SARS-CoV-2 spike S1 subunit induces neuroinflammatory, microglial and behavioral sickness responses: Evidence of PAMP-like properties

Abstract

SARS-CoV-2 infection produces neuroinflammation as well as neurological, cognitive (i.e., brain fog), and neuropsychiatric symptoms (e.g., depression, anxiety), which can persist for an extended period (6 months) after resolution of the infection. The neuroimmune mechanism(s) that produces SARS-CoV-2-induced neuroinflammation has not been characterized. Proposed mechanisms include peripheral cytokine signaling to the brain and/or direct viral infection of the CNS. Here, we explore the novel hypothesis that a structural protein (S1) derived from SARS-CoV-2 functions as a pathogen-associated molecular pattern (PAMP) to induce neuroinflammatory processes independent of viral infection. Prior evidence suggests that the S1 subunit of the SARS-CoV-2 spike protein is inflammatory in vitro and signals through the pattern recognition receptor TLR4. Therefore, we examined whether the S1 subunit is sufficient to drive 1 ) a behavioral sickness response, 2 ) a neuroinflammatory response, 3 ) direct activation of microglia in vitro , and 4 ) activation of transgenic human TLR2 and TLR4 HEK293 cells. Adult male Sprague-Dawley rats were injected intra-cisterna magna (ICM) with vehicle or S1. In-cage behavioral monitoring (8h post-ICM) demonstrated that S1 reduced several behaviors, including total activity, self-grooming, and wall-rearing. S1 also increased social avoidance in the juvenile social exploration test (24h post-ICM). S1 increased and/or modulated neuroimmune gene expression ( Iba1, Cd11b, MhcIIα, Cd200r1, Gfap, Tlr2, Tlr4, Nlrp3, Il1b, Hmgb1 ) and protein levels (IFNγ, IL-1β, TNF, CXCL1, IL-2, IL-10), which varied across brain regions (hypothalamus, hippocampus, and frontal cortex) and time (24h and 7d) post-S1 treatment. Direct exposure of microglia to S1 resulted in increased gene expression ( Il1b, Il6, Tnf, Nlrp3 ) and protein levels (IL-1β, IL-6, TNF, CXCL1, IL-10). S1 also activated TLR2 and TLR4 receptor signaling in HEK293 transgenic cells. Taken together, these findings suggest that structural proteins derived from SARS-CoV-2 might function independently as PAMPs to induce neuroinflammatory processes via pattern recognition receptor engagement.

The route of SARS-CoV-2 to brain infection: have we been barking up the wrong tree?

“…First, as already mentioned, Rhea et al [ 10 ] established that the cleaved S1 subunit can be transported from the olfactory epithelium to the hypothalamus, apparently by binding to ACE2. Second, Frank et al [ 14 ] showed that the S1 subunit, but not the S2 subunit, is sufficient to elicit neuroinflammation, including microglia activation and gene expression of multiple pro-inflammatory cytokines, as well as altered animal behavior reminiscent of neurological and cognitive symptoms in COVID-19 patients. Since microglia do not express ACE2 [ 14 ], these effects are likely mediated by binding of the S1 protein to pattern recognition receptors such as those of the toll-like receptors, especially TLR4 [ 14 ].…”

“…Second, Frank et al [ 14 ] showed that the S1 subunit, but not the S2 subunit, is sufficient to elicit neuroinflammation, including microglia activation and gene expression of multiple pro-inflammatory cytokines, as well as altered animal behavior reminiscent of neurological and cognitive symptoms in COVID-19 patients. Since microglia do not express ACE2 [ 14 ], these effects are likely mediated by binding of the S1 protein to pattern recognition receptors such as those of the toll-like receptors, especially TLR4 [ 14 ]. Accordingly, emerging evidence suggests that the cleaved S1 subunit travels along the ACE2-expressing nervus terminalis neurons and their axons into the brain, and the spike protein subunit 1 may then activate microglia by binding to TLR4, resulting in enhanced expression of pro-inflammatory cytokines such as IL1b and antigen-presenting molecules such as MHCII – molecules that are also upregulated in postmortem brains of COVID-19 patients.…”

“…Accordingly, emerging evidence suggests that the cleaved S1 subunit travels along the ACE2-expressing nervus terminalis neurons and their axons into the brain, and the spike protein subunit 1 may then activate microglia by binding to TLR4, resulting in enhanced expression of pro-inflammatory cytokines such as IL1b and antigen-presenting molecules such as MHCII – molecules that are also upregulated in postmortem brains of COVID-19 patients. Thus, a full-blown infection of the brain with replicating SARS-CoV-2 is not necessary to induce neuroinflammation with neurological, cognitive, and neuropsychiatric symptoms that can be caused by microglia activation, synapse stripping and neuronal death [ 14 ].…”

“…On the other extreme, SARS-CoV-2 in the brain – if it does not replicate and does not induce inflammation – may have little negative consequences. Microglial activation could be responsible for the neurological and neuropsychiatric effects in COVID and enhance the risk of dementia and other neurodegenerative diseases, even when the microglia in the brain become activated not by infection with replicating SARS-CoV-2 virus, but rather by exposure to the virus’ spike protein [ 14 ].…”

Does SARS-CoV-2 affect neurodegenerative disorders? TLR2, a potential receptor for SARS-CoV-2 in the CNS

“…However, increasing evidence supports that TLRs might play a role during SARS-CoV-2 pathogenesis 112 . It has been shown that the surface proteins of SARS-CoV-2 could behave as a PAMP, thereby inducing the upregulation of inflammatory factors in the rodent model through TLR2 and TLR4 113 . Prophylactic administration of a TLR2 agonist showed a protective effect against SARS-CoV-2 infection and decreased virus transmission through the activation of the innate immune system 114 .…”