For 2023, two years after 2021, we checked the final experimental results and found that they matched precisely within the seven pathways. The experimental results for 2022 and 2023 are underlined along with the cited papers. Papers published before 2020 were excluded from the analysis. Only papers published from 2020 to 2021 are described (Table 1).
1. Of the eight papers cited on the ACE2 and TLR pathways, five were used, and three were not used. Six papers were added to the last version. Nineteen paper was added while explaining ACE2, TLRs, and inflammasome activation. Six papers were successful and rediscovered between 2022 and 2023.
2. In the neuropilin‑1 pathway, all seven cited papers were used. Twenty four papers were ultimately added. Papers 55 and 59 were added while explaining the engrams. These findings provide insight into the distribution of NRP1 in brain cells. It was inherited and rediscovered by seven papers in 2022-2023.
3. In the SAMHD1 tetramerization pathway, two papers were used. Thirteen papers were ultimately added. Papers 65 and 83 were added while explaining the characteristics of the inflammatory response and IFN action caused by the spike protein. These findings were inherited and rediscovered by seven papers from 2022-2023.
4. In the inflammasome activation pathway, three of the five cited papers were still used. Paper 207 was not used, and paper 120 was used to explain why the spike protein activates the inflammasome. Thirteen papers were added to the last version, and the findings were successfully rediscovered by six papers from 2022-2023.
5. In the cGAS–STING signaling pathway, three of the six cited papers were still used. Papers 209 and 210 were not used, and paper 122 went to explain the generation of an immune response by the spike protein. Eleven papers were ultimately added, and the findings were successfully rediscovered by five papers from 2022-2023.
6. In the spike protein pathway, seven out of fourteen papers were continuously used. Papers 213, 214, 215 and 216 were not used; 17 went to explain the mechanism by which ACE2 activates the inflammasome; and 65 and 83 went to describe SAMHD1, which prevents virus invasion. Twenty three papers were ultimately added, of which paper 120 explaining from inflammasome and 122 from cGAS–STING; these findings were successfully and rediscovered by eight papers from 2022-2024.
7. In the immunological memory engram pathway, nine out of the thirteen papers were continuously used. Papers 217 and 218 were not used, and papers 55 and 59 were used to explain the clinical correlation between NRP1 in the brain and ARDS in the lung. Eighteen papers were added and succeeded, and the findings were rediscovered by five papers published between 2022 and 2024.
8. Three papers were added to the excess acetylcholine pathway in 2022, and new results were rediscovered by nine papers from 2022-2023.
1. ACE2 and TLR pathways
The SARS-CoV-2 life cycle begins after binding to ACE2 in the epithelium of the oral mucosa, lung, heart, and kidney, and the expression of ACE2 increases with age 12 13. Smoking affects ACE2 expression and induces mineral dust‑induced gene (MDIG) expression, which alters the transcription of several essential proteins implicated in exacerbating COVID-19 14 15. This type of epigenetic gene expression alters gene locus function without changing the underlying DNA sequence. Instead, it relies on posttranslational chemical changes in chromatin, RNA, and DNA. These changes include acetylation, methylation, phosphorylation, ubiquitination and SUMOylation. These changes are linked to genotype and phenotype 16. The interaction of ACE2 with the SARS-CoV-2 SP in tiny numbers of embryonic-like stem cells (VSELs) and hematopoietic stem cells (HSCs) activates the NLRP3 inflammasome. The exposure of human umbilical cord blood (UCB)-purified VSELs to recombinant SP can lead to upregulation of NLRP3 mRNA expression 17. Human VSELs in adult tissues can be damaged by SARS-CoV-2, which has downstream and subsequent effects on tissue/organ regeneration 17. SARS-CoV-2 activates mitochondrial reactive oxygen species (ROS) production and the glycolytic shift. SP alone can damage vascular endothelial cells by downregulating ACE2 and inhibiting mitochondrial function 18.
ACE2 and Toll-like receptor 4 (TLR4, CD284) on the cell surface belong to the pattern recognition receptor (PRR) family. ACE2 and TLR4 are highly expressed in hematopoietic stem and progenitor cells. These cells are highly susceptible to the spike protein (SP). ACE2 and TLR4 produce inflammatory cytokines and activate innate immune responses. Erythroid precursor cells (from CD 34+) differentiate into red blood cell (RBC) precursors and subsequently express ACE2. The SARS-CoV-2 spike (S) protein (SP) interacts with RBC precursors, leading to dysregulation of hemoglobin and degradation of Fe-heme 19 20. Blocking the interaction of SP with cell surface-expressed ACE2 and TL4 decreased the activation of the downstream mediator of the NLR family PYRIN domain containing-3 (NLRP3) inflammasome, caspase-1. This suppression was even more noticeable after blocking the interaction of the SP with both receptors. Exposure to SP upregulates the expression of proteins participating in the positive stimulation of the TLR4 signaling pathway 19. The SP has been proposed to have the most substantial protein‒protein interaction with TLR4. TLR2 and TLR4 are expressed intracellularly in dendritic, epithelial, and endothelial cells 21 22. The molecular influence of TLR4 is understood as a prime regulatory factor associated with immunity 23. TLR4 mediates anti-gram-negative bacterial immune responses by recognizing lipopolysaccharide (LPS) from bacteria 24. Staphylococcus aureus triggers an inflammatory response in innate immune cells via TLR4 and the inflammasome 25. SARS-CoV-2 infection results in viral sepsis and provokes an antibacterial-like response at the very early stage of infection via TLR4 26 (Fig. 1).
TLRs are a class of membrane pattern recognition receptors that detect microbes on the cell surface and in the cytoplasm, and a cytokine surge is induced by TLRs, mainly through the activation of TLR3, TLR4, TLR7, and TLR8 27. SARS-CoV-2 spike protein 1 (S1) subunit induces sickness behavior and a subacute neuroinflammatory response for approximately 24 hours and a chronic neuroinflammatory response for approximately 7 days. Moreover, S1 directly induces a proinflammatory response in primary microglia and activates TLR4 signaling 28. The induction of neuroinflammation by microglia is mediated through the activation of nuclear factor kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) due to TLR2 and TLR4 activation 27 28 29. TLR4 has been shown to play a critical role in mediating the neurotoxicity induced by α-synuclein oligomers. Misfolded α-synuclein induces inflammatory responses, and extracellular α-synuclein can activate the proinflammatory TLR4 pathway in astrocytes; however, α-synuclein uptake is independent of TLR4 30. The interaction between TLR4 and the SARS-CoV-2 SP can trigger an intracellular TLR4 signaling cascade. The NF-kB-mediated transcriptional activation of specific genes induces the release of proinflammatory cytokines, which can cause neuronal damage and pathological modification of α-synuclein 31. The final sequence of NF-κB activation involves a range of cytokine receptor- and TLR-mediated signaling cascades. SARS-CoV-2 induces TLR4-mediated NF-κB activation, and erythroreticulum (ER) stress induces NF-κB activation and the production of immature IL-1β (pro-IL-1β) 32.
2. The neuropilin‑1 pathway
Neuropilin-1 (NRP1) is a pleiotropic single-transmembrane coreceptor for class 3 semaphorins and vascular endothelial growth factors. It is well established that NRP1 facilitates the entry of SARS‑CoV‑2 into host cells in conjunction with ACE2. NRP1 is a highly conserved transmembrane receptor lacking a cytosolic protein kinase domain 33 34. In combination with host TMPRSS2, SARS-CoV-2 uses the ACE2 receptor for cell entry, which cleaves the viral S glycoprotein 35 36. The expression of ACE2 and NRP1 with TMPRSSs has been observed in various tissues and organs in the human body, thus facilitating viral activation and representing the essential host factors for SARS-CoV-2 pathogenicity. These viruses contribute to the tropism of SARS-CoV-2 in diverse tissues and organs and its related symptoms.
NRP1 is expressed in all vertebrates. NRP1 is the primary coreceptor for ACE2. NRP1 contributes to the primary tissue or organ tropism of SARS-CoV-2. NRP1 and NRP2 are involved in angiogenesis, axon control, cell proliferation, immune function, neuronal development, and vascular permeability because NRP1 is a coreceptor for vascular endothelial growth factors 37. SARS-CoV-2 targets ciliated cells in the respiratory mucosa. NRP1 is expressed in olfactory‑related neuronal regions, but in the olfactory mucosa, the primary target is nonneuronal sustentacular cells 38 39. In addition, NRP1 has been confirmed to be a coreceptor that facilitates SARS-CoV-2 infection in cells and may be expressed in the brainstem 33,34. SARS-CoV-2 may exhibit tropism because the brainstem has relatively high expression of the ACE2 receptor compared with other brain regions. The recognized pathways involve transsynaptic transfer via peripheral, olfactory, or cranial nerves and blood‒brain barrier (BBB) penetration from the systemic circulation to invade the brainstem. NRP1 plays a complex role in the secondary CD8+ T-cell response to control VRDs and tumors 40. A complete understanding of NRP1 or NRP2 and its associated mechanical pathways will facilitate understanding of SARS-CoV-2 infectivity and improve patient treatments; however, ACE2 is the primary receptor for entry of SARS-CoV-2 into cells. (Fig. 2) 41 42 43.
The genetic susceptibility locus in respiratory failure patients with COVID-19 is located on chromosomes 3p21.31 and 9q34.2 and is related to severity; the a 3p21.31 gene cluster can be found on chromosome 3 44. The risk locus is inherited from Neanderthals, which are segmented by a genomic size of approximately 50 kilobases, and there is no evidence that genetic haplotypes progressed from Neanderthals into African populations 32. According to the protein docking crystal structures, the SARS-CoV-2 spike RBD has a potentially high affinity for dipeptidyl peptidase-4 (DPP4). The present genetic variants from a Neanderthal heritage plant were located in six genes on chromosome 3p21.31, which is in the proximal promoter region of DPP4. The DPP4 gene encodes the enzyme dipeptidyl peptidase IV. Dipeptidyl peptidase IV serves as a receptor for MERS-CoV 45, but a genetic variant in the promoter region of the DPP4 gene has been shown to double the risk of developing critical COVID-19 pathogenesis. Moreover, DPP9, a homolog of DPP4, was significantly associated with severe COVID-19. This finding suggested a potential role for DPP4 in COVID-19 46. A haplotype on chromosome 12 from Neanderthals is associated with an approximately 22% decrease in the relative risk of developing severe illness 47. MDIGs are mainly responsible for the expression of inflammatory cytokines, the critical component of the inflammasome, and most of the genes involved in glycan metabolism for hyaluronan generation and glycosylation. MIDGsare a crucial determinant of viral infection and cytokine storms 48. MDIG is an environmentally induced lung cancer oncogene whose entry into MDA-MB-231 and A549 cells depends on NRP1 and NRP2 expression in the cell membrane 48. MDIG is also an essential regulator of NRP1 and NRP2. In MDIG knockout cells, researchers observed strong H3K9me3 and H4K20me3 upstream of the DPP4 gene from the Neanderthal haplotype region at chromosome 3p21.31 and chromosome 2q24.2 on the proximal promoter region of DPP4, which is another Neanderthal variant gene 48. Moreover, these effects are attributable to pulmonary fibrosis in some COVID-19 survivors. Among patients with a history of environmental exposure, MDIG plays a critical role in preventing SARS-CoV-2 infection and reducing the severity of COVID-19. The MDIG-dependent expression of NRP1 or NRP2 enhances SARS-CoV-2 infection in cells with lower ACE2 expression 48. Knockout of MDIG does not affect the enrichment of the repressive histone trimethylation markers H3K9me3, H3K27me3, or H4K20me3 on the protective Neanderthal haplotypes on chromosome 12, which reduces the risk of exacerbating COVID-19 47.
NRP1 is a tissue-specific marker of lung 2 ILC2s and is induced postnatally and sustained by lung-derived transforming growth factor-β1 (TGFβ1). TGFβ1–NRP1 signaling enhances ILC2 functions and type 2 immunity, suggesting that NRP1 is a tissue-specific regulator of lung-resident ILC2s and that an NRP1 regulator is a potential therapeutic agent for pulmonary fibrosis 49. NRP1 and NRP2 support competent viral entry into host cells in the lung. MDIG access to two additional cell lines (MDA-MB-231 and A549) depends on NRP1 and NRP2. Moreover, MDR-2 plays a critical role in SARS-CoV-2 infection and the severity of COVID-19 in patients who are experiencing environmental exposure to toxins. These effects explain the observed pulmonary fibrosis in some COVID-19 survivors; MDIG-dependent expression of NRP1 or NRP2 increases SARS-CoV-2 infection despite reduced ACE2 expression 48. ILC2s are involved in virus-induced exacerbation of airway inflammation and are critical in pulmonary fibrosis and autoimmune disease 50 51. Human ILC2s are flexible and adapt to the cytokine microenvironment by changing cytokine outputs to meet existing requirements 52. ILC2s and eosinophils play vital roles in pulmonary arterial hypertrophy 53.
ILC3s produce greater amounts of cytokines than ILC3s that do not express NRP1. NRP1+ ILC3s are present in fetal tissues and ectopic lymphoid aggregates and play a role in inflammation and vascularization 54. NRP1 facilitates SARS-CoV-2 infection of cells and is expressed in the brainstem 21 34. The recognized pathways invade the brainstem and involve transsynaptic transfer via peripheral, olfactory, or cranial nerves (Fig. 3) 41.
Clinically, COVID-19-related acute respiratory distress syndrome (ARDS) is characterized by relatively preserved aeration on chest computed tomography (CT) despite severe respiratory hypoxemia. However, this early, high-compliance phenotype can develop into a low-compliance phenotype with poor aeration in some patients. We described the clinical presentation as L-type, characterized by low elastance, high compliance, and preserved aeration; and H-type, characterized by high elastance, low compliance, and poor aeration 55. Patients with cryptococcus-associated immune reconstitution inflammatory syndrome can suffer from pulmonary dysfunction caused by T-cell-driven neurodegeneration in the vital medullary nucleus responsible for respiratory control 56 57. L-type ARDS and paralysis of the pre-Bötzinger complex affect COVID-19-associated fatality 58. NRP-1 might induce the tropism of SARS-CoV-2 in the brainstem 42 58 59.
3. The SAMHD1 tetramerization pathway
VRDs produce interferon (IFN)-1, which exacerbates their pathological course, but interferon treatment reduces inhibitory M2 muscarinic receptor function 60. The genesis of the acetylcholine (ACh) receptor requires interferon 61. Muscarinic and nicotinic ACh receptors regulate immune function 62. The sterile alpha motif (SAM) and histidine-aspartate domain (HD)-containing protein 1 (SAMHD1) operate at stalled replication forks to prevent the induction of IFN, a significant regulator of deoxynucleotide triphosphate (dNTP) concentrations in human cells 63. The concentrations of dNTPs, substrates for DNA-polymerizing enzymes, are limited in cells. However, the SAMHD1 is a dNTPase that cleaves dNTPs to deoxynucleosides and triphosphates. The induction of SAMHD1 in differentiated cells requires low levels of dNTPs in nonproliferating cells to mediate DNA repair and maintain mitochondria. High dNTP levels can cause problems in maintaining mitochondrial function, which might occur in Aicardi–Goutières syndrome (AGS) patients. This genetic inflammatory encephalopathy resembles congenital viral infections and certain autoimmune disorders. AGS mutations in the SAMHD1 gene reduce catalytic activity or allosteric activation by dGTP. They also increase intracellular dNTP levels. These mutations may contribute to the dysfunctional differentiation of innate immune cells. The phenotype of SAMHD1 mutations is consistent with that of AGS, which increases dNTP levels. This could lead to a more robust viral infection because of the loss of the dNTP triphophohydrolase activity of SAMHD1. Viruses can replicate their viral genome with their polymerase 64. Elevated intracellular levels of dNTPs are biochemical markers of cancer cells. Many multifunctional dNTPase and SAMHD1 mutations have been reported in various cancers. The SAMHD1 R366C/H mutant has been found in colon cancer and leukemia, as shown in Supplemental Fig. 1 of Supplement 4 65. R366C/H mutants retain dNTPase-independent functions, such as mediating dsDNA break repair, interacting with C-terminal binding protein 1 interacting protein (CtIP) and cyclin A2, and suppressing innate immune responses. The SAMHD1 R366 mutation does not alter the cellular protein levels of the enzyme but does inhibit the dNTPase activity and nucleotide density at the catalytic site on the X-ray structure. R366C/H does not restrict HIV-1 replication, which is a function of SAMHD1 that is dependent on the ability to hydrolyze dNTPs 65. Eliminating SAMHD1 activates cellular innate immunity and suppresses SARS-CoV-2 replication 66. Suppressing innate immune responses is essential for the survival of SARS-CoV-2 and HIV-1. Viral protein X (Vpx) performs several functions during infection, including downregulating SAMHD1 67 68 69. This function of Vpx is conserved among the HIV-2/simian immunodeficiency virus (SIV) accessory protein Vpx 70. The lentiviral Vpx variant suppresses SARS-CoV-2 RNA expression in primary human monocyte–derived macrophages 66. Moreover, Vpx inhibits STING signalosomes and interferes with the nuclear translocation of NF-κB and the induction of innate immune genes 69.
Mutations in SAMHD1 are implicated in the pathogenesis of chronic lymphocytic leukemia (CLL) and AGS. SAMHD1 has a target motif for cyclin-dependent kinase 1 (CDK1) (592TPQK595: the CDK-targeted motif driving threonine 592 (T592) phosphorylation) 71. CDKs are protein kinases that play key roles in cell division, transcriptional regulation, and viral infections 72. SARS-CoV-2 infection triggers the redistribution of cyclin D1 and D3 from the nucleus to the cytoplasm and subsequent proteasomal degradation. Cyclin D3 prevents the efficient incorporation of the envelope protein into virions during assembly and is exhausted during SARS-CoV-2 infection to rebuild the efficient assembly and release of newly produced virions 73.
Phosphorylation of SAMHD1 on residue T592 modulates the ability of SAMHD1 to mutation surrounding T592-generated electrostatic repulsive movement from a distinct negatively charged block retroviral infection without affecting its ability to reduce cellular dNTP levels. However, SAMHD1 can still decrease cellular dNTP levels 71. A phosphomimetic environment. This repulsive force significantly reduced the population with active SAMHD1 tetramer structures. Hence, this repulsion by mutation substantially decreases dNTPase activity and may modify antiviral functions 74. The genetic loss of SAMHD1 elevated the innate immune response and IFN activation and effectively suppressed SARS-CoV-2 replication 66.
Inactive apo-SAMHD1 interconverts between monomers and dimers 75. The binding of dGTP to four allosteric sites stimulates tetramerization and causes a conformational change in the substrate-binding pocket to yield the catalytically active tetramer 76. The binding sites are plastic, and the allosteric binding sites can adjust oligonucleotides instead of the allosteric activators GTP and dNTPs. The binding of G-nucleotide-containing oligonucleotides in the presence of GTP and dNTPs promotes the formation of a specific tetramer with mixed occupancy of the allosteric sites responsible for the antiretroviral activity of SAMHD1 77. SAMHD1 can restrict retroviruses and protect cells from viral infections by catalyzing the hydrolysis of dNTPs in the dNTP pool. SAMHD1 depletes intracellular dNTPs into 20-deoxynucleoside and triphosphate products 71 78. SAMHD1 limits virus-induced IFN production in myeloid cells. SAMHD1 reduces the induction of virus-specific cytotoxic T cells and controls viral infection through innate and adaptive immunity at the level of the infected cell 79. SAMHD1 mutations result in autoinflammatory AGS, and AGS secretes chronic IFN-I despite the absence of viral infections and is characterized by early-stage brain disease 80 81. SARS-CoV-2 encounters a response that requires the strong induction of a subclass of cytokines, including IFN I, IFN III, and a few chemokines 82. The SAMHD1 tetramer structure could provide the basis for a mechanistic understanding of its rapid function in SARS-CoV-2 restriction and the pathogenesis of COVID-19. IFN-I-mediated antiviral gene expression mediated by cyclic-GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) (cGAS–STING) is decreased during radiotherapy in cancer patients but promptly recovers after radiotherapy. SAMHD1, which suppresses viral replication and viral response genes, occurs more frequently in severe ventilation-associated COVID-19 patients than in nonventilated patients 83. The degradation of SAMHD1 in human primary-activated/dividing CD4+ T cells, the increase in cellular dNTP levels, and the loss of dNTPase activity contribute to the increase in commonly observed dNTP levels 65. SARS-CoV-2 aggravates a reaction in which SAMHD1 controls the innate immune response 84. SAMHD1 in cells inhibits NF-κB activation and IFN-I induction 85. Therefore, low levels of IFN-I could drive more severe SARS‐CoV‐2 infection 86.
4. Inflammasome activation pathway
Active NLRP3 inflammasomes were found in peripheral blood mononuclear cells (PBMCs) and tissues from postmortem patients in moderate and severe COVID-19 studies. The serum levels of caspase-1, caspase-4/11, and IL-18, including inflammasome-derived products such as IL-6 and LDH, are correlated with the severity of COVID-19. Moreover, higher Caspase-1, Caspase-4/11, and IL-18 levels are associated with poor clinical COVID-19 outcomes 87. SARS-CoV-2 causes pyroptosis in human monocytes. Pyroptosis is associated with caspase-1, caspase-4/11, interleukin 1β (IL-1β), and gasdermin D expression and cytokine levels in primary monocytes 88 89. Microglial NLRP3 inflammasome activation is a major driver of neurodegeneration 90 91 92, and purified SP activated the NLRP3 inflammasome in LPS-primed microglia in an ACE2-dependent manner 93.
There are four main types of inflammasomes: NLRP1, NLRP3, NLRC4, and AIM2, which are classified after being regarded as distinct sensing proteins. Inflammasomes consist of at least three components: the inflammasome caspase (caspase-1, Caspase-4/11), an adapter molecule (apoptosis-associated speck-like protein with caspase recruitment domain (ASC)), and a sensor/receptor protein (NLRP1, NLRP3, NAIP1/2/5, NLRP12, AIM2, etc.). The NLRP3 inflammasome contains NLRP3 as a sensor protein, ASC as an adaptor protein, and caspase-1 as an effector protein. The NLRP3 protein has three domains: the pyrin domain (PYD), nucleotide-binding domain, and leucine-rich repeat domain. PYD interacts with ASC PYD and subsequently promotes ASC oligomer formation. The ASC platform induces caspase-1 activation, which catalyzes the conversion of pro-IL-1β to mature IL-1β. NLRP3 deubiquitination and self-aggregation occur after ASC recruitment and oligomerization. Active caspase-1 cleaves pro-IL-1β and pro-IL-18 into mature IL-1β and IL-18, respectively. Excessive IL-1β activates various signaling pathways, such as the NF-κB and JNK signaling pathways, and as a result, it stimulates systemic inflammatory responses. IFN-α, IFN-β, IL-6, tumor necrosis factor (TNF), and TGFβ1 can lead to cytokine storms. The SARS-CoV-2 genome is enclosed by a nucleocapsid (N) protein in phospholipid bilayers. The membrane and envelope proteins are located among the SPs in the virus envelope 94. SARS-CoV-2 engages in Caspase 4/11-mediated noncanonical activation of NLRP3 and contributes to COVID-19 exacerbation 89. When recombinant baculoviruses displaying SP or N protein were constructed and transfected into lung epithelial A549 cells and a spontaneously immortalized monocyte-like cell line (THP-1)-derived macrophages, N protein triggers A549 cells to release more serum cytokines levels than the SP 95.
Blocking the NLRP3 inflammasome reduces the cytokine storms and lung injury caused by SARS-CoV-2 infection. The N protein facilitated ASC oligomerization by increasing the interaction between NLRP3 and ASC. The N protein, NLRP3, and ASC form a complex and activate the NLRP3 inflammasome 96. In addition, mitochondrial antiviral signaling protein (MAVS) associates with NLRP3 and regulates its inflammasome activity 97. The SARS-CoV-2 ORF10 antagonizes STING-dependent IFN activation and autophagy 98. ORF9b and nonstructural protein 7 (NSP7) antagonize the production of type I and III IFNs by targeting the retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5), TLR3-TIR-domain-containing adapter-inducing interferon-β (TRIF), and cGAS-STING signaling pathways. The ectopic expression of NSP7 blocks innate immune activation and facilitates virus replication 99 100 101. The detection of cytosolic DNA via the cGAS–STING axis induces a cell death program that initiates potassium efflux upstream of NLRP3. Activated STING triggers membrane permeabilization and thus lysosomal cell death. The NLRP3-cGAS-STING pathway constitutes the primary inflammasome response during viral and bacterial infections in human myeloid cells. The NLRP3-cGAS-STING-lysosomal cell death pathway ameliorates the pathology of inflammatory conditions linked with cytosolic DNA sensing 102.
5. cGAS–STING signaling pathway
SARS-CoV-2, an RNA virus, activates the cytosolic DNA sensor cGAS–STING signaling in endothelial cells. Mitochondrial DNA is released and leads to IFN-I production. Blocking STING reduces severe lung inflammation and disease severity 103. Conversely, a STING agonist protects against SARS-CoV-2 infection 104.
cGAS catalyzes the conversion of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) to cytosolic DNA. It triggers STING–tank-binding kinase 1 (TBK1)–interferon regulatory factor 3 (IRF3) signaling 105. cGAS also appears in the nucleus, where cGAS in an inactive state is isolated from chromatin. Nuclear cGAS recruits protein arginine methyltransferase 5 (PRMT5) upon viral infection. In innate immunity, nucleus-localized cGAS interacts with PRMT5 to catalyze the symmetric dimethylation of histone H3 arginine 2 at IRF3-responsive genes such as interferon beta 1 (IFNβ1) and interferon alpha 4 (IFNα4). As a result, PRMT5 facilitates IRF3 access 106. Activated cGAS releases cyclic GMP-AMP (cGAMP), and cGAMP binds to STING; as a result, STING relocalizes and forms a clustered platform at the perinuclear Golgi. TBK1 kinase phosphorylates IRF3, and phosphorylated IRF3 enters the nucleus. At that time, NF-κB triggers the expression of IFN-1 and proinflammatory cytokine genes (Fig. 4) 107 108. cGAS–STING responds to foreign DNA from viruses and bacteria and to mitochondrial and genomic self-DNA in the cytosol from senescent or dying cells. The pathway evokes microglial activation to resolve inflammation in the brain. However, excessive engagement can lead to neuroinflammation and neurodegeneration 109. The SARS-CoV-2 ORF3a can interact with STING. It selectively blocks cGAS–STING-induced autophagy by disrupting the STING-light chain 3 (LC3) interaction 110.
Severe COVID-19-related inflammation is associated with excessive lung tissue damage and syncytial pneumocyte formation. Cultured epithelial cells expressing ACE2 and SP formed multinucleated syncytial cells. The fused cells exhibited DNA damage and micronuclei expressing cGAS-STING, which colocalized with and stimulated IFNs and IFN-stimulated genes 111. The useful cellular functions of cGAS-STING are mediated by canonical and a few noncanonical pathways, but dysfunction of cGAS-STING-mediated cellular functions and noncanonical signaling underlie disease pathogenesis 112. Activated STING triggers membrane permeabilization and thus lysosomal cell death. cGAS–STING–lysosomal cell death combined with NLRP3 ameliorates the pathology of inflammatory conditions through cytosolic DNA sensing 102. cGAS–STING signaling is strongly linked to the pathogenesis of CNS diseases, which are characterized by neuroinflammation-driven disease progression. Innate immune recognition is facilitated by a vast array of germline-encoded innate immune receptors, regularly called PRRs. Due to cellular senescence, autoimmune disorders, and mitotic stress in cancers, cytosolic DNA levels are increased (Fig. 5). These events lead to the activation of cGAS–STING and the exacerbation of pathological courses 113.
6. Spike protein pathway
Glycoproteins are required for viral entry and fusion. The SP is a trimeric glycoprotein that is encoded by ORF2 in the viral genome. The SP is shaped by a membrane-distal S1 subunit and a membrane-proximal S2 subunit, which form homotrimers in the virus envelope 114. Glycoproteins derived from SARS-CoV-1, SACR-Co-V-2, human cytomegalovirus, and hepatitis C virus potentially trigger NLRP3 inflammasome activation and pyroptosis in THP-1 macrophages 115 116. SP binding to ACE2 induces NF-κB activation and inflammation via ACE2 in endothelial cells 117. The furin cleavage product of SP uses the vascular endothelial growth factor A (VEGF-A) binding site on NRP-1 as an entry point 118.
The SARS-CoV-2 SP S1 subunit activated the NF-κB and c-Jun N-terminal kinase (JNK) signaling pathways. Furthermore, SP interacts with and activates TLR4 26 119. SP induces neuroinflammation in BV-2 microglia, a microglial cell line derived from C57BL/6 mice. Immunofluorescence microscopy revealed increased TLR4 expression in BV-2 microglia when stimulated with S1 29. After SARS-CoV-2 infection, the augmented immunogenicity of the SP results from macrophage reprogramming. SP-driven IL-1β secretion in macrophages requires nonspecific monocyte preactivation in vivo. Then, macrophages trigger NLRP3 inflammasome signaling 120. SP drives inflammasome activation in macrophages isolated from convalescent COVID-19 patients, correlating with distinct epigenetic and gene expression signatures 120. The SP is a PAMP that requires macrophage preactivation for NLRP3 inflammasome formation, and vigorous SP-driven inflammasome activity releases IL-1β in the convalescent macrophages of COVID-19 patients 120. However, it is not released in macrophages from healthy SARS-CoV-2-naive patients. SARS-CoV-2 infection causes profound and long-lived reprogramming of macrophages, resulting in augmented immunogenicity of the SARS-CoV-2 SP, an effective vaccine antigen and potent driver of adaptive and innate immune signaling 120.
SARS-CoV-2 infection can cause syncytium formation within cells. The syncytia express ACE2 and SP, producing approximately four micronuclei per syncytium. Remarkably, these micronuclei are highly expressed during the DNA damage response or cGAS–STING signaling 121 122. These signaling pathways are associated with cellular devastation and poor immune reactions 122. Pathogenic platelet factor 4 (PF4)-dependent syndrome can occur after ChAdOx1 nCoV-19 vaccination 123. Various side effects caused by the vaccine’s SP appear in a real-time setting. The SP has a unique pathological mechanism: there are strange similarities with amyloid-disease-associated blood coagulation and fibrinolytic disturbances together with neurologic and cardiac problems. The protease neutrophil elastase (NE) efficiently cleaves SP, exposes amyloidogenic segments, and accumulates the most amyloidogenic synthetic spike peptide, but full-length folded SP does not form amyloid fibrils 124 125. SARS-CoV-2 SP vaccination establishes SP–specific long-lived plasma cells reservoirsin the bone marrow of nonhuman primates 126. The SP might have induced immune reactions to human.
Lipid nanoparticles of the formulated nucleoside-modified mRNAs of SPs are stabilized in their prefusion conformation and induce an immune reaction involving IL-2+ CD8+ and CD4+ T helper type 1 cells or IFNγ+ cells 127. Lipid nanoparticles encode the prefusion conformation of SP. General adverse reactions include redness, pain, swelling, muscle pain, fever, headache, and chills after vaccination. Serious adverse events included death and anaphylaxis: AESI, ICU admission, life-threatening, and permanent disability/sequelae. The overall seroprevalence of Korean anti-SARS-CoV-2 was very low on September 6, 2021, but the incidence of AESI was very high, as was the case in England during the pandemic. This finding suggested that AESI might originate from inflammasomes induced by lipid nanoparticles, including the SARS-CoV-2 SP, in mRNA vaccines 13.
Like other RNA viruses, SARS-CoV-2 undergoes genetic evolution and develops mutations over time, resulting in the emergence of multiple variants that may have different characteristics than their ancestral strains 128 129. The wild-type/Wuhan variant S1 is highly proinflammatory in zebrafish, but the SP of the SARS-CoV-2 variants of interest shows differential proinflammatory effects 130. SP signals through TLR2 and activates NLRP3 in human macrophages from convalescent patients with COVID-19 but not from healthy SARS-CoV-2–naïve individuals 120. We compared adverse events of special interest (AESI) after vaccination in England and South Korea (Fig. 6). SP could prime the NLRP3 inflammasome and enhance caspase-1 activity through NF-κB signaling. S1 interacts with amyloid-beta, prion protein, α-synuclein, and tau, presumably through heparin-binding domains, to form homopolymers or heteropolymers resembling amyloid fibrils in the neurodegenerative process of misfolded protein disorders in the brain and increases the protein level of p38 MAPK in BV-2 microglia. S1 also binds to transactive response DNA-binding protein 43 (TDP-43) and RNA-binding motifs (RRMs); TDP-43 RRM is involved in amyotrophic lateral sclerosis (ALS) and AD. The interaction of the SP with the prion protein is more robust than that with amyloid-beta, tau, or α-synuclein 29 93 131. The hyperinflammatory state of COVID-19 triggers CNS neuroinflammation by activating astrocytes and microglia. This condition could facilitate prion-like pathology 132. Like other prion proteins, the SP contains several prionogenic domains. SP triggers a neurodegenerative condition known as prion-disease-like pathology 133. SP can catalyze the aggregation of aggregation-prone proteins in the brain, and spike-derived peptides can act as functional amyloids. Cross-reactive antibodies can originate from many reported complications, such as the worsening of demyelinating diseases, Guillain–Barré syndrome, immune thrombotic thrombocytopenia, and stroke 134. As a result, rare hypersensitivity reactions to mRNA-based SARS-CoV-2 vaccines develop, such as anaphylaxis, chest pain, chills, flushing, hypertension, and tachycardia 13 135. In addition, two distinct self-limiting syndromes, myocarditis and pericarditis, occur only on one patient after COVID-19 vaccination. Specifically, myocarditis develops rapidly in younger patients. It occurred mainly after the second vaccination. However, pericarditis, including after receiving mRNA vaccines, affects older people after the first or second vaccination 136.
7. Immunological memory engram pathway
A single or double layer of invaginated pia in the perivascular space in the brain forms an interstitial fluid-filled space representing an extension of the extracellular fluid space around the intracranial vessels as they descend into the brain parenchyma. Human sensory stimuli and abnormal muscular sensations affect breathing via the cerebral cortex and hypothalamus 137 138 139. The substrate of information is stored in cells termed engram cells 140. The brain can trigger immune reactions in patients with COVID-19. The subsequent reactivation of the engram stimulates memory retrieval of immune-related information in the insular cortex 141. Chemogenetic reactivation reflects the inflammatory conditions described in the insular cortex 142. The immunological memory engram pathway can restore the initial disease state during COVID-19 pathogenesis 143. SARS-CoV-2 infection during the fetal period may alter the normal development of the brain region where memory engrams are generated and affect neuronal progenitor cells. The massive infection rate in young people leads to the possibility of an increase in the incidence of congenital infections and originating cognitive alterations in terms of new variants; consequently, neuronal circuit anomalies may indicate vulnerability to mental problems throughout life 144 145 146 (Fig. 7).
SARS-CoV-2 does not appear to be a neurotropic virus, and transient insufficient support in sustentacular cells of the olfactory bulb causes temporary dysfunction 39. SARS-CoV-2 may disrupt BBB dysfunction by damaging the choroid plexus epithelium through cytokine, chemokine, and adhesion molecule storms 147 148. Neuroinflammation in COVID-19 brains was significantly greater than microgliosis and T-cell infiltration in COVID-19-free patients 149. This might trigger inflammasomes and pyroptosis in the CNS. Brainstem involvement could explain sudden deaths by respiratory failure 150 151 152. COVID-19 is characterized by the rapid development of acute lung injury, ARDS, death due to dysregulated cytokine release, disseminated intravascular coagulation (DIC), multisystem failure, and pneumonia. However, COVID-19 causes unique type L or H phenotype lung injury and requires different ventilatory approaches, depending on the underlying physiology 55. Often, preexisting neurological disease may become clinically evident or worsen with COVID-19. This explains why various neuronal manifestations react favorably to immune suppression or modulation 153. ACE2 expression in the lungs is modest compared to that in other organs, such as the heart, kidneys, and small intestines. However, TLR4 is expressed intracellularly on the whole body's dendritic, epithelial, and endothelial cells 154 155. Conventional dentritic cells are highly specialized antigen-presenting cells, key initiators, and regulators of T-cell-mediated immunity and their absence in the murine line which lack the conventional dendritic dendritic cells resulted in the consequent impaired CD8 T-cell responses, and subsequently in significant increase in SARS-CoV-2 viral load in the lungs 156. Microglia and astrocytes participate in immune-to-brain communication during immune activation. Glia, microglia, and astrocytes propagate inflammatory signals and influence physiological responses in the body 157. Janus kinase (JAK)1-dependent type 2 cytokines develop atopic dermatitis and asthma, and human JAK1 gain-of function variant (JAK1GoF) into mice developed spotanous atopic dermatitis and stigger JAK1 in vagus nerve to induce lung inflammation, subsequent genetic expressions suppress group 2 ILC function and allergic airway inflammation. 158 ACE2 is localized to the cytoplasm, and its expression appears to be highly regulated by other renin-angiotensin system components. In transgenic mouse brains, ACE2 is present in the cytoplasm of neuronal cell bodies but not in glial cells. ACE2 in transgenic mice was significantly increased in an area lacking the blood‒brain barrier and sensitive to blood-borne angiotensin II 159. Activating the peripheral immune system via the immunologically coordinated engram pathway elicits exaggerated COVID-19 symptoms. This immunological memory engram pathway is activated during COVID-19 pathogenesis.
8. Excess acetylcholine pathway in dementia patients with anti-Alzheimer’s disease
T and B cells in the mucosa play pivotal roles in maintaining immune homeostasis by suppressing responses to harmless antigens and ensuring the integrity of the barrier functions of the gut mucosa. Position-specific phenotypes and functions are influenced by the microbiota. Imbalances in the gut microbiota can trigger several immune disorders through the activity of T cells that are both near and distant from the site of induction 160. The gut microbiota drives systemic antiviral immunity via IFN-I priming, and microbiota-driven IFN-I priming involves the cGAS–STING axis. The microbiota mediates systemic IFN-I priming via DNA-containing membrane vesicles 161. Nevertheless, the nucleotide-binding oligomerization domain containing 2 (NOD2) in the hypothalamus recognizes neuropeptides and fragments of bacterial cell walls, which change temperature regulation and feeding behavior in mice, particularly older female mice 162. These findings explain the multidimensional roles of human IFN in regulating senescence, autophagy, apoptosis, antitumor effects, and cell metabolism 163. Group 3 ILCs (ILC3s) are essential for host defense against infection and tissue homeostasis. ILC3s depend on the transcription factor retinoic acid-related orphan receptor-gamma γt (RORγt). It plays a role in angiogenesis, initiating ectopic pulmonary lymphoid aggregates 54. NRP1+ ILC3s are found in ectopic lymphoid aggregates in patients with chronic lung disease. NRP1+ ILC3s also potentially contribute to inflammation and vascularization 52. NRP1+ ILC3s are present in the lymphoid tissues and lung tissues of smokers and chronic obstructive pulmonary disease (COPD) patients. ILC3s with NRP1 produce more cytokines than ILC3s without NRP1 54. Moreover, transcriptomic signatures of olfactory mucosa cells at the air-liquid interface from individuals with AD were highly were infected with the SARS-CoV-2 and showed increased transcriptomic signatures of neuroinflammation (increased levels of oxidative stress, desensitized inflammation and immune responses), and alterations to genes associated with olfaction 164. SARS-CoV-2 N protein triggers a cytokine storm originating from lung epithelial cells 95, and dendritic cells regulates CD8 T-cell response in the lung viral load 156, and SARS-CoV-2 triggers neuroinflammation in the olfactory mucosa of AD 164, and an Anticatalysis agent against neuroinflammation prevents and treats AD exacerbation in the fifteen year-cohort study 165. The cohort study indicated another exacerbation factor from the endemic on Sorok Island.
We observed the effects of excess acetylcholine in anti-Alzheimer’s disease (AD) drug (AAD) on the sustained viral RNA interferon response. VRDs cause lung inflammation and inflammatory cytokine production. Participants were randomized to VRDs after prescribing dapsone as a standard treatment or AADs as AD symptom treatments from 2005 to 2019 in an RCT. The incidence of endemic diseases on Sorok Island, South Korea, sharply increased from 2008 to 2009; that of COPD increased rapidly in 2012 and 2013; that of acute bronchitis increased from 2012 to 2014; and that of pneumonia increased in 2013 compared to earlier years. The equation for the use of dapsone with acetylation effects by as a preventive treatment for VRDs and excess ACh in AADs (AA equation) was strongly negatively correlated with the incidence of bronchitis and COPD. ACh excess by AADs exacerbates bronchitis and COPD 166. The muscarinic (M) and nicotinic ACh receptors play life-threatening roles in regulating immune function. However, viral infection and interferon treatment cause the release of IFN-gamma, decrease M2 receptor gene expression at parasympathetic nerve endings, and ultimately inhibit M2 receptor gene expression 60 62. COVID-19 is regarded as a hyperinflammatory disease characterized by cytokine release by harmful immune cells. However, the plasma concentrations of these viruses are close to those provoked by classical viral respiratory infections (VRDs), such as influenza 167 168. VRDs and their IFN-1-induced loss of inhibitory M2 receptor function and gene expression in cultured airway parasympathetic neurons. Moreover, the excess of ACh produced by AADs appears to inhibit the production of the ACh receptor, which prevents virus invasion (Fig. 8) 166. As of April 17, 2022, there were 55,841 cases of COVID-19 reinfection in South Korea, for an incidence rate of 0.35%. Ninety days after the initial diagnosis, there were 53,301 cases of reinfection, which accounted for 95.5% of the total cases. A total of 99.0% of reinfection cases occurred during the period when the omicorn virus was dominant. There were 72 cases of exacerbated COVID-19 , 70 (97.2%) patients were exacerbated after 50 years of age, and 52 (100%) patients died after 50 years of age 169. Sixty-seven of the 72 cases (93.1%) occurred in nursing hospitals and homes. Patients in nursing hospitals and homes take the following AD drugs: donepezil, choline alfoscerate, rivastigmine, galantamine, and memantine 170. We compared higher risk subjects who had elapsed since receiving the third vaccination because the fourth COVID-19 vaccine dose was first released from February 16 to April 30, 2022. We analyzed the data of 1,509,970 participants in the high-risk groups, namely, residents of patients in elderly care hospitals and facilities (E1) and immunocompromised individuals (E2). Standardizing per 100,000, infection after the third vaccine was 71.7% (E1) and 28.3%, and severity was 89.4% (E1) and 10.6% (E2), death was 92.0% (E1) and 8.0% (E2), and infection after the fourth vaccine was 74.2% (E1) and 25.8% (E2). The severity of infection was 91.5% (E1) and 8.5% (E2), and the mortality rate was 93.8% (E1) and 6.2% (E2) 171. One major factor is that E1 has excess ACh, which increases susceptibility to infection and exacerbates severity and mortality compared with E2. Excess ACh appears to inhibit ACh receptors for IFN production against viral invasion 166 172. Excess ACh related to AD and related dementias is an underlying or contributing cause of excess mortality in nursing homes, long-term care settings, homes, and medical facilities 173 174.