Spike protein and its receptor-binding domain stimulate different proinflammatory mediators by activating different receptors in cultured microglia

Researchers from the United States investigated the effects of the full-length recombinant SARS-CoV-2 spike (S) protein and its receptor-binding domain (RBD) on the release of proinflammatory mediators in human microglia. The results showed that S protein and RBD stimulate the secretion of different proinflammatory molecules via activation of distinct receptors.

SARS-CoV-2 infection can lead to a new disease, known as post-acute sequelae of SARS-CoV-2 infection or long COVID syndrome. Long COVID includes neurological, cardiovascular and thrombotic disorders. However, the most frequent, persistent and disabling symptoms of long COVID are neurological. Neurological manifestations of long COVID include headaches, sleep disturbances, visual and olfactory dysfunction, gait disturbances, paresthesia, coordination problems, and cognitive impairments. The neurological sequelae that are less prevalent include cranial naturopathies, peripheral naturopathies and tremor. Their presence is associated with a long-term impairment of functional ability. No significant progress has been made in comprehending the underlying mechanisms, despite extensive mapping of the spectrum of neurological sequelae in patients with long COVID.

The S protein is a key pathogenic factor that contributes to the unique pathogenesis of SARS-CoV-2. The S protein is composed of two subunits: the S1 subunit which contains N-terminal domain, RBD with a receptor binding motif, and two C-terminal domains, and the S2 subunit which contains a transmembrane anchor that mediates the fusion of viral and host cell membranes. RBD is a short immunogenic fragment that facilitates the binding of S protein to the angiotensin-converting enzyme-2 (ACE2) receptor. Multiple conformations showing RBD in either an ‘up’ or a ‘down’ orientation have been observed by cryogenic electron microscopy. It has been reported that the RBDs of S protein binds the ACE2 receptor only in an ‘up’ conformation.

However, SARS-CoV-2 binding is not restricted to the ACE2 receptor. The S protein interacts with other cell surface factors that are thought to serve as attachment factors and promote SARS-CoV-2 entry.

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. Microglia express toll-like receptors (TLRs), which are transmembrane receptors activated by damage associated molecular patterns (DAMPs). The postmortem examination of brains from deceased individuals infected with SARS-CoV-2 revealed extensive microglial activation and neuroinflammation associated with brain pathology.

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, the chemokine CXCL8, IL-6, tumor necrosis factor-alpha  (TNF-α), IL-18, matrix metallopeptidase 9 (MMP-9) and S100B protein in the immortalized human microglia-SV40 cell line, which was derived from primary human microglia.


The results showed that 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.

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

According to these findings, both, the full-length S protein and RBD stimulated the secretion of TNF-α and S100B.

The authors then investigated whether the proinflammatory responses triggered by the full-length recombinant S protein in human microglia were mediated by either TLR2 or TLR4 signaling. They preincubated human microglia with anti-TLR2, anti-TLR4, and anti-ACE2 antibodies.

As mentioned previously, full-length S protein (10 ng/ml) significantly increased the secretion of IL-1β, CXCL8, IL-6, S100B, and MMP9 in human microglia after 24 hours of stimulation. Only pretreatment with 2 μg/ml of anti-TLR4 antibody completely suppressed this effect. The release of TNF-α, stimulated by the full-length S protein, was completely suppressed by pretreatment with anti-TLR2 antibody or anti-TLR4 antibody (2 μg/ml).

RBD alone significantly increased the secretion of TNF-α, IL-18 and S100B in human microglia, and only pretreatment with anti-ACE2 antibody (2 μg/ml) completely suppressed this effect.

These results show that S protein stimulates 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 release of TNF-α, IL-18 and S100B via ACE2 signaling.

In summary, this study demonstrates that recombinant full-length S protein and RBD can stimulate human microglia to secrete various proinflammatory mediators via activation of different receptors. These results confirm that SARS-CoV-2 spike protein contributes to neuroinflammation via several mechanisms that may be involved in CNS pathologies associated with long-COVID.

The results of the study have been published on a preprint server and are 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).