Role of SARS-CoV-2 Spike-Protein-Induced Activation of Microglia and Mast Cells in the Pathogenesis of Neuro-COVID

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). About 45% of COVID-19 patients experience several symptoms a few months after the initial infection and develop post-acute sequelae of SARS-CoV-2 (PASC), referred to as “Long-COVID,” characterized by persistent physical and mental fatigue. However, the exact pathogenetic mechanisms affecting the brain are still not well-understood. There is increasing evidence of neurovascular inflammation in the brain. However, the precise role of the neuroinflammatory response that contributes to the disease severity of COVID-19 and long COVID pathogenesis is not clearly understood. Here, we review the reports that the SARS-CoV-2 spike protein can cause blood–brain barrier (BBB) dysfunction and damage neurons either directly, or via activation of brain mast cells and microglia and the release of various neuroinflammatory molecules. Moreover, we provide recent evidence that the novel flavanol eriodictyol is particularly suited for development as an effective treatment alone or together with oleuropein and sulforaphane (ViralProtek®), all of which have potent anti-viral and anti-inflammatory actions.

A systematic review of the literature using the MEDLINE data base (1 January 1990-1 January 2023) was conducted to identify peer-reviewed publications relevant to the diagnosis, pathogenesis and treatment of neuro-COVID using the search terms angiotensin-ceptors for neurotensin (NT) [146] and corticotropin-releasing hormone (CRH), secreted under stress [147], which are especially associated with COVID-19 [148]. Microglia are typically characterized as resting (M0), pro-inflammatory (M1), and anti-inflammatory and neuroprotective (M2) phenotypes with different cytokine expressions associated with neuroinflammatory response. We reported that cultured human microglia can be activated by neuropeptides such as NT to release IL-1β and CXCL8 [149] that induces proinflammatory response. Microglial-derived proinflammatory cytokines and chemokines induce astrogliosis, amyloid deposition, and subsequently, further worsening neuroinflammation [122]. Psychological stress can increase microglial reactivity to other challenges [150] and lead to cognitive decline [151] and neuroinflammatory response.

Microglia Communicate with Mast Cells
Mast cells communicate with microglia [32,164], leading to their activation [33, [164][165][166] and contributing to neuroinflammation [32,33] and neurodegenerative diseases [32,167]. This effect is not seen in mast-cell-deficient mice [168,169]. In fact, mast cell proteases can trigger astrocytes and glia/neurons and release IL-33 [170]. Stabilization of mast cells was shown to inhibit lipopolysaccharide (LPS)-induced neuroinflammation by suppressing the activation of microglia [171]. Activation of mast cells and microglia in the hypothalamus and brain [172] could lead to cognitive dysfunction [173] and neuronal apoptosis ( Figure 1) [173]. In addition, mast cells can activate the hypothalamic-pituitary-adrenal (HPA) axis [174][175][176][177] through the release of histamine [178], IL-6 [179], and CRH [180]. It is interesting that stress has been linked to the possible priming of immune cells thus contributing to neuroinflammation in AD [181,181]. Furthermore, NT [182,183] and substance P (SP) [2] induce CRH receptor-1 (CRHR1) expression in mast cells. Mast-cell-derived histamine [184] and tryptase [185] can trigger microglia and induce neuroinflammation [33]. Mast cells have been shown to be an early activator of LPS-induced neuroinflammation and BBB damage in the hippocampus [172]. In addition, food allergy that depends on mast cell activation has been shown to increase activated microglia and TNF in the hippocampus, associated with behavioral and learning impairments [186]. Another paper reported that early stress in mice and humans disrupted interactions between mast cells and glia via the involvement of histamine [187]. As such, mast cells can participate in neuroinflammation [188,189] by releasing histamine and several inflammatory cytokines and chemokines [190]. A variety of triggers including toxins and viruses such as SARS-CoV-2 can reach the hypothalamus, mostly through the nose and olfactory nerve tract. There, they can disrupt the BBB via activation of perivascular mast cells, which then further increase BBB permeability and activate microglia. Proinflammatory molecules released from microglia can damage neurons, disrupt homeostasis, and contribute to the pathogenesis of neuro-COVID.

Mast Cells in the CNS
Mast cells are ubiquitous in the body [191]. They are mostly known for mediating allergic and anaphylactic reactions [192], and several other diseases such as mastocytosis [193]. The functions of mast cells in health and several pathologic conditions were reviewed recently [194][195][196][197]. Mast cells respond to allergic but also to various other nonallergic stimuli [193]. Activated mast cells can secrete as many as 100 biologically powerful mediators, including pro-inflammatory molecules [190] such as bradykinin, chymase, histamine, tryptase, chemokine (C-C motif) ligand 2 (CCL2), CXCL8, [198] IL-6, [199] IL-1β, and TNF-α [200]. A particular potent stimulus of the mast cells is the peptide SP, especially when primed by the "alarmin" cytokine IL-33 [201][202][203][204]. In addition, we showed that SP can induce expression of the IL-33 receptor (ST2) [200], thus further increasing mast cell stimulation. Mast cells can also be stimulated to secrete mitochondrial DNA (mtDNA) [205], which serves as an additional "alarmin" and can trigger an auto-inflammatory reaction [206,207]. Mast cells are also found in the CNS perivascularly [29,208], especially in the meninges [28,209] and the median eminence of the hypothalamus [122,209,210], where they could have numerous functions (Table 1). We have called brain mast cells the "immune gate to the brain" [29]. Functional interactions have been reported between mast cells and neurons [209,211] that are often positive for CRH [183,209]. Mast cells are the

Mast Cells in the CNS
Mast cells are ubiquitous in the body [191]. They are mostly known for mediating allergic and anaphylactic reactions [192], and several other diseases such as mastocytosis [193]. The functions of mast cells in health and several pathologic conditions were reviewed recently [194][195][196][197]. Mast cells respond to allergic but also to various other nonallergic stimuli [193]. Activated mast cells can secrete as many as 100 biologically powerful mediators, including pro-inflammatory molecules [190] such as bradykinin, chymase, histamine, tryptase, chemokine (C-C motif) ligand 2 (CCL2), CXCL8 [198], IL-6 [199], IL-1β, and TNF-α [200]. A particular potent stimulus of the mast cells is the peptide SP, especially when primed by the "alarmin" cytokine IL-33 [201][202][203][204]. In addition, we showed that SP can induce expression of the IL-33 receptor (ST2) [200], thus further increasing mast cell stimulation. Mast cells can also be stimulated to secrete mitochondrial DNA (mtDNA) [205], which serves as an additional "alarmin" and can trigger an auto-inflammatory reaction [206,207]. Mast cells are also found in the CNS perivascularly [29,208], especially in the meninges [28,209] and the median eminence of the hypothalamus [122,209,210], where they could have numerous functions (Table 1). We have called brain mast cells the "immune gate to the brain" [29]. Functional interactions have been reported between mast cells and neurons [209,211] that are often positive for CRH [183,209]. Mast cells are the richest source of histamine in the CNS, particularly in the amygdala, hippocampus, hypothalamus, and thalamus [212,213]. Stimulated brain mast cells contribute to postoperative cognitive dysfunction (POCD) through the release of inflammatory and neurotoxic mediators from activated microglia [86,173]. Activation of mast cells [183] and microglia in the hypothalamus [49] could cause cognitive dysfunction [173] that is also seen in patients with mastocytosis [47, 214,215] and may be related to brain abnormalities [216]. Allergic stimulation of nasal mast cells resulted in stimulation of the HPA axis [174][175][176][177], possibly via mast cell release of histamine [178], IL-6 [178,217], and CRH [180]. The influence of stress on mast cells has also been reviewed [140,218]. Restraint stress in rodents increased BBB permeability [210,219,220] via CRH [219,221,222]. Mast-cell-released cytokines [223,224] increased BBB permeability [210,219] and permitted mammary adenocarcinoma brain metastases in mice [221]. This process could worsen with stress, acting via CRH stimulation of mast cells [219,221] and an increase in dura vascular permeability. Meningeal mast cells affected the integrity of the BBB and promoted T-cell brain infiltration [225]. Inflammation mediated by mast cells and microglia disrupted the BBB [226]. Mast cell responsiveness may be regulated not only by the neuroimmune stimuli but also by the effects of the different receptors involved. For instance, mast cells express high-affinity neurokinin-1 (NK-1) receptors for SP [2]. Moreover, SP and NT [182] induced the expression of CRHR-1 in human mast cells. Secretion of mediators can occur by utilizing different signaling [227][228][229][230] and secretory [228,230] pathways. The regulation of mast cells by neurotransmitters and neuropeptides has been reviewed [231][232][233], with emphasis on CRH [177], hemokinin-1 (HK-1) [234], nerve growth factor (NGF [235], NT [236], SP [237], and somatostatin [238,239] acting via activation of high-affinity receptors. Activated mast cells could release a number of pro-inflammatory and vasoactive mediators that could contribute to long COVID syndrome symptoms [177,240]. Some mediators are pre-stored in secretory granules (e.g., histamine, tryptase, and TNF-α) [241,242] and are released immediately following stimulation, while others are newly synthesized and then released, such as chemokines (e.g., CCL2, CCXL8) [198], and cytokines (IL-6 [199], IL-1β [243], TNF-α [200]). Apart from allergic triggers acting via IgE, mast cells are stimulated by non-allergic agents [192,203,244], especially neuropeptides [231], such as SP [237,243] and the SP-related HK-1 [234], which have pro-inflammatory properties. Under such conditions, especially when primed by IL-33 [203,204], mast cells can release various inflammatory mediators without the release of histamine or tryptase [245], thus contributing to inflammatory disorders [189,192]. Moreover, mouse mast-cell proteases 6 (MMCP 6) and MMCP 7 stimulated the release of IL-33 from mouse fetal-brain-derived cultured primary astrocytes in vitro [170]. A case in point is the selective release of IL-6 [199,246], which is elevated in systemic mastocytosis and correlated with disease severity [247][248][249] and can increase mast cell numbers [250].

Mast Cells in Long COVID
Mast cells are activated by viruses [251,252] such as SARS-CoV-2 [17,18,20,53,55,57, [253][254][255][256][257][258][259][260][261]. Recent studies have also reported mast cell activation in the lungs [254] and perivascular inflammation in the brains [75] of COVID-19 patients. We hypothesized that the spike protein can get into the brain either directly or through the activation of mast cells, which then disrupts the integrity of the BBB (Figure 1) [79]. Two studies reported elevated serum levels of chymase in patients with COVID-19 [253,260]. Moreover, a recent study demonstrated that mast cells enhance cellular entry of SARS-CoV-2 through the generation of chymasespike complexes [52]. Chymase converts angiotensin I to angiotensin II and may act in an autocrine fashion to increase the expression of ACE2, which then facilitate viral entry. Another paper reported that mast-cell-derived histamine can increase SARS-CoV-2 entry into endothelial cells [90]. Mast cells also release extracellular mtDNA [205], which was shown to be significantly elevated in COVID-19 patients [262]. Extracellular mtDNA can then stimulate the secretion of pro-inflammatory mediators from other immunocytes [206,207].

Neuroimmune Biomarkers
While a number of molecules are elevated in the blood of patients with COVID-19 [34-36,263], the results have been inconsistent and have focused primarily on pro-inflammatory mediators. A few studies have investigated blood biomarkers that may reflect brain injury in COVID-19 patients [264,265]. Anti-receptor antibodies and autoimmune gene expression [266] have also been reported. IL-15 is implicated in viral clearance with anti-viral properties, including in COVID-19 [267,268]. We showed elevated IL-18 in the serum of patients with COVID-19 [269]. IL-18 remains elevated longer than other cytokines in inflammatory and autoimmune disorders [270,271], including COVID-19 [269]. Calprotectin (S100A8/A9) was associated with microglia activation [272] and was elevated in the serum of patients with COVID-19 [269]. Calprotectin was also in the CSF of patients with Multiple Sclerosis (MS) [273] and demyelinating polyneuropathy [274]. Neuroligins (NLGs) and neurexins are implicated in synaptic function and cognitive disease [275]. NLG1 levels were reduced in the cortex and the CSF of AD patients [276] or those with mild cognitive impairment (MCI) [277]. NLG4 was associated with cognitive decline [278], while neuropilin-1 (NRP-1) was shown to facilitate SARS-CoV-2 entry by binding to the spike protein [279]. Moreover, S100β was shown to be associated with COVID-19 severity [280] and promote microglia activation [281][282][283] and has been linked to neuroinflammation and cognitive decline [284]. Neurofilament light chain (NfL), microtubule-associated protein-2 (MAP-2), and glial fibrillary acidic protein (GFAP) indicate axonal/neuronal damage and brain injury [264,[285][286][287][288]. Elevated levels of osteopontin have been associated with reduced cognition [289,290]. A recent study indicated that COVID-19 was associated with brain pathology in the UK Biobank [291] and was associated with neuroinflammation involving primarily the chemokine CCL11 in a mouse model [292]. CCL11 has been implicated in neuroinflammatory disorders [293], while osteopontin was reported to disrupt the BBB [294]. Chemokine CCL19 and its receptor C-C chemokine receptor type 7 (CCR7) axis are involved in the immune response to viral infections [268,295]. Increased levels of CCL19 were associated with disease severity in COVID-19 patients [296].

Lack of Effective Treatments
To date, there are no effective drugs to either treat long COVID or mitigate the release of inflammatory mediators from microglia. Understanding how neuro-immune and toxic triggers contribute to long COVID and how to regulate this response is of clinical importance (Figure 2). One of the major impediments has been the lack of appropriate disease surrogates either in vivo or in vitro [297], as well as the lack of effective inhibitors of neuroinflammation. Apparently, there have been therapeutic considerations of "stabilizing" the BBB [226,298].  (Figure 2). One of the major impediments has been the lack of appropriate dis ease surrogates either in vivo or in vitro [297], as well as the lack of effective inhibitors o neuroinflammation. Apparently, there have been therapeutic considerations of "stabiliz ing" the BBB [226,298]. Neuro-COVID can activate mast cells, and several inflammatory mediators released from activated mast cells ca activate microglia and other brain cells to release inflammatory and neurotoxic mediators that ca cause neuroinflammation and neurodegeneration and exacerbate neuro-COVID disease severity Eriodictyol could inhibit neuro-COVID-associated mast cell activation-mediated inflammatory me diator release as well as inflammatory mediators released from activated microglia. These inhib tions could reduce the disease severity or treat neuro-COVID.
For inflammation, non-steroidal anti-inflammatory drugs (NSAIDs) did not improv COVID-19 [297]. Biologics have also been tried in COVID-19. Even though IL-6 has bee reported to be elevated and possibly an independent risk factor, clinical trials using ILinhibitors did not show any consistent benefit in COVID-19 [299]. One study reported tha a clinically available IL-1β antagonist significantly improved COVID-19 with secondar hemophagocytic lymphohistocytosis (sHLH) that was characterized by pancytopenia, hy For inflammation, non-steroidal anti-inflammatory drugs (NSAIDs) did not improve COVID-19 [297]. Biologics have also been tried in COVID-19. Even though IL-6 has been reported to be elevated and possibly an independent risk factor, clinical trials using IL-6 inhibitors did not show any consistent benefit in COVID-19 [299]. One study reported that a clinically available IL-1β antagonist significantly improved COVID-19 with secondary hemophagocytic lymphohistocytosis (sHLH) that was characterized by pancytopenia, hyper-coagulation, and acute kidney injury [300]. Glucocorticoids have been used extensively in severe, hospitalized patients with COVID-19 [301], but the results are confusing. One paper reported a reduction in mechanical ventilation and a 20 percent reduction in the mortality rate of COVID-19 patients but also longer hospital stays and longer viral clearance time [302]. A more recent systematic review and meta-analysis showed a trend toward a higher discharge rate, but the effect was minimal and not significant [301]. Another analysis of 16 randomized control trials reported that systemic corticosteroids slightly reduced 30-day mortality in severe patients, but there was no benefit up to 120 days [303]. A multicenter observational cohort study conducted in 55 Spanish intensive care units reported that early administration of high doses of dexamethasone since symptom onset could actually prove harmful for 90-day mortality [304]. In fact, it has been argued that even though glucocorticosteroids may improve outcomes in severe, intubated patients with COVID-19, they could also reduce the production of antiviral IgG antibodies [305], thus hampering protection from other infections and worsening long-term outcomes [306].

Conclusions
Neuro-COVID is a common presentation of long COVID patients and could be at least partly caused by the activation of brain mast cells and microglia, leading to perivascular inflammation and disruption of neuronal connectivity and neuronal signal transmission. In the absence of any approved drugs, a combination of certain natural compounds could help minimize these processes and associated symptoms.

Conflicts of Interest:
The authors declare no conflict of interest.