 antibodies. Only pretreatment with 2 μg/ml of anti-TLR4 antibody completely suppressed the increase in the secretion of IL-1β, CXCL8, IL-6, S100B, and MMP9 in human microglia after 24 hours of stimulation with the full-length S protein. The pretreatment with an anti-TLR2 antibody or anti-TLR4 antibody (2 μg/ml) completely suppressed a release of TNF-α, stimulated by the full-length S protein. In addition, only pretreatment with anti-ACE2 antibody (2 μg/ml) completely suppressed the increase in the secretion of TNF-α, IL-18, and S100B in human microglia observed after the incubation with RBD alone.


In summary, SARS-CoV-2 S protein stimulated the release of IL-1β, CXCL8, IL-6, and MMP9 in human microglia by activating the TLR4 receptor. The S protein stimulates the secretion of TNF-α through the activation of the TLR2 and TLR4 receptors, whereas recombinant RBD alone stimulates the release of TNF-α, IL-18, and S100B via ACE2 signaling.

These data show that recombinant full-length S protein and RBD alone stimulated human microglia to secrete various proinflammatory mediators through the activation of different receptors. They confirm that SARS-CoV-2 S protein contributes to neuroinflammation via several mechanisms that are very likely involved in CNS pathologies associated with long-COVID.

This study has been published on a preprint server and is currently being peer-reviewed.

Journal Reference

Tsilioni I, Theoharides TC. 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. Research Square preprint. (Open Access).

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