SARS-CoV-2 と COVID-19 に関する備忘録 Vol.14――mRNA系ワクチンの濫用による免疫干渉、SARS-CoV-2ウイルスは老化を促進する…etc.

SARS-CoV-2 と COVID-19 に関するメモ・備忘録

Viral-like TLR3 induction of cytokine networks and α-synuclein are reduced by complement C3 blockade in mouse brain【nature scientific reports 2023年9月13日】


Inflammatory processes and mechanisms are of central importance in neurodegenerative diseases. In the brain, α-synucleinopathies such as Parkinson’s disease (PD) and Lewy body dementia (LBD) show immune cytokine network activation and increased toll like receptor 3 (TLR3) levels for viral double-stranded RNA (dsRNA). Brain inflammatory reactions caused by TLR3 activation are also relevant to understand pathogenic cascades by viral SARS-CoV-2 infection causing post- COVID-19 brain-related syndromes. In the current study, following regional brain TLR3 activation induced by dsRNA in mice, an acute complement C3 response was seen at 2 days. A C3 splice-switching antisense oligonucleotide (ASO) that promotes the splicing of a non-productive C3 mRNA, prevented downstream cytokines, such as IL-6, and α-synuclein changes. This report is the first demonstration that α-synuclein increases occur downstream of complement C3 activation. Relevant to brain dysfunction, post-COVID-19 syndromes and pathological changes leading to PD and LBD, viral dsRNA TLR3 activation in the presence of C3 complement blockade further revealed significant interactions between complement systems, inflammatory cytokine networks and α-synuclein changes.


Viral infections and consequent brain inflammation increase neurodegenerative disease risk

Inflammatory processes and mechanisms have emerged to be of central importance in the genesis of neurodegenerative diseases. Neuroinflammation, characterized by reactive microglia and astrocytes, and elevated levels of inflammatory mediators in the brain, was traditionally viewed as secondary to neuronal death and dysfunction in Alzheimer’s disease (AD) and Parkinson’s disease (PD). Contrary to this conventional view, there is now robust evidence from preclinical and clinical studies that immune activation can contribute to and drives disease pathogenesis. Drivers of such inflammation can be cell-autonomous or the result of network interaction between neurons, glia, vascular and blood-derived agents. We and others have previously shown that bacterial, viral, lipid, or metabolic activators are potent initiators of such neuroinflammation. Of particular interest are the dsRNA toll like receptor 3 (TLR3) receptor activators from viral sources that can initiate such inflammatory cascades.

Viral and other inflammatory conditions are linked to increased risk of neurodegenerative diseases and cognitive dysfunction. Systemic cytokine elevations following viral infection, such as those that cause cytokine storm in patients with COVID-19, are associated with cytokine and glial activations in the brain. Infection with Epstein-Barr virus (EBV) is associated with a 32-fold increased risk for developing multiple sclerosis, with onset of disease symptoms beginning approximately 10 years after infection16. Furthermore, recent epidemiological studies provide compelling evidence that repeated viral infections causing influenza drastically increase the probability (more than tenfold) of developing neurodegenerative diseases. Viral outbreaks, including the 1918 influenza pandemic and periodic mosquito-borne flavivirus epidemics such as Japanese encephalitis virus and West Nile virus, have led to subsequent diagnoses of post-encephalitic parkinsonism among survivors of the incident viral infection. Given the risk of neurological “long-COVID” symptoms following SARS-CoV-2 infection, evidence of COVID-induced changes in brain regions that are related to cognition and neurological disorders, and association of COVID-19 with onset of neurodegenerative disease, it is highly significant to establish the underlying biological cellular brain responses that are associated with TLR3 activation and viral inflammation. In the context of historical viral outbreaks and the current COVID-19 pandemic, understanding how the long-term consequences of such viral infections could precipitate degenerative changes within vulnerable brain regions is of current and critical importance.

Several inflammatory processes have been shown to be linked to the classic pathological hallmarks of neurodegenerative diseases at post-mortem. For example, α-synuclein, which is associated with neuropathology of PD and Lewy body dementia (LBD), is increased and aggregates in response to cellular stressors, such as mitochondrial dysfunction, lipid accumulation, and inflammation. Injection of the toll like receptor 4 (TLR4) agonist, lipopolysaccharide (LPS), into the midbrain of rodents induces activation of inflammatory cytokine networks, accumulation of insoluble and aggregated α-synuclein and dopamine neuron degeneration, and long-term systemic administration of low-dose LPS administration in mice induces significant reductions of dopaminergic neurons. It has also been shown that α-synuclein is upregulated by certain viral infections. Deficiency of α-synuclein in mice increases susceptibility to infection by viral or bacterial causes, suggesting that α-synuclein can participate in anti-microbial functions. Importantly, α-synuclein accumulation has been reported in the brain of rodents and non-human primates following SARS-CoV-2 systemic infection. These connections between acute α-synuclein functional responses to potent inflammatory stimuli, and pathological α-synuclein increases that lead to aggregation associated with chronic inflammatory cytokine and glial activations in brain regions vulnerable to degeneration, highlight that inflammation is a critical driver of pathology in neurodegenerative disease.

Brain responses to viral-like TLR3 activation

The cellular milieu of the brain and periphery carries many receptors for immune activation, and it is important to understand how such receptor systems and cells can trigger neuroinflammation. Typically, evolutionarily conserved immune responses are linked to receptors specialized in recognizing bacterial and viral components. TLRs recognize specific pathogen and damage-associated molecular patterns. The function of TLRs is to surveil extracellular and intracellular environments for signs of infection or damage and to signal the activation of innate immune and inflammatory responses to clear infection and remove apoptotic cells. The up-regulation of TLRs has been demonstrated in brains with α-synucleinopathies, such as PD, LBD, and multiple system atrophy (MSA). For these reasons, it is of critical importance to experimentally model conditions that mimic viral-like activation of receptors designated for immune activation. TLR3 specifically recognizes and is activated by dsRNA, which are intermediates of viral replication, and signals downstream anti-viral immune and inflammatory responses. Brain TLR3 activation has previously been modelled in vivo by direct administration of the synthetic viral-like dsRNA mimetic, Poly(I:C), into rodent brain. TLR3 activation by the administration of Poly(I:C) into substantia nigra and striatum led to a timeline of neuroinflammatory responses, characterized by activation of astrocytes and microglia within 4 days that continued to increase until peak activation at 12 days during cytokine storm, and persisted for 33 days. While TLR3 activation by itself was not sufficient to induce neurodegeneration, priming with such viral mimetics exacerbated the degeneration of nigral dopaminergic neurons to subsequent injection of 6-OHDA and to a small molecule inducer of α-synuclein fibril formation, showing that brain inflammation increases the vulnerability of neurons to additional stress. Neutralizing the Poly(I:C) induced brain inflammation by systemic administration of IL-1 receptor antagonist protected the nigral dopaminergic neurons from the damage caused by TLR3 activation combined with 6-OHDA, demonstrating that persistent inflammation of the brain increases neuronal vulnerability to additional damage, while reducing inflammation promotes neuronal cell resilience. The clear evidence for long-term brain inflammatory and degenerative sequelae to viral infections, which has led to subsequent neurological diagnoses such as post-encephalitic parkinsonism, supports the hypothesis and findings that preventing brain inflammatory responses caused by viral-like TLR3 activation could be protective.

Targeting the complement pathway to reduce inflammatory activations

In the sequence of events that typically follow viral or bacterial perturbations, there are well-known activation cascades including the innate acute response involving complement factors. The complement system, an integral part of the innate immune system, plays a vital role in clearance of pathogens by mounting an inflammatory response. Additionally, two components of this pathway, C1q and C3, have been identified to play beneficial role in microglial regulation of synaptic pruning during neural development and excessive pruning in adult brain have been implicated in neurodegeneration. Complement can be activated by three different pathways, a C1q-dependent classical pathway, and C1q-independent alternative and lectin pathways. All three activation axes converge on the enzymatic cleavage of complement factor C3 into C3a and C3b fragments. C3b in conjunction with other proteins lead to cleavage of C5 into C5a and C5b. C5b along with C6 through C9, constitute the terminal lytic pathway that leads to the formation of the membrane attack complex (MAC). The three main effector functions of the complement cascade include opsonization of C3b, C4 and C1q tagged target cell, immune cell recruitment to the site of injury by the anaphylatoxins C3a and C5a, and lysis of the target cell by the formation of MAC. TLRs and the complement cascade are both part of the innate immune system and respond to pathogenic infections, and interactions between them are plausible.

It is of great interest to explore the influence of complement system blockade in reducing brain inflammatory processes and factors, such as α-synuclein in PD and LBD. With the presence of multiple activation pathways and downstream effects, the complement system can be targeted at several levels depending on the desired outcome. While blocking action of the anaphylatoxins C3a by the C3aR antagonist SB290157, and C5a by the C5aR antagonists PMX53 and PMX205, will inhibit inflammation and opsonization of target cells, targeting C5b, and or C6 through C9 would block the terminal lytic pathway. Being at the convergence point of multiple complement activating pathways, C3 is a good target to manipulate all branches of the complement cascade, and this was the approach utilized in this study.

The current study utilized a rodent model of TLR3 activation in combination with a C3 splice-switching antisense oligonucleotide (ASO) specific to complement C3, to reduce C3 expression in the brain in vivo. The aim of this study was to experimentally model innate brain immune responses following TLR3 viral-like activation, and to examine how blocking complement C3 activation during TLR3 stimulation can reduce acute cellular and inflammatory responses downstream of TLR3. Mice received two brain injections of C3 ASO or control ASO at separate timepoints, followed by injection of dsRNA (PolyI:C) to activate brain TLR3 receptors. Changes in brain levels of complement pathway proteins, cytokines and α-synuclein were examined in the acute phase of TLR3 activation at 2 days following injection of dsRNA.


Dysregulation of intracellular redox homeostasis by the SARS-CoV-2 ORF6 protein【Virology Journal 2023年10月18日】


SARS-CoV-2 has evolved several strategies to overcome host cell defenses by inducing cell injury to favour its replication. Many viruses have been reported to modulate the intracellular redox balance, affecting the Nuclear factor erythroid 2-Related Factor 2 (NRF2) signaling pathway. Although antioxidant modulation by SARS-CoV-2 infection has already been described, the viral factors involved in modulating the NRF2 pathway are still elusive. Given the antagonistic activity of ORF6 on several cellular pathways, we investigated the role of the viral protein towards NRF2-mediated antioxidant response. The ectopic expression of the wt-ORF6 protein negatively impacts redox cell homeostasis, leading to an increase in ROS production, along with a decrease in NRF2 protein and its downstream controlled genes. Moreover, when investigating the Δ61 mutant, previously described as an inactive nucleopore proteins binding mutant, we prove that the oxidative stress induced by ORF6 is substantially related to its C-terminal domain, speculating that ORF6 mechanism of action is associated with the inhibition of nuclear mRNA export processes. In addition, activation by phosphorylation of the serine residue at position 40 of NRF2 is increased in the cytoplasm of wt-ORF6-expressing cells, supporting the presence of an altered redox state, although NRF2 nuclear translocation is hindered by the viral protein to fully antagonize the cell response. Furthermore, wt-ORF6 leads to phosphorylation of a stress-activated serine/threonine protein kinase, p38 MAPK, suggesting a role of the viral protein in regulating p38 activation. These findings strengthen the important role of oxidative stress in the pathogenesis of SARS-CoV-2 and identify ORF6 as an important viral accessory protein hypothetically involved in modulating the antioxidant response during viral infection.