4. Activation of TLR4
While it is well established that the SARS-CoV-2 virus gains entry into human cells via the ACE2 receptor, there is another activation pathway that may be responsible for the cytokine storm associated with severe disease. A gene expression assay study involving peripheral blood mononuclear cells drawn from 48 subjects, including 28 COVID-19 patients (8 severe vs. 20 mild) revealed that severe cases were associated with activation of TLR4 signaling and a response that bore a strong resemblance to bacterial sepsis [30]. Furthermore, in vitrostudies on both human and mouse macrophages demonstrated that the S1 subunit of the spike protein alone activates TLR4 receptors and induces a strong inflammatory response via the NF-κB and JNK pathways [12]. The spike protein has also been shown to activate TLR2 [31]. This receptor is specifically associated with induction of IL-6 [32]. A case study involved four individuals who died of an ”unknown cause” following a second dose of an mRNA vaccine. RNA sequencing revealed that genes involved in neutrophil degranulation and a cytokine storm were sharply upregulated in the cases compared to controls, suggesting that the vaccines induced an excessive inflammatory response [33]. Another experiment showed that the S1 subunit of the SARS-CoV-2 spike protein interacts specifically with the extracellular leucine rich repeat domain of TLR4 to activate NF-κB [34].
TLR4 is a receptor that often responds to bacterial infections. The best-known stimulator of the TLR4 response is bacterial lipopolysaccharide (LPS). There is an acidic four-amino-acid sequence (PRRA) in the S1 segment of the spike protein, just above the furin cleavage site, unique among coronaviruses, that is also found inStaph aureus enterotoxin B (SEB), an extremely toxic enterotoxin. SEB is a potent inducer of TNF-α, and it induces an expansion of the pool of CD16+ monocytes. SARS-CoV-2 entry into cells can be inhibited by a monoclonal antibody against SEB [35,36]. It is possible that toll like receptor activation by spike depends in part on this unique sequence.
CD16+ cells are known for their more mature stage compared to other circulating monocytes. They are the primary cell type that infiltrates inflammatory tissues and launches the TLR4 signaling cascade [37].
When inflammation is occurring outside the CNS, there is a systemic response that takes place in the brain, whereby microglia become activated and upregulate TNF-α signaling. Subsequently, circulating monocytes are recruited into the brain through enhanced expression of cerebral monocyte chemoattractant protein (MCP)-1 [38]. Through such a mechanism, it is possible that CD16+ monocytes deliver spike protein to the brain, causing neuronal injury and explaining cognitive issues linked to long COVID.
A case study involved a 76-year-old man with Parkinson’s disease who died three weeks after his third immunization against COVID-19 (the BNT162b2 mRNA vaccine) [39]. Histopathological analyses of the brain revealed acute lymphocytic vasculitis and multifocal necrotizing encephalitis. Immunohistochemistry analysis identified the spike protein but not the nucleocapsid protein in the foci of inflammation in both the brain and the heart. The patient had not been previously diagnosed with COVID-19, so there is strong evidence that the vaccine caused this condition.