Pathways toward deterioration in SARS-CoV-2 VI: how COVID deploys IFN-suppression, mitochondrial invasion and metabolic reprogramming to evade immunity

In this contribution, I will discuss the role of SARS-CoV-2 proteins in evasion strategies to prevent immunity against COVID. Two major mechanisms are infiltration of mitochondria ("hijacking mitochondria") and metabolic reprogramming.

In this message

1.       Mitochondria
1.1.1. Connecting mitochondria with adaptive immunity;
1.1.2 The innate immune system detecting viruses;
1.1.3 Transcribing the Interferon response: from cGAMP synthase cGAS to STING translocation in the Golgi apparatus

2     SARS-1 and SARS-2 (COVID): built to evade the first-line immune defense
2.2.1 Learning from the first epidemic: SARS-Coronavirus-1 evades immunity by destruction of mitochondria and targeting of MAVS;
2.2.2  ORF9b: killing NEMO;
2.2.3  SARS-1 and SARS-2 effectively delay the IFN-I-response;
2.2.4  ORF10 attenuates STING-autophagy to disrupt viral clearance

3.        Mitochondrial metabolism in (Long) COVID
3.1.1   Mitochondrial ATP generation;
3.1.2   Substrate feeding through the TCA and Oxydative Phosphorylation (OXPHOS);
3.1.3   The Warburg Effect in disease: shifting from TCA & OXPHOS to aerobic glycolysis or FAS;
3.2       Depletion and alteration of substrates
3.2.1   Tryptophan, glutamine and arginine depletion are hallmarks of COVID-progression, immune suppression and inflammaging;
3.2.2   Cholesterol: different (antiviral) functions for different metabolites;
3.2.3   Lipid alterations;
3.2.4   Arginine;
3.2.5   Glutathione: a role for GlyNAC (N-acetylcysteine) to alleviate GSH deficiency?
3.2.6   The PEA potential

4.  SARS and Post-Acute SARS (= Long COVID) metabolic alterations
4.1 Acetylcholine
4.2 Fatty acid dysregulation
4.3 Iron metabolism: ferroptosis

 
1. Mitochondria

1.1.1 Connecting mitochondria with adaptive immunity
Mistakenly, mitochondria are mainly memorized as powerhouses of the cell. The dynamics of mitochondria are more than just a way of supplying energy to cells through ATP generation and OXPHOS. In the lungs, mitochondria are involved in airway and smooth muscle regulation in order to regulate gas exchange, ventilation and blood flow, but mitochondria also protect pulmonary cells and lung tissue (Mitochondrial Dysfunction in Lung Pathogenesis, Annual Review of Physiology Vol. 79, February 2017).

Mitochondrial metabolism is necessary for each type of T-cell in the immune system. Naïve T cells, lymphocytes, generate ATP by oxidative phosphorylation (OXPHOS). During the process of T cell receptor activation (TCR), these cells go into an anabolic state to consume glucose for cell proliferation (glutamine flux). CD4 T cells differentiate into pro-inflammatory T helper cells (Th17) or regulatory cells (Treg). CD8 T cells differentiate into memory cells and effector cells. Tregs increase OXPHOS and decrease glycolytic flux, while fatty acid oxidation is activated (Mitochondria Drive Immune Responses in Critical Diseases, Cells 2022, 11).

CD8 T memory cells increase OXPHOS and decrease glycolytic flux, while fatty acid oxidation is also activated. TRAF6 regulates fatty acid oxidation to promote CD8 T memory cells during infection.
A proposed pharmacological treatment to improve mitochondrial function is nicotinamide dinucleotide (NAD+) administration (Mitochondria: in the Cross Fire of SARS-CoV-2 and Immunity, Cell Reports iScience Vol. 23, Issue 10, October 23, 2020).

1.1.2 The innate immune system detecting viruses
PAMPs > TLRs > RIG-I > TOM70 > MAVS > TANK > IRF3 > HSP90 > JAK1 > TYK > STAT1 & 2 > TRAF3 & 6 > NF-kB (NEMO) & IFN I
The Interferon response is a first-line defense against pathogens. When a pathogen such as a virus enters the body, Pathogen-associated patterns (PAMPs) are recognized by PRRs such as Toll-like receptors (TLRs) and RIG-I-receptors such as LGP2, RIG-I and MDA5. Through Caspase, RIG-I activates the mitochondrial MAVS-signal.
The outer mitochondrial membrane (MAVS) is the cellular antiviral system. In response, the TANK-kinase phosphorylates the Interferon regulatory IRF3. In coordination with HSP90, IRF3 enhances a strong antiviral response.

Further downstream, the Janus Kinase (JAK1), Tyrosine Kinase (TYK1 and TYK2) STAT1 and STAT2 are involved. During this process, Interferon-stimulated genes are expressed to eliminate a pathogen. MAVS also recruits TRAF3 and TRAF6, thereby inducing the antiviral signal NF-kB through essential modulator NEMO. The Mitochondrial Outer Membranes TOM70 and TOM20 mediate MAVS formation (Mitochondria: in the Cross Fire of SARS-CoV-2 and Immunity, CellPress iScience, 23, October 2020).

Binding of mitochondrial DNA (mtDNA) to TLR9 on neutrophils, induces the release of inflammatory IL-6 and TNF-alpha. mtDNA also activates the inflammasome NLRP3, thereby activating Caspase1, IL-1beta, IL-18 and pyroptosis (cell death). In addition, activation of purinoreceptor 7 (P2X7), ATP generated by mitochondria activates NLRP3 (Mitochondria Drive Immune Responses in Critical Diseases, Cells 2022, 11).

1.1.3 Transcribing the Interferon response: from cGAMP synthase cGAS to STING translocation in the Golgi apparatus
The cGAMP synthase cGAS senses virus DNA. Following the binding of virus DNA, cGAS effects synthesis of cGAMP from ATP and GTP. cGAMP binds to STING. STING translocates from the endoplasmic reticulum (ER) to the Golgi, through which STING activates TBK1. TBK1 phosphorylates STING, TBK1 and Interferon Regulatory Factor 3 (IRF 3), transcribing IFN III and IFN I (SARS-CoV-2 ORF10 antagonizes STING-dependent interferon activation and autophagy, Journal of Medical Virology 2022;94).
 
2 SARS-CoV-1 and SARS-CoV-2 (COVID) are built to evade the first-line immune defense

2.2.1 Learning from the first epidemic: SARS-Coronavirus-1 evades immunity by destruction of mitochondria and targeting of MAVS
From SARS-CoV-1, it was already known that virus proteins effectively target mitochondria and the first-line immune defense. ORF9 localizes to the mitochondria and depletes DRP1, accompanied by mitochondrial elongation. Mitochondrial damage as such, prevents the healthy maintenance of mitochondrial morphology and number through fusion and fission. Degradation of mitochondrial MAVS signaling by SARS-ORF9b is accompanied by a loss of TRAF3 and TRAF6. Functional loss of TRAF3 and TRAF6 impairs both the IFN I response and induction of NF-kB activated B cells. ORF9b targets mitochondrial fusion to induce structural changes to mitochondrial biogenesis and antiviral activities functions of fusion and fission. In addition, SARS-CoV-1 promotes autophagosome formation in order to destroy mitochondria (SARS-Coronavirus ORF9b Suppresses Innate Immunity by Targeting Mitochondria and the MAVS/TRAF3/TRAF6 Signalosome, Journal of Immunology 2014; 193).

SARS-CoV Nsp2 impairs mitochondrial biogenesis via PHB1 and 2. ORF9b promotes mitochondrial fusion to modulate the antiviral defense. Degradation of the fission factor DRP1 promotes fusion to enhance viral replication in mitochondria. Nsp10 alters NADH-cytochrome activity in lung cells and depolarizes the mitochondria to cause damage to lung cells.

With ORF7a of SARS-CoV-2 showing similarity to SARS-CoV-1 protein ORF7a, this protein manipulates Bcl-Xl to induce apoptosis, invade the Golgi body and endoplasmic reticulum of mitochondria. CD4+ and CD8+ depletion is indicative of apoptosis. ORF7b localizes to the Golgi, inducing apoptosis.

ORF3a targets Bax, p53 and p38MAPK and enhances apoptosis through Caspase 8 and Caspase 9. The result is leakage of Cytochrome C, indicating mitochondrial destruction. ORF3a stimulates pro-inflammatory IL-1beta through K+ and ROS production. Disruption of the ionic concentration leads to activation of inflammasome NLRP3.

ORF6 localizes to the Golgi body and endoplasmic reticulum. In coordination with ORF3b and the N-protein, ORF6 disrupts the IFN response. Transporting to the Golgi and ER, ORF6 diminishes the antiviral STAT1 signal. ORF6 engages in activation of Caspase 3 to induce mitochondrial apoptosis.

ORF8a localizes inside mitochondria, alters the mitochondrial membrane potential (MMP) and induces ROS production. ORF8b damages lysosomes and acticates autophagy. ORFb was found to cause cell death and inflammasome NLRP3 in epithelial cells and macrophages (Severe acute respiratory syndrome coronaviruses contributing to mitochondrial dysfunction: implications for post-COVID complications, Mitochondrion 69(2023)).

Mitochondrial damage with subsequent mitokine Fibroblast Growth Factor-21 (FGF-21) secretion is reported to be high in severe COVID patients. ATP respiration is induced in COVID patients, while glycolysis is induced and mitochondria were found to use glucose as the main feeding substrate during COVID (Mitochondrial metabolic manipulation by SARS-CoV-2 in peripheral mononuclear blood cells of patients with COVID, Am. Journal of Cell Physiology 320:2021).

2.2.2  SARS-CoV-2 protein ORF9b: killing NEMO to suppress the NF-kB-B cell response
ORF9b suppresses IFN-I by targeting TOM70. While ORF9b interacts with TOM70 to disrupt MAVS-signaling, this SARS-CoV-2 protein was also found to impair cGAS-STING and TLR3-pathways. ORF9b interrupts K63 to target NEMO, an essential pathway for the NF-kB response. NEMO is not a mitochondrial protein and the subunit is not necessary for inducing the IFN I-response. The K63-NEMO target disrupted by SARS-CoV-2 indicates that SARS suppresses the NF-kB-B cell response in addition to the suppression of IFN I throught mitochondrial dysruption (SARS-CoV-2 inhibits RIG-I-MAVS antiviral signaling by interrupting K63-linked ubiquitination of NEMO, Cell Reports 34, Februari 16 2021).

2.2.3 SARS-1 and SARS-2 effectively delay the IFN-I-response to undermine the first line-defence
In 2007, it was found that SARS already reached peak titers at day one after inoculation with the virus. Where the IFN type I-response would begin after day 1, mice would die within 3-5 days from Diffuse Alveolar Damage (DAD), similar to human patients with either SARS-CoV-1 or SARS-CoV-2 (A Mouse-Adapted SARS-Coronavirus Causes Disease and Mortality in BALB/c Mice, PloS Pathogens, January 12 2007).

SARS-1 and SARS-2 (COVID) are able to effectively delay the immune reponse by downregulation of IFN I through evasion of PRR-MDA5-sensing. It does so by hiding dsRNA within double membrane vesicles, capping of mRNA5' and attacking antiviral pathways. The delayed IFN-response enhances the sudden onset of monocyte-macrophage influx, inflammatory factors CCL2, CCL7 and CCL12.
It was found that T cells were primed to apoptosis, resulting in depletion of CD8 and CD4 T cells (Dysregulated Type I Interferon and Inflammatory Monocyte-Macrophage Responses Cause Lethal Pneumonia in SARS-CoV-1 infected mice, Cell Host & Microbe Vol. 19, Issue 2, 10 Februari 2016).

SARS-CoV-2 proteins suppress the IFN-I response. Nonstructural proteins Nsp1, Nsp6, Nsp12, Nsp13, Nsp14, Open-reading frames ORF3, ORF3a, ORF6, ORF7b and the M-protein are involved in IFN-1 inhibition. From this follows, that SARS-CoV-2 is able to evade the first-line defense (Evasion of type I Interferon by SARS-CoV-2, Cell Reports Vol. 33, Issue 1, October 2020).

2.2.4 SARS-CoV-2 protein ORF10 attenuates STING-autophagy to disrupt viral clearance and degrade antiviral MAVS
ORF10 impairs STING-induced IRF3 phosphorylation and translocation, inhibits STING-TBK1 interaction, prevents translocation of the STING signal to the Golgi in the mitochondria and attenuates STING-induced autophagy to clear the virus from cells (SARS-CoV-2 ORF10 antagonizes STING-dependent interferon activation and autophagy, Journal of Medical Virology 2022;94). In addition, ORF10 degrades mitochondrial MAVS signaling by inducing mitophagy though binding to NIX and interaction with LC3B (SARS-CoV-2 suppresses the antiviral innate immune response by degrading MAVS through mitophagy, Nature Cellular & Molecular Immunology 19(2022)).

A possible treatment to enhance MAVS is D-glucosamine.

3. Mitochondrial metabolism in (Long) COVID

3.1.1 Mitochondrial ATP generation
Mitochondria are located on and in most cells, except for mature erythrocytes (COVID-19 sepsis: revisiting mitochondrial dysfunction in pathogenesis, aging, inflammation and mortality, Inflammation Research, 7 August 2020). A 2015 study revealed that mitochondria located on the edge of muscle cells are optimized to generate membrane voltage (power supply), while interconnected mitochondria inside muscle cells are optimized to use voltage in order to produce ATP (High-resolution 3D images reveal the muscle mitochondrial power grid, NIH News, 30 July 2015).

Mitochondria contribute to cellular homeostasis through generation of ATP and low levels of ROS, required for cell signaling. Endothelial cells are supplied with ATP by glycolysis, whilst ROS generation towards endothelial cells depends on mitochondria.

3.1.2  Substrate feeding through the TCA and Oxydative Phosphorylation (OXPHOS)
Glucose (metabolized via glycolysis and pyruvate oxidation), fatty acids (metabolized via fatty acid-beta-oxidation) and amino acids (via oxidative deamination) feed into the TCA cycle (or Krebs cycle) before entering the Electron Transport Chain (ETC) within the mitochondrial matrix, in order to undergo Oxidative Phosphorylation (OXPHOS).

Under normoxic conditions, cells metabolize glucose into pyruvate in order to feed the TCA. The TCA cycle produces Nicotinamide adenine dinucleotide (NADH) to substrate OXPHOS in order to generate ATP. Beta-oxidation of fatty acids or pyruvate form substances to produce Acetyl-CoA within the mitochondrial matrix. Citrate synthase converts acetyl-CoA to citrate. Succinyl-CoA is hydrolized (Mitochondrial ETC: OXPHOS, oxidant production and methods of measurement, Redox Biology 37 (2020)).

Five protein complexes are located inside the inner mitochondrial membrane, close to the TCA cycle. Complexes I-IV make up the Electron Transport Chain; Complex V is part of the ATP Synthase. The TCA cycle provides NADH and FADH2 to the ETC. NADH and FADH2 donate a pair of electrons to Complex I and II of the Electron Transport Chain. Through a coupling synthase, Complex V is tied to the generation of ATP from ADP (Mitochondrial electron transport chain: Oxidative phosphorylation, oxidant production and methods of measurement, Redox Biology 37 (2020); Feeding Mitochondria: Potential Role of nutritional components to improve critical illness convalescence, Clinical Nutrition 38 (2019)).

3.1.3 The Warburg Effect in disease: shifting from TCA and OXPHOS to aerobic glycolysis or FAS
The Warburg Effect marks a shift from the TCA cycle and oxidative phosphorylation to aerobic glycolysis by glucose uptake and increased fermentation of glucose to lactose, even in the presence of abundant oxygen (Neoplasia, Robbins & Cotran Pathologic Basis of Disease, 2021). Aerobic glycolysis is exploited by rapidly replicating cells and necessary for cell proliferation, viral replication and drug resistance. Initially, aerobic glycolysis is necessary for proliferation of neutrophils and M1 macrophage activation. As a first line of defense, neutrophils and M1 macrophages depend on glycolysis and Fatty Acid Synthesis (FAS).

Under hypoxic conditions, pyruvate is converted to lactate. Hypoxia-inducible factor-1 (HIF-1), consisting of HIF-1a and HIF-1-beta, shifts metabolism from mitochondrial respiration towards aerobic glycolysis. COVID/SARS-CoV-2-infection is marked by increased levels of pyruvate, pyruvate kinase and lactate dehydrogenase (LDH), indicating glycolysis with lactate fermentation.

3.2 Depletion and alteration of substrates

3.2.1 Tryptophan, glutamine and arginine depletion are hallmarks of COVID-progression, immune suppression and inflammaging
The metabolic pathways of Tryptophan (Trp) are kynurenine, decarboxylation, transamination and serotonin pathways (Tryptophan availability for kynurenine pathway metabolism across the life span: Control mechanism and focus on aging, exercise, diet and nutritional supplements, Neuropharmacology Vol. 112, Part B, January 2017). Indoleamine 2,3 (IDO1) catabolizes L-Tryptophan into kynurenine (KYN). Both KYN and Kynurenine acid (KYNA) activate the aryl hydrocarbon receptor (AhR).

L-Tryptophan depletion inhibits the proliferation of immune cells and reduces serotonin synthesis. While IDO1 suppresses immunity via its tyrosine-based inhibitory motifs (ITIM), the activation of IDO1-KYN-AhR suppresses the function of effector immune cells, impairs autophagy, manipulates the extracellular matrix and enhances vascular diseases and osteoporosis (Role of indoleamine 2,3-dioxygenase 1 (IDO1) and kynurenine pathway in the regulation of the aging process, Ageing Research Reviews Volume 75, March 2022). While effector T cells are depressed, regulatory T cells (Tregs) are enhanced.

COVID patients show upregulation of IFN-$$\gamma$$ or IFN-β in alveolar epithelial cells, which activate the IDO1-KYN-AhR pathway, thereby accumulating mucins to trigger hypoxia. The process delays virus clearance.

SARS-CoV-2 uses glutamine for virus assembly. An increased glutamine-to-glutamate ratio indicates enhanced glutamine metabolism, corresponding with purine, folate and one carbon metabolism for viral replication. Depletion of glutathione (GSH, a glutamine synthesis) leads to ROS, cell death and viral spread (Metabolic Reprogramming in COVID, International Journal of Molecular Sciences 2021, 22).

Arginine regulates T cell-cycle progression through cyclin D3. In COVID, arginine shortage is associated with T cell defects (SARS-CoV-2 Induced ARDS Associates with MDSC Expansion, Lymphocyte Disfunction and Arginine Shortage, Journal of Clinical Immunology Vol. 41(2021)).

In addition, phenylalanine and tyrosine are decreased in COVID.

3.2.2 Cholesterol: different (antiviral) functions for different metabolites
SARS-CoV-2 uses lipid droplets (LDs), containing cholesterol, to gain entry. Depletion of LDs suppresses replication.

In COVID patients, the interferon-stimulated gene cholesterol 25-hydroxylase (CH25H) is increased in macrophages and epithelial cells, indicating antiviral properties of CH25H. In addition, oxysterol 27-hydroxysterol (27HC) inhibits SARS-CoV-2 and is reported to be significantly decreased in COVID patients (The cholesterol metabolite 27-HC inhibits SARS-CoV-2 and is markedly decreased in COVID patients, Redox Biology Vol. 36, September 2020, 101682).

Desmosterol, lanosterol and lathosterol were found to be descreased during COVID. ApolipoproteinA-1 (APOA1) is downregulated. Decreased HDL-cholesterol, LDL-C, TC, Fatty Acid-Binding Proteins (FABPs) and apoA-1 levels are indicative of a poor prognosis (Lipid metabolism changes in patients with severe COVID, Clinica Acta Chimia Volume 517, June 2021).

3.2.3 Lipid alterations
Two main enzymes are involved in fatty acid metabolism: Acetyl-CoA-carboxylase (ACC) and fatty acid synthase (FASN). Fatty acids undergo fatty acid oxidation (FAO). The process of ACC and FASN increases viral replication.

A 2020 study revealed that COVID patients had higher levels of acylcarnitines, essential for fatty acid oxidation. Accumulation of acylcarnitines inhibits ion channels, disrupts calcium signaling and impairs ATP production in mitochondria.

Higher levels of lysophospholipids (LPCs) were associated with viral replication. Plasma LPCs are produced by the phospholipase A2-pathway (PLA2). Levels of 2-hydroxy-3-methylbutyric acid, palmitic acid, succinic acid, pyroglutamic acid and myristic acid were elevated. Biosynthesis of the unsaturated fatty acid pathway (arachidonic acid, oleic acid, palmatic acid and stearic acids) was upregulated.

Upregulated LPCs reduce the production of Nitric Oxide (NO) and Prostaglandin in endothelial cells. LPC is involved in the attraction of adhesion molecules through production of IL-8, ROS production, CXCR4 expression in CD4+ T cells and macrophage activation.

Glycerophospholipids are generally downregulated, while PCs were found to be the most downregulated of this class. Downregulation of L-valine, L-proline and isoleucine was also considered to be a hallmark of infection. Dysregulation of panthotenate and CoA biosynthesis for the production of panthotenic acid, causes vitamin B5 deprivation (Large-Scale Plasma Analysis Revealed New Mechanisms and Molecules Associated with the Host Response to SARS-CoV-2, International Journal of Molecular Sciences 2020, 21).

3.2.4 Arginine
Among the discriminate metabolites in a 2020 study, L-ornithine and L-glutamine were involved in arginine metabolism, xanthine and adenine in purine metabolism and 4-guanidinobutanoate and L-ornithine in arginine and purine metabolism. The most clinically relevant metabolic pathways found were spermidine/spermine synthesis and two common metabolites were thymine and xanthine.

Arginase 1 (ARG1) is upregulated in COVID patients, impairing arginine levels and shunting the arginine metabolism away from nitric oxide synthesis (NO) towards ornithine production, which is synthesized into polyamines and proline via OCD and OAT. M2 macrophages are involved in ARG1 expression via Th2 cytokines, IL-4 and IL-13. The skewing of NO synthesis enhances endothelial dysfunction, immune impairment, vasoconstriction, platelet aggregation, smooth muscle cell proliferation, thrombosis and fibrosis. Arginine and citrulline, inhalation of NO, sGC, PDE5 inhibitors and homoarginine are proposed therapeutic interventions to restore arginine and NO synthesis in COVID patients (Targeting Arginine in COVID-induced Immunopathology and Vasculopathy, Metabolites 2022,12, 240).

3.2.5 Glutathione: a role for GlyNAC (N-acetylcysteine) to alleviate GSH deficiency?
Glutathione (GSH) is an intracellular antioxidant, present in mitochondria, nucleus and endoplasmic reticulum. While declining levels of GSH are part of the aging process, GSH levels are decreased in young COVID patients, a finding of relevance for the occurence of oxidative stress and oxidative damage in COVID.

It is proposed to supplement patients with GlyNAC: GSH precursor amino acids glycine and cysteine provided as N-acetylcysteine (available as NAC in commercial form). Not only does GlyNAC alleviate GSH deficiency, it is also an anti-inflammatory agent that improves endothelial function, mitochondrial function, insulin resistance, musculature and mitophagy (Severe Glutathione Deficiency, Oxidative Stress and Oxidant Damage in Adults Hospitalized with COVID: Implications for GlyNAC (Glycine and N-Acetylcysteine) Supplementation, Antioxidants 2022, 11, 50).

3.2.6 The PEA potential
Palmitoyl-ethanolamide (PEA) is known to inhibit mast cell activation and inflammation. Its anti-inflammatory properties are exerted via PPAR-alpha receptors. It was discovered that PEA also decreases the SARS-CoV-2 Spike RBD binding to ACE2. PEA acting on the PPAR-alpha also disrupts Lipid Droplet (LD) formation in monocytes, used by SARS-2 to defend itself against the cellular defense (Palmitoylethanolamide (PEA) Inhibits SARS-CoV-2 Entry by interacting with S protein and ACE2 Receptor, Viruses 2022, 14, 1080).

4. SARS and Post-Acute SARS (= Long COVID) metabolic alterations

4.1 Acetylcholine
Projection neurons and interneurons release acetylcholine (ACh) in the Central Nervous System (CNS).  Acetylcholine receptors (AChRs) are metabotropic muscarinic or nicotinic (nAChRs). It is hypothesized that SARS-CoV-2 not only binds to ACE2, but is also able to bind to nicotinic receptors. Virus particles may compete with acetylcholine to bind to nAChRs. As nAChRs have a high affinity for nicotine, nicotine administration is prosposed to treat neurological Long COVID. Cholinergic signaling and the release of neurotransmitters such as dopamine, glutamate and gamma-aminobutyric acid (GABA) are increased to adapt neuronal activity.

Of special interest is that nicotinic receptors (a7nAChR) stimulate the nervus vagus through inflammation. Autonomic regulation and exercise intolerance are reported to be improved in case reports on administration of nicotine patches (Is the post-COVID syndrome a severe impairment of acetylcholine-orchestrated neuromodulation, that responds to nicotine administration?, Bioelectronic Medicine (2023) 9:2).

4.2 Fatty acid and amino dysregulation in Long COVID
Exercise intolerance in Long COVID is accompanied by elevated levels of arterial lactate and slowed rates of fatty acid oxidation (FAtOx) during graded exercise. For extended exercise, ATP (adenosine triphosphate) is usually generated through oxidative phosphorylation (OXPHOS) in the TCA cycle. The supply consists of fatty acids and carbohydrates, which have to undergo beta-oxidation, FAtOx and CHOx. Lactate is a substrate for gluconeogenesis (biosynthesis of glucose), muscle glycogenesis and a regulator of FAtOx.

Carnitine palmitoyl transferase (CPT) has to form fatty acids (acyl-CoAs) to acylcarnitine. Acetyl-CoA has to be generated to supply energy. Low levels of FAtOx and elevated levels of lactate indicate metabolic shifts and impairment. Patients who recover from moderate to critical COVID show higher levels of acylcarnitines and lower levels of TCA products such as succinate, mono-pyruvate and malate.

Elevation of carnitine and poly- and highly unsaturated fatty acids in Post-COVID and Long COVID is indicative of decreased fatty acid oxidation in mitochondria. This is accompanied by changes in respiratory gas exchange. Carnitine and free fatty acids are associated with erythrocyte dysfunction and impaired oxygen delivery.

In both acute COVID and Post COVID, alanine, asparagine, methionine, serine and threonine are decreased. In Long COVID, leucine/isoleucine, tryptophan, tyrosine, proline and valine are significantly decreased (Signatures of Mitochondrial Dysfunction and Impaired Fatty Acid Metabolism in Plasma of Patients with Post-Acute Sequelae of COVID (PASC), Metabolites 2022, 12(11)).

4.3 Iron metabolism: ferroptosis
COVID causes cardiological disease, among which alterations to the human pacemaker, through ferroptosis (The potential role of ferroptosis in COVID-related cardiovascular injury, Biomedicine & Pharmacotherapy 168 (2023)). Ischemia/reperfusion injury (I/RI) is shown to induce mitochondrial iron accumulation and ferroptosis. Ferroptosis is a form of cell death that depends on iron accumulation. Iron accumulation is induced by imbalance of elimination of lipid hydroperoxide (LOOH) and L-ROS accumulation. Inhibitors are iron depletion or prevention of lipid peroxidation.

Two systems inhibit ferroptosis. The cystine/glutamate system/glutathione peroxidase 4 (GPX4) system catalyzes the reduction of lipid peroxides. The ferroptosis suppressor protein (FSP)/Coenzyme 10 (CoQ10) removes lipid hydrogen peroxide radicals, whilst dihydroorotate dehydrogenase (DHODH)/CoQ10H2 removes lipid hydrogen peroxide radicals from mitochondria. Ferroptosis inhibitors target Fe2+ accumulation and lipid peroxide accumulation.

SARS-CoV-2 inhibits expression of GPX4. Levels of prostaglandin-endoperoxide synthase 2 (PTGS2) are elevated. Upon infection, a cytokine cascade is released, among which IL-6 increases hepicidin and ferritin. IL-6 also upregulates the transferrin receptor TfR. Fe3+ is transported in its transferritin (TF)-form, entering the cell via TfR, reduced to Fe2+ and released into the cytosol by DMT1. Fe2+ is also stored in ferritin. Accumulation of ferritin causes NCOA4 to transport ferritin for ferritinophagy, a process that leads to the release of iron in unstabile form. Unstabile iron induces ferroptosis through lipoxygenase (LOX).

Erythrocytes express ACE2 and CD147 and are therefore a target in COVID. Upon infection, erythrocytes release hemoglobin and divide to create heme iron. Increased expression of HMOX-1 breaks down heme to form carbon monoxide (CO), Fe2+ and biliverdin. Ceruloplasmin transforms Fe2+ to Fe3+. Entering the cell via TfR1 and transferrin, Fe3+ is again broken down to Fe2+, which causes ferroptosis.

Increased hepcidin synthesis via upregulation of HAMP expression impairs heme production and iron bio-availability, leading to oxygen-binding dysfunction. The result, ROS production, is a survival strategy for SARS.

Acyl-Coenzyme A 4 (ACSL4) is found to be involved in viral replication via ferroptosis. ACSL4 catalyzes polyunsaturated fats (PUFAs), among which arachidonid acid (AA), to acyl-CoA. PUFA-CoA is esterified to phospholipids containing PUFAs (PUFA-PLs). Depletion of glutathione (GHS) and inhibition of GPX4 causes ferroptosis through ROS. Active iron or ROS cause PUFA-PLs to form phospholipid hydroperoxides (PLOOH) (Acyl-Coenzyme A Synthetase Long-Chain Family Member 4 is Involved in Viral Replication Organelle Formation and Facilitates Virus Replication via Ferroptosis, mBio Jan/Feb 2022, Vol. 13, Issue 1).

GPX4 catalyzes the reduction of phospholipid polyunsaturated fatty acid peroxides (PL-PUFA-OOH). CoQ10, DHODH and FSP1 can decrease PL peroxidation, thereby blocking ferroptosis caused by GPX4 deficiency (Phospholipase iPLA2β acts as a guardian against ferroptosis, Cancer Communications 2021;41).

Activation of TLR4 and increase in NOX4 are involved in ferroptosis, while NOX2 induces thrombotic activity and TfR1 dysfunction induces coronary syndrome. TLR4 promotes cardiac injury through leukocyte trafficking. Ferroptotic cell death triggers TLR4 through the release of DAMPs. Hsp60, a DAMP released during ischemia, activated inflammation via TLR4/MyD88. Ferroptosis induces neutrophil recruitment to cardiac vascular endothelial cells through TLR4/Trif signaling (Ferroptotic cell death and TLR4/Trif signaling initiate neutrophil recruitment after heart transplantation, JCI February 26, 2019). Ferrostatin-1 (Fer-1) inhibits TLR4 and scavenges peroxidated lipids, specifically 15-HpETE-PE, an oxidized PUFA-PL (Insight into the mechanism of ferroptosis inhibition by Fer-1, Redox Biology Vol. 28, January 2020).

DFO, Lip-1 and NAC are other potential therapeutics for cardiovascular disease due to COVID. Specifically, Lip-1 prevents myocardial I/RI by descreasing VDAC1 and restoring GPX4 (Liproxstatin-1 protects the mouse myocardium against ischemia/reperfusion injury by decreasing VDAC1 levels and restoring GPX4 levels, Biochemical and Biophysical Research Communications Vol. 520, Issue 3, 10 December 2019).

5 Effects on coagulation, platelet health, cardiology and function of monocytes

5.1 The Warburg Effect in COVID via PI3K/AKT/mTOR and MAPK/ERK pathways induce microthrombosis
Pyruvate Dehydrogenase (PDH) inhibition by Pyruvate kinase dehydrogenase 1 (PDK1) is stimulated by HIF-1a, PI3K/AKT/mTOR and the MAPK/ERK pathway, activated by loss of P53. This means that the PI3K/AKT pathway, by activating PDK1, blocks off pyruvate from feeding into mitochondria.

PDK1 and ERK in platelets stimulate aerobic glycolysis, which leads to thromboxane activation and microthrombosis, while Platelet-derived Growth Factor (PDGF) activates glycolysis via PI3K and HIF-1a. The PI3K/AKT pathway activates ATP Citrate Lyase (ACLY), thereby enhancing Acetyl-CoA to sustain Fatty Acid Synthesis (FAS). Acetyl-CoA Carboxylase (ACC) sustains arachidonic acid synthesis, necessary for generation of thromboxane. 

AMP-activated protein kinase (AMPK) counteracts the Warburg effect and the PI3K/AKT/mTOR pathway. In addition, AMPK acts on production of the vasodilator Ang 1-7 in endothelial cells and stabilizes ACE2, decreasing vasoconstriction and platelet-derived microthrombosis (The Key role of the Warburg Effect in SARS-CoV-2 replication and associated inflammatory response, Biochimie 180 (2021)).

SARS-CoV-2 increases glucose carbon entry into the TCA cycle, increases pyruvate carboxylase (PC) expression and decreases glutamine metabolism. Synthesis of aspartate from oxalo-acetate is likely maintained. Aspartate and asparagine are used to replicate viral RNA, while mTORC1 activity is increased. As mTORC1 is involved in the anabolic metabolism to enhance viral replication, mTORC1 inhibitors (Rapamycin, everolimus, temsirolimus) are proposed to treat COVID progression (SARS-CoV-2 Infection rewires host cell metabolism and is potentially susceptible to mTORC1 inhibition, Nature Communications (2021)12:1876).

Pathways toward deterioration in SARS-CoV-2/COVID V: SARS causes mitochondrial dysfunction, loss of membrane potential (ΔΨ m) and impaired OXPHOS under hypoxic conditions

Mitochondria are located inside the cell. Their function is to maintain cellular homeostasis and "aerobic respiration": by generating Adenosine Triphosphate (ATP) from ADP, mitochondria supply cells with energy. Through generation of low levels of Reactive Oxygen Species (ROS), mitochondria are involved in cell signaling. Imbalance in one of the processes involved in energy supply and ROS generation contributes to pathology, as is the case with infectious diseases such as COVID. Mitochondrial dysfunction is also involved in systemic disease. 

In this part of the "Deterioration in SARS-CoV-2 series", I will discuss the key role of mitochondria in the energy production process and ROS generation. As is known since SARS-CoV-1 (2003), the proteins of the coronavirus are able to "hack" mitochondria and get access to host cells via the "hijacking of mitochondria". I will offer insight into the properties of the virus that impair mitochondrial health which by doing so, contribute to viral replication and suppression of the immune response. I will also discuss possible treatment options to effectuate "mitochondrial redox".

1.   Mitochondrial function
1.1 Substrate feeding through the TCA and OXPHOS
1.2 What impaired mitochondrial function amounts to
1.3 The Warburg effect

2.  COVID-mitochondriopathy and hypoxia
2.1 Hypoxic conditions in COVID
2.2 The Warburg Effect in COVID via PI3K/AKT/mTOR and MAPK/ERK pathways induce microthrombosis
2.3 Inhibition of Bcl-2 family members leads to necrotic cell death
2.4 NSP4 and ORF9b of SARS-CoV-2 damage mitochondrial membrane potential (ΔΨ _m) and induce release of mtDNA into the cytosol

2.5 Fibrosis and mitochondrial dysfunction
2.5.1 Alleviating pulmonary fibrosis through restoration of mitochondrial integrity and REDOX (Metformin)
2.5.2 Mitophagy impairment in spite of enhanced PINK1/Parkin
2.6 Mitochondrial damage leading to mismatched ventilation/perfusion (V/Q) in COVID
3. Mitchondrial DNA and TLR-9 contribute to SARS-endothelial damage

1. Mitochondrial function

Mitochondria are located on and in most cells, except for mature erythrocytes (COVID-19 sepsis: revisiting mitochondrial dysfunction in pathogenesis, aging, inflammation and mortality, Inflammation Research, 7 August 2020). A 2015 study revealed that mitochondria located on the edge of muscle cells are optimized to generate membrane voltage (power supply), while interconnected mitochondria inside muscle cells are optimized to use voltage in order to produce ATP (High-resolution 3D images reveal the muscle mitochondrial power grid, NIH News, 30 July 2015).

Mitochondria contribute to cellular homeostasis through generation of ATP and low levels of ROS, required for cell signaling. Endothelial cells are supplied with ATP by glycolysis, whilst ROS generation towards endothelial cells depends on mitochondria.

1.1 Substrate feeding through the TCA and OXPHOS
Glucose (metabolized via glycolysis and pyruvate oxidation), fatty acids (metabolized via fatty acid-beta-oxidation) and amino acids (via oxidative deamination) feed into the TCA cycle (or Krebs cycle) before entering the Electron Transport Chain (ETC) within the mitochondrial matrix, in order to undergo Oxidative Phosphorylation (OXPHOS).

Under normoxic conditions, cells metabolize glucose into pyruvate in order to feed the TCA. The TCA cycle produces Nicotinamide adenine dinucleotide (NADH) to substrate OXPHOS in order to generate ATP. Beta-oxidation of fatty acids or pyruvate form substances to produce Acetyl-CoA within the mitochondrial matrix. Citrate synthase converts acetyl-CoA to citrate. Succinyl-CoA is hydrolized (Mitochondrial ETC: OXPHOS, oxidant production and methods of measurement, Redox Biology 37 (2020)).

Five protein complexes are located inside the inner mitochondrial membrane, close to the TCA cycle. Complexes I-IV make up the Electron Transport Chain; Complex V is part of the ATP Synthase. The TCA cycle provides NADH and FADH2 to the ETC. NADH and FADH2 donate a pair of electrons to Complex I and II of the Electron Transport Chain. Through a coupling synthase, Complex V is tied to the generation of ATP from ADP (Mitochondrial electron transport chain: Oxidative phosphorylation, oxidant production and methods of measurement, Redox Biology 37 (2020); Feeding Mitochondria: Potential Role of nutritional components to improve critical illness convalescence, Clinical Nutrition 38 (2019)).

1.2 What impaired mitochondrial function amounts to
Mitochondrial function is essential for neurological functions. Age-related neurodegeneration is a consequence of age-affected mitochondrial degeneration. Pathogens, such as viruses, impair mitochondrial health as well. During the ATP synthase through oxidative phosphorylation (OXPHOS), oxidative stress is generated. Excess Reactive Oxygen Species (ROS) is harmful to mitochondria. ROS damages lipids, proteins and nucleic acids, leading to impaired metabolism, cell death, impaired glucose metabolism, impaired calcium homeostasis and DNA damage. Damage Associated Patterns (DAMP) generate ROS in response to the detection of viral DNA. Oxidized mitochondrial DNA, cardiolipin and cytochrome from damaged mitochondria are released into the cytoplasm,leading to enduring systemic inflammation (COVID: a Mitochondrial Perspective, DNA and Cell Biology Vol. 40 Number 6, 2021).

Hyperferritinemia, accumulation of iron occurring due to COVID, induces ROS formation. Iron homeostasis dysregulation incites platelet destruction. The cytokine cascade contributes to ROS formation through TNF-α, IFN-, IL-6 and IL-10, stimulating the pro-inflammatory profile. IL-6 and TNF-α impair the processes of ATP and OXPHOS. This amounts to the loss of mitochondrial membrane potential, increasing mitochondrial permeability ("leaky mitochondria").

Upon losing integrity, mitochondrial DNA is released into the intracellular fluid, inducing an inflammatory profile of IL1-β and IL-6 through stimulation of the inflammasome NLRP3. Damage to mitochondria in epithelial alveolar cells contributes to the release of inflammatory CXCL-8, CCL3, CCL4, CCL20, IL-6 and IL-12. Hyperferritinemia drives mitochondrial respiration from aerobic to an anaerobic state.

In SARS-CoV-2 infection, TCA cycle/Krebs cycle metabolites citrate, malate, fumarate and aconitate are shown to be decreased through depression of TCA cycle genes. TCA cycle and OXPHOS are depressed in COVID, indicating altered cellular metabolism. Furthermore, choline uptake is increased by polarized macrophages during SARS-CoV-2 infection, subsequently leading to choline downregulation. On the other hand, homocysteine upregulation in COVID contributes to endothelial damage (Metabolic reprogramming and epigenetic changes of vital organs in SARS-CoV-2-induced systemic toxicity, JCI Insight 2021; 6(2)).

1.3 The Warburg effect
The Warburg Effect marks a shift from the TCA cycle and oxidative phosphorylation to aerobic glycolysis by glucose uptake and increased fermentation of glucose to lactose, even in the presence of abundant oxygen (Neoplasia, Robbins & Cotran Pathologic Basis of Disease, 2021). Aerobic glycolysis is exploited by rapidly replicating cells and necessary for cell proliferation, viral replication and drug resistance. Initially, aerobic glycolysis is necessary for proliferation of neutrophils and M1 macrophage activation. As a first line of defense, neutrophils and M1 macrophages depend on glycolysis and Fatty Acid Synthesis (FAS).

2. COVID-mitochondriopathy and hypoxia

2.1 Hypoxic conditions in COVID
Under hypoxic conditions, pyruvate is converted to lactate. Hypoxia-inducible factor-1 (HIF-1), consisting of HIF-1a and HIF-1-beta, shifts metabolism from mitochondrial respiration towards aerobic glycolysis. COVID/SARS-CoV-2-infection is marked by increased levels of pyruvate, pyruvate kinase and lactate dehydrogenase (LDH), indicating glycolysis with lactate fermentation.

Hypoxia occurs when leukocytes are activated in response to Interferon activation, as well as in response to Pathogen-Associated Patterns (PAMP) and Damage-Associated Patterns (DAMP). DAMP activate the inflammatory NF-kB-pathway and STING (Interferon Genes) pathway. Moreover, fragmented damaged mitochondria are secreted as DAMP. Vascular Endothelial Growth Factor (VEGF) is upregulated under hypoxic conditions, notably by HIF-1-alpha. HIF-1-alpha can accumulate in inflammatory cells through the Prolyl Hydroxylase-pathway or TLR4-mTOR.

Increased glycolysis in monocytes sustains inflammatory cytokine production (IL-1β, TNF-α and IL-6), T cell impairment and pulmonary epithelial cell death (Metabolic reprogramming in COVID, International Journal of Molecular Sciences 2021, 22, 11475).

The hyper-inflammatory stage of hypoxia is marked by activation of NF-kB, NLRP3, mTOR and MAPK (mitogen-activated protein kinase), which induce the cytokine cascade. The NAD+ (Nicotinamide Adenine Dinucleotide) mitochondrial homeostatic regulators Sirtuins 3, 4 and 5 are downregulated. Downregulated Sirtuins enhance ROS formation, which in turn downregulates PHD, a regulator of HIF-1-alpha. HIF-1-alpha thus stabilizes. Enzymes Glut1, LDH, PDHK, HK and COX-2 indicate the shift to anabolic glycolysis (COVID sepsis: revisiting mitochondrial dysfunction in pathogenesis, aging and inflammation, Inflammation Research, 3 August 2020). Glut2 uptake is stimulated by the EGFR.

In addition, activation of complement C5a is involved in activating pro-inflammatory neutrophils and macrophages by activation of PI3K/Akt and MAPK pathways, stimulating endothelial damage and thrombosis. HIF-1a in alveolar epithelial cells activates the NF-kB pathway, mediates cell inflammation through CD4+ and CD8+ and enhances inflammatory cytokines IL-2 and TNF-a, driving complement-activated endothelial damage (COVID-driven endothelial damage: complement, HIF-1 and ABL2 are potential pathways of damage and targets for cure, Annals of Hematology 9 June 2020).

Cells under oxygen deprivation induce adaptive responses through AMP-kinase. When presented with high O2 tension, Prolyl Hydroxylases (PHDs) oxidize HIF-1a under normoxic conditions. Under low O2 tension, impaired succinate dehydrogenase leads to accumulation of succinate with subsequent inhibition of PHDs. Disproportional lactate production through glycolysis lowers cellular pH. HIF-1 activates transcription of PDK1, encoding a kinase that inactivates pyruvate dehydrogenase. Since pyruvate dehydrogenase is essential for metabolizing acetyl-CoA in the mitochondrial matrix, mitochondrial respiration is decreased (Hypoxia and mitochondrial oxidative metabolism, Biochimica et Biophysica Acta 1797 (2010)).

2.2 The Warburg Effect in COVID via PI3K/AKT/mTOR and MAPK/ERK pathways induce microthrombosis
Pyruvate Dehydrogenase (PDH) inhibition by Pyruvate kinase dehydrogenase 1 (PDK1) is stimulated by HIF-1a, PI3K/AKT/mTOR and the MAPK/ERK pathway, activated by loss of P53. This means that the PI3K/AKT pathway, by activating PDK1, blocks off pyruvate from feeding into mitochondria.

PDK1 and ERK in platelets stimulate aerobic glycolysis, which leads to thromboxane activation and microthrombosis, while Platelet-derived Growth Factor (PDGF) activates glycolysis via PI3K and HIF-1a. The PI3K/AKT pathway activates ATP Citrate Lyase (ACLY), thereby enhancing Acetyl-CoA to sustain Fatty Acid Synthesis (FAS). Acetyl-CoA Carboxylase (ACC) sustains arachidonic acid synthesis, necessary for generation of thromboxane. 

AMP-activated protein kinase (AMPK) counteracts the Warburg effect and the PI3K/AKT/mTOR pathway. In addition, AMPK acts on production of the vasodilator Ang 1-7 in endothelial cells and stabilizes ACE2, decreasing vasoconstriction and platelet-derived microthrombosis (The Key role of the Warburg Effect in SARS-CoV-2 replication and associated inflammatory response, Biochimie 180 (2021)).

2.3 Inhibition of Bcl-2 family members leads to necrotic cell death
Bcl-2 family members tightly regulate Bax/Bak activation. Through Bax/Bak activation, Bcl-2 family members regulate apoptosis by regulating mitochondrial outer membrane permeability (BAX/BAK macropores regulate the mtDNA extrusion). Upon permeabilization of the mitochondrial outer membrane, cytochrome-C is released in order to activate Caspase, leading to necrotic cell death. Right after SARS-CoV-2 infection, pro-apoptotic genes are upregulated, among which the BCL2L11, leading to apoptosis by inhibition of anti-apoptotic Bcl-2 and activation of Bax-Bak (SARS-CoV-2 Mitochondriopathy in COVID-19 Pneumonia Exacerbates Hypoxemia, Redox Biology 58 (2022)).

2.4. NSP4 and ORF9b of SARS-CoV-2 damage mitochondrial membrane potential (ΔΨ _m) and induce release of mtDNA into the cytosol
Mitochondrial DNA (mtDNA) released into the cytosol enhances inflammation. mtDNA is the result of mitochondrial damage following loss of mitochondrial membrane potential. mtDNA are DAMPs, therefore promoting an inflammatory cascade through the cGAS/STING1-mediated interferon signaling.

It was found that transfection with SARS-CoV-2 proteins NSP2, NSP4, NSP6, NSP8, ORF3a, ORF6 and ORF9b upregulate mitochondrial Reactive Oxygen Species and downregulate mitochondrial membrane potential. Notably, NSP4, ORF6 and ORF9b induce mtDNA release from epithelial cells.

MCL-1 regulates inner membrane vesicle formation and packaging of mtDNA. Vesicles enclosing mtDNA are derived from the inner membrane and extruded through BAX/BAK macropores. NSP4 acts on BAX/BAK to induce macropore formation. ORF9b acts on MCL-1 to impair the inhibitory (protective) effect on BAX/BAK macropore formation and to impair the regulatory effect of MCL-1 on inner membrane stability and vesicle formation (NSP4 and ORF9b of SARS-CoV-2 induce pro-inflammatory mitochondrial DNA release in inner membrane-derived vesicles, Cells 2022, 11).

Extracellular mitochondrial DNA induces the release of pro-inflammatory cytokines (PICs). The loss of mitochondrial (membrane) integrity and the release of mtDNA induce a highly inflammatory IL1-β release. Moreover, loss of ΔΨ m and mtDNA circulating in the cytosol contributes to chronic inflammation, one of the appearances of Post-Acute SARS Syndrome/Long COVID.

2.5 Fibrosis and mitochondrial dysfunction
Excess ROS generation under hypoxia, leads to damage of mitochondrial DNA, proteins and lipids. The transition pore in the mitochondrial inner membrane loses integrity, inducing mitochondrial depolarization and swelling and loss of Electric Chain Transport (energy generation).
Increased mitochondrial permeability enhance the release of Cytochrome C into the cytosol.

Tissue Growth Factor-beta (TGF-β), induced by anti-inflammatory macrophages, stimulates fibroblasts through Smad signaling. Fibroblasts activate fibro-collagen, Extracellular Matrix Molecules (ECM) and inhibit the degradation of ECM molecules. The deletion of mitochondrial DNA, induced by Angiotensin II, is reported in cardiac fibrosis. Mitochondrial damage in alveolar epithelial cells contributes to pulmonary fibrosis. Impaired mitophagy, the removal of dysfunctional and defective mitochondria, contributes to fibrotic disease through activation of the platelet PDGFR/PI3K/AKT pathway (Mitochondrial function in fibrotic diseases, Cell Death Discovery (2020)6:80).

2.5.1 Alleviating pulmonary fibrosis through restoration of mitochondrial integrity and REDOX
Targeting mitochondrial Sirtuin-3 (SIRT3) could alleviate pulmonary fibrosis (Mitochondrial Sirtuin 3: New emerging biological function and therapeutic target, Theranostics 2020;10(18)). Furthermore, as hydrogen peroxide generation dependent on NOX4 contributes to fibroblast formation, LYCAT and NOX4-inhibition through activation of the AMPK-pathway could attenuate lung fibrosis.

Metformin attenuates lung fibrosis through activation of the AMPK-pathway, which inhibits TGF-β induced NOX4 expression and ROS. Furthermore, AMPK activation attenuates mTOR-activation (Metformin attenuates lung fibrosis development via NOX4 suppression, Respiratory Research 17, Art. 107(2016)). Mitoquinone is an antioxidant that inhibits TGF-β and NOX4 expression; mitoquinone also prevents Nrf2 downregulation (Mitoquinone ameliorates pressure overload-induced cardiac
fibrosis and left ventricular dysfunction in mice, Redox Biology 21, 101100 (2019)).

2.5.2 Mitophagy impairment in spite of enhanced PINK1/Parkin
Infection with SARS-CoV-2 causes mitochondrial lesion. In spite of PINK1/Parkin activation and mitochondrial P62 accumulation, the normal process of mitophagy of damaged mitochondria was inhibited. SARS-CoV-2 was found to inhibit P62 and LC3 binding. The mitochondrial outer membrane protein Tom20 was found to be used by SARS-CoV-2 to provide SARS-CoV-2 dsRNA entry. Interestingly, Chaperonin HSP60 remained high following SARS-CoV-2 infection, indicative of impaired clearance.

Knockdown of Tom20 and application of Cyclosporin D inhibitor Cyclosporin A, each decrease viral replication, indicating that loss of loss of mitochondrial membrane integrity and MPTP opening are vital in progression towards COVID (SARS-CoV-2 causes Mitochondrial Dysfunction and Mitophagy Impairment, Frontiers in Microbiology Vol. 12 (ahead of print)).

2.6 Mitochondrial damage leading to mismatched ventilation/perfusion (V/Q) in COVID
SARS-CoV-2 damages airway epithelial mitochondria (AEC) and pulmonary artery smooth muscle cells (PASMC), thereby triggering apoptosis and impairing hypoxic pulmonary vasoconstriction (HPV).

The M protein of SARS-CoV-2 was found to depolarize the mitochondrial membrane potential and increase the opening of the mitochondrial permeability transition pore (mPTP). Nsp7, Nsp9 and the M protein induced apoptosis of airway epithelial cells. While Ca2+ levels rise in response to hypoxia, M and Nsp9 were found to inhibit the rise of Ca2+, reducing an accurate HPV under hypoxic circumstances.

Increased Drp1-mediated fission and Nsp7-induced inhibition of ETC electron transport chain Complex I oxidative metabolism by SARS-CoV-2, contribute to mitochondrial dysfunction in COVID. Through expression of Caspase7 and Annexin V, apoptosis is induced. These factors contribute to impairment of oxygen-sensing. As a result, impaired HPV leads to mismatched ventilation/perfusion (V/Q) and leakage into the capillary (SARS-CoV-2 mitochondriopathy in COVID pneumonia exacerbates hypoxemia, Redox Biology 58 (2022) 102508).

3. Mitchondrial DNA and TLR-9 contribute to SARS-endothelial damage
SARS-CoV-2 was found to infect HUVECs, endothelial cells, through their expression of ACE2 and TMPRSS2. The sera of COVID patients evidenced release of mitochondrial cytochrome B and NAD dehydrogenase. Mitochondrial Complex I protein levels were significantly decreased in COVID. Complex I is necessary to maintain healthy ROS levels. A reduction of Complex I levels induces mitochondrial damage. Abnormal levels of intracellular Ca2+ concentrations indicate endothelial and mitochondrial dysfunction. Reduction of Ca2+ disrupts vasodilation and induces endothelial damage.

From SARS-CoV-1 (2003), it was known that the mitochondrial antiviral signaling protein (MAVS) was disabled in order to manipulate mitochondrial function.
The E-ORF and ORF10 of SARS-CoV-2 activates TLR-9 through multiple cells.

TLR-9 activates a hyperinflammatory state. Through an increase of mtDNA release in HUVECs, TLR-9 becomes activated, which increases the inflammatory pathways My88 and NF-kB, decreases eNOS and induces the release of high levels of cytokine IL-6, thus creating a feedback loop into endothelial damage and thrombosis (Mitochondrial DNA and TLR-9 activation contribute to SARS-CoV-2 induced endothelial cell damage, Vascular Pharmacology 142(2022)). 

Pathogenesis and structure of SARS-CoV-2 and therapeutic options for COVID-19: a research collection

Coagulopathy characterizing COVID-19
The unique characteristics of COVID-19 coagulopathy, Critical Care 2020; 24: 360;

IL-6 and central cytokine storms are not characteristic for COVID-19
Severe COVID-19 Infections- Knowledge gained and remaining questions, JAMA Internal Medicine, 18 September 2020;

Immune profile
Doctors may have found a new way to predict and treat severe coronavirus cases (IL-7 to treat severe COVID-19), BGR Science, May 31, 2020;

Pathogenesis and viral tropism of SARS-Coronaviruses
Role of SARS-CoV Viroporins E, 3a and 8a in Replication and Pathogenesis, mBio 2018 May-Jun; 9(3);
Secondary structure of the SARS-CoV-2 5'-UTR, RNA Biology 2020:1-10;
SARS-CoV-2 uses a second doorway into cells: Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity, Science 20 October 2020;
The SARS-coronavirus papain-like protease: Structure, function and inhibition by designed antiviral compounds, Antiviral Research 2015 Mar;115:21-38;
Molecular epidemiology, evolution and phylogeny of SARS-coronavirus, Infection, Genetics and Evolution 2019 Jul;71:21-30;
SARS-CoV-2 ORF3b is a Potent Interferon Antagonist whose activity is increased by a naturally occurring elongation variant, Cell Reports 2020 Sep 22;32(12);
Cardiovascular Implications of Fatal Outcomes of Patients with COVID-19, JAMA Cardiology 2020 Jul; 5(7): 1-8;
Outcomes of Cardiovascular MRI in Patients Recently discovered from COVID-19, JAMA Cardiology 2020, 27 July 2020;
Patient-derived SARS-CoV-2 mutations impact viral replication dynamics and infectivity in vitro and with clinical implications in vivo, Cell discovery 2020;6:76;
Lung tissue in fatal COVID-19 shows broad cell tropism and extensive damage, News Medical Life Sciences, September 30, 2020;
The Natural History, Pathobiology and Clinical Manifestations of SARS-CoV-2 Infections, Journal of Neuroimmune Pharmacology 2020 Jul 21: 1-28;
The PDZ-Binding Motif of SARS-Coronavirus envelope protein is a determinant of viral pathogenesis, PLoS Pathogens 2014 Aug; 10(8);
In silico identification of Tretinoin as a SARS-CoV-2 envelope (E) protein ion channel inhibitor, Computers in Biology and Medicine 2020 Dec; 127: 104063;
SARS-CoV-2 strategically mimics proteolytic activation of human ENaC, eLife Sciences 2020;9;
Dying with SARS-CoV-2 infection-- an autopsy study of the first consecutive 80 cases in Hamburg, Germany, International Journal of Legal Medicine 2020 Jun 4: 1-10;

Possible treatment options for COVID-19
Kallikrein-kinin blockade in patients with COVID-19 to prevent Acute Respiratory Syndrome, eLife 2020; 9;
Mechanisms of Protective Effects of SGLT2 Inhibitors in Cardiovascular Disease and Renal Dysfunction, Current topics in medicinal chemistry 2019;19(20): 1818-1849;
Outcomes Associated with use of a Kinin B2 Receptor Antagonist among patients with COVID-19, JAMA Infectious Diseases 2020;3(8);
Abelson Kinase Inhibitors Are Potent Inhibitors of SARS-CoV and MERS-CoV Fusion, Journal of Virology 2016 Oct 1; 90(19):8924-8933;
Salvia miltiorrhiza Injection alleviates LPS-induced Acute Lung Injury by adjusting the balance of MMPs/TIMPs ratio, Evidence Based Complement Alternative Medicine 2020;2020;
Sonic Hedgehog Signaling: Evidence for its Protective Role in Endotoxin Induced ALI in mouse model, PLoS One 2015; 10(11);
Extracorporeal membrane oxygenation for SARS associated with COVID-19: a retrospective cohort study, Lancet Respiratory Medicine Vol. 8, Issue 11, November 01, 2020;
Novel hybrid antiviral VTRRT-13V2.1 against SARS-CoV-2 main protease: retro-combinatorial synthesis and molecular dynamics analysis, Heliyon Vol. 6, Issue 10, October 2020;
A perspective on Erythropoietin as a potential adjuvant therapy for ALI/ARDS in patients with COVID-19, Archives of Medical Research 2020 Aug 11;
Association between chronic ACE receptor inhibitor exposure and decreased odds of severe disease in patients with COVID-19, Anatolian Journal of Cardiology 2020; 24(1): 21-29;
Vorolanib and everolimus: Lenvatinib and everolimus part deux, or something new?, EBioMedicine Vol. 56, June 2020;
MMP-8 Inactivates Macrophage Inflammatory Protein-1alpha to Reduce Acute Lung Inflammation and Injury in Mice, Journal of Immunology 2010 Feb 1;184(3): 1575-1588;
Sofosbuvir for COVID-19 infection: a potential candidate, Indian Journal of Pharmacology 2020 May-Jun; 52(3): 232-233;
Broad spectrum coronavirus antiviral drug discovery, Expert Opin Drug Discovery 2019; 14(4): 397-412;
Apirin Use is Associated with Decreased Mechanical Ventilation, ICU Admission, and In-Hospital Mortality in Hospitalized Patients with COVID-19, Anesthesia & Analgesia: October 21, 2020;
Is Acetylsalicylic Acid a Safe and Potentially Useful Choice for Adult Patients with COVID-19?, Drugs 80, 1383-1396(2020);
Ac2-26 mimetic peptide of annexin A1 to treat severe COVID-19: A hyptothesis, Medical Hypotheses 2020 Dec; 145: 110352;
Umbilical cord: an allogenic tissue for potential treatment of COVID-19,
Ebselen, Disulfirum, Carmofur, PX-12, Tideglusib and Shikonin are nonspecific promiscious SARS-CoV-2 main protease inhibitors, ACS Pharmacological Translational Sciences 2020 Oct 9;

Preprints (warning: never draw conclusions based on MedRxiv and BioRxiv research papers: preprints from the Rxiv server still have to undergo peer review, conclusions are preliminary and might turn out to be invalid)
Ethacridine inhibits SARS-CoV-2 by inactivating viral particles in cellular models, October 28, 2020;
A neutrophil activation signature predicts critical illness and mortality in COVID-19, September 02, 2020;

Immunological and inflammatory factors in COVID-19/SARS-CoV-2 infection: a research collection

1. Questions on COVID-19 to be answered
ACE2, COVID-19 Infection, Inflammation and Coagulopathy: Missing Pieces in the Puzzle, Frontiers in Physiology 2020; 11: 574753;

ADAM
ADAM9 is a Novel Product of Polymorphonuclear Neutrophils: Regulation of Expression and Contributions to Extracellular Matrix Protein Degradation During Acute Lung Injury, Journal of Immunology 2014 Sep 1; 193(5): 2469-2482;
The Role of Metalloproteinase ADAM17 in Regulating ICOS Ligand-Mediated Humoral Immune Responses, The Journal of Immunology Vol. 193, Issue 6, 15 September 2014;
B cell ADAM17 controls T cell independent humoral immune responses through regulation of TACI and CD138, Biochemical and Biophysical Research Communications Vol. 552, Issue 2, 5 February 2020;
ACE2/ADAM17/TMPRSS2 Interplay may be the Main Risk Factor for COVID-19, Frontiers Inflammation, 07 October 2020;
ADAM17, the TNF-alpha convertase, ScienceDirect

ALI (Acute Lung Injury) due to COVID-19
Inhibition of metalloproteinases in therapy for severe lung injury due to COVID-19, Medicine in Drug Discovery 2020 Sep; 7: 100052;

Autophagy
Can endolysosomal deacidification and inhibition of autophagy prevent severe COVID-19?, Life Sciences Vol. 262, 1 December 2020;

Caveolin-1, ARDS and ALI
Caveolin-1: a critical regulator of lung injury, American Journal of Physiology, Lung Cellular and Molecular Physiology 2011 Feb; 300(2);
Inflammation-induced caveolin-1 and BMPRII depletion promotes endothelial dysfunction and TGF-β-driven pulmonary vascular remodeling, Lung Cellular and Molecular Physiology 2017 May 1; 312(5);

Clinical Immunology
Long-term infection of SARS-CoV-2 changed the body's immune status, Clinical Immunology 2020 Sep; 218: 108524;

Contact system involvement
The contact activation system as a potential therapeutic target in patients with COVID-19, Research and Practice in Thrombosis and Haemostasis Vol. 4, Issue 4, May 2020;

Cytokine profile
Cytokine elevation in severe and critical COVID-19: a rapid systematic review, meta-analysis and comparison with other inflammatory syndromes, Lancet Respiratory Medicine October 16, 2020;

Elevation of liver enzymes in COVID-19
Serum Activity of Liver Enzymes is Associated with Higher Mortality COVID-19: A Systematic Review and Meta-Analysis, Frontiers Gastroenterology, 22 July 2020;

Ethnic profiles
Ethnic differences in alpha-1 antitrypsin deficiency allele frequencies may partially explain national differences in COVID-19 fatality rates, FASEB Journal 2020 Sep 22;

FOXP3 (Forkhead family of transcription responsible for Treg development)
FOXP3- an overview, ScienceDirect;
T-cell Hyperactivation and Paralysis in Severe COVID-19 Infection Revealed by Single-Cell Analysis, (CD25+ hyperactivation facilitates coronavirus entry by producing Furin; FOXP3-mediated negative feedbacks are impaired) Frontiers Immunological Tolerance and Regulation, 08 October 2020;
The many functions of FOXP3+ Regulatory T cells in the intestine, Frontiers T cell Biology, 20 October 2020;

Genetic profile
Host/genetic factors associated with COVID-19 call for precision medicine (NETs, NETosis and DAMPS), Precision Clinical Medicine 2020 Vol. 3, Issue 3, September 2020;

Interferons (IFN-)
SARS-CoV-2 ORF3b Is a Potent Interferon Antagonist Whose Activity is Increased by a Naturally Occurring Elongation Variant (anti-IFN I and NLS), Cell Report Vol. 32, Issue 12, 108185, September 22, 2020;
The ORF6, ORF8 and nucleocapsid proteins of SARS-CoV-2 inhibit type I Interferon (IFN) signaling pathway (SARS-CoV-2 inhibits IFN-β activation and the NF-kB pathway, ORF6 and ORF8 suppress innate immune functions), Virus Research Vol. 286, September 2020;
Heightened circulating Interferon-Inducible Chemokines and activated Pro-Cytolytic Th1-cell phenotype features COVID-19 aggravation in the second week, Frontiers Cytokines and Soluble Mediators in Immunity, 20 October 2020;

Interleukins (IL- )
Elevated IL-33 promotes expression of MMP2 and MMP9 via activating STAT3 in alveolar macrophages during LPS-induced acute lung injury (ALI), Cellular & Molecular Biology Letters 2018; 23:52;
IL-1 induces thromboxane-A2 (TxA2) in COVID-19 causing inflammation and micro-thrombi: inhibitory effect of the IL-1-Receptor antagonist (IL-1Ra), Journal of Biological Regulators and Homeostatic Agents 2020 Aug 3;34(5);
Imperfect storm: is interleukin-33 the Achilles heel of COVID-19?, Lancet Rheumatology, 9 October 2020;

Macrophages
Targeting macrophages as a therapeutic option for COVID-19 (M1 and M2), Frontiers Inflammation Pharmacology, to be published;
Activated protein C and PAR1-derived peptides are anti-inflammatory by suppressing macrophage NLRP3 inflammasomes, Journal of Thrombosis and Haemostasis, 13 October 2020;
A critical role for suppressor of cytokine signalling 3 in promoting M1 macrophage activation in vitro and in vivo, Immunology 2014 Jan;141(1):96-110;
Protective effect op suppressing STAT3 activity in LPS-induced lung injury, Lung Cellular and Molecular Physiology 2016 Nov 1; 311(5);
Macrophage responses associated with COVID-19: A pharmacological perspective, European Journal of Pharmacology Vol. 887, 15 November 2020;
Alveolar macrophage dysfunction and cytokine storm in the pathogenesis of two severe COVID-19 patients, EBioMedicine Vol. 57, July 2020;
The lung macrophage in SARS-CoV-2 Infection: A Friend or a Foe?, Frontiers in Immunology 2020; 11: 1312;

MAPK signalling pathways
MAPK signalling pathways as molecular targets for anti-inflammatory therapy- from molecular mechanisms to therapeutic benefits, Biochimica et Biophysica Acta (BBA)- Proteins and Proteomics, Vol. 1754, Issue 1-2, 30 December 2005, p253-262;

Megakaryocytes
Development and characterisation of a novel, megakaryocyte NF-kB reporter cell line for investigating inflammatory responses, Journal of Thrombosis and Haemostasis, 09 October 2020;

Matrix Metalloproteinases (MMPs) and their role in Acute Lung Injury (ALI)
Elevated IL-33 promotes expression of MMP2 and MMP9 via activating STAT3 in alveolar macrophages during LPS-induced acute lung injury (ALI), Cellular & Molecular Biology Letters 2018; 23:52;
Upregulation of matrix metalloproteinase 9 (MMP9)/tissue inhibitor of metalloproteinase 1 (TIMP1) and MMP2/TIMP2 ratios may be involved in lipopolysaccharide-induced acute lung injury (LPS-induced ALI), Journal of International Medical Research 2020 Apr; 48(4);
Inhibition of metalloproteinases in therapy for severe lung injury due to COVID-19, Medicine in Drug Discovery 2020 Sep; 7: 100052;
Matrix Metalloproteinases in ALI: mediators of injury and drivers of repair, European Respiratory Journal Vol. 38 Issue 4, 2011;
Serum MMP-8 and TIMP-1 in critically ill patients with acute respiratory failure: TIMP-1 is associated with increased 90-day mortality, Anesthesia and analgesia 2014 Apr;118(4):790-8;

Natriuretic peptide in healthy individuals
Subclinical elevated B-type natriuretic peptide (BNP) indicates endothelial dysfunction contributing to hypoxia susceptibility in healthy individuals, Life Sciences Vol. 260, 1 November 2020;

Natural Killer Cells (NK-)
KLRD1 Killer Cell lectin-like receptor (CD94), gene id;

Neutrophil Extracellular Traps (NETs) and NETosis
Host/genetic factors associated with COVID-19 call for precision medicine (NETs, NETosis and DAMPS), Precision Clinical Medicine 2020 Vol. 3, Issue 3, September 2020;
Neutrophil Extracellular Traps (NETs) and Damage-Associated Molecular Patterns (DAMPs): Two Potential Targets for COVID-19 Treatment, Mediators of Inflammation 2020;2020;
Devilishly radical NETwork in COVID-19: Oxidative Stress, neutrophil extracellular traps (NET) and T cell suppression, Advances in Biological Regulation 2020 Aug; 77;
Tissue damage from neutrophil-induced oxidative stress in COVID-19, Nature Reviews Immunology 2020 Jul 29: 1-2;
Neutrophils and Neutrophil Extracellular Traps Drive Necroinflammation in COVID-19, Cells 2020 Jun; 9(6): 1383;
SARS-CoV-2-triggered NETs mediate COVID-19, Journal of Experimental Medicine 2020 Dec 7; 217(12);

NLRP3 Inflammasome
Severe COVID-19: NLRP3 Inflammasome Dysregulated, Frontiers Immunology 2020; 11: 1580;

PAMPs (Pathogen-Associated Molecular Patterns)
Host/genetic factors associated with COVID-19 call for precision medicine (NETs, NETosis and DAMPS), Precision Clinical Medicine 2020 Vol. 3, Issue 3, September 2020;

Perspective from scientists
Surviving COVID-19: A disease tolerance perspective, Science Advances 2020 May; 6(18);

ROS (Reactive Oxidative Species) and Oxidative Stress (= biophysics)
Reactive Oxidative Species-Modulated Ca2+ release Regulates β₂ integrin activation on CD4+CD28null T Cells of Acute Coronary Syndrome Patients, Journal of Immunology Vol. 205, Issue 9, 1 November 2020 (see edit);
Mild SARS-CoV-2 infections in children might be based on evolutionary biology and linked with host Reactive Oxidative Stress and antioxidant capabilities, New Microbes and New Infections Vol. 36, July 2020;

Suppressor of Cytokine Signalling (SOCS)
SOCS Proteins Participate in the Regulation of Innate Immune Response Caused by Viruses (feedback through Janus kinase and STAT), Frontiers in Immunology 2020; 11;
Suppressor of cytokine signalling (SOCS1) is a key determinant of differential macrophage activation and function, Journal of Leukocyte Biology Vol. 90, Issue 5, November 2011, p845-854;
SOCS3 Attenuates GM-CSF/IFN-y-Mediated Inflammation During Spontaneous Spinal Cord Regeneration, Neuroscience Bulletin 2020 Jul; 36(7): 778-792;

T cells
Decreased T cell populations contribute to the increased severity of COVID-19 (CD3+, CD4+ and CD8+ lymphocytes decreased in severe COVID), Clinica Chimica Acta Vol. 508, September 2020, P110-114;
T-cell Hyperactivation and Paralysis in Severe COVID-19 Infection Revealed by Single-Cell Analysis, Frontiers Immunological Tolerance and Regulation, 08 October 2020;
Tumor-derived soluble MIC ligands impair expression of NKG2D and T-cell activation, Nature 419, 734-738(2002), 17 October 2002;
COVID-19 and the Path to Immunity (boosting CD8⁺ and T_H1 CD4⁺ T cells), JAMA 2020;324(13):1279-1281;
Imbalance of Regulatory and Cytotoxic SARS-CoV-2-Reactive CD4+ T Cells in COVID-19, Cell 5 October 2020;

Th cells and Treg cells
Th17 and Treg cells function in SARS-CoV-2 patients compared with healthy controls, Journal of Cellular Physiology, 14 September 2020;
Heightened circulating Interferon-Inducible Chemokines and activated Pro-Cytolytic Th1-cell phenotype features COVID-19 aggravation in the second week, Frontiers Cytokines and Soluble Mediators in Immunity, 20 October 2020;

Therapeutic targets
Treatment of COVID-19 With Conestat Alfa, a Regulator of the Complement, Contact Activation and Kallikrein-Kinin System, Frontiers Molecular Innate Immunity, 14 August 2020;
Nutraceuticals have potential for boosting the type I Interferon (IFN) response to RNA viruses including influenza and coronavirus (downregulation of NOX2 to enhance Toll-like Receptor 7 (TLR7) activity), Progress in Cardiovascular Diseases 2020 May-June; 63(3): 383-385;
Cathepsin L-selective inhibitors: A potentially promising treatment for COVID-19 patients, Pharmacology & Therapeutics 2020 Sep; 213: 107587;
Caveolin-1: a critical regulator of lung injury, Lung Cellular and Molecular Physiology 2011 Feb; 300(2);
MMP8 Inactivates Macrophage Inflammatory Protein-1alpha to Reduce ALI in mice, Journal of Immunology 2010 Feb 1; 184(3);
Protective Effect of Epigallocatechin-3-Gallate (EGCG) in Diseases with Uncontrolled Immune Activation: Could Such a Scenario Be Helpful to Counteract COVID-19?, International Journal of Molecular Science 2020 Jul;21(14):5171;
Inhibition of metalloproteinases in therapy for severe lung injury due to COVID-19, Medicine in Drug Discovery 2020 Sep; 7: 100052;
IL-1 induces thromboxane-A2 (TxA2) in COVID-19 causing inflammation and micro-thrombi: inhibitory effect of the IL-1-Receptor antagonist (IL-1Ra), Journal of Biological Regulators and Homeostatic Agents 2020 Aug 3;34(5);
New putative insights into neprisylin (NEP)-dependent pharmacotherapeutic role of roflumimast in treating COVID-19, Vol. 889, 15 December 2020;
Can endolysosomal deacidification and inhibition of autophagy prevent severe COVID-19?, Life Sciences Vol. 262, 1 December 2020;

Neuroinvasie van het Centraal Zenuwstelsel door SARS-CoV-2: een bespreking van klinische casus en neuroinvasieve mechanismen van SARS-CoV-2

In mijn bericht van 7 maart 2020 besprak ik het neuro-invasieve potentieel van SARS-CoV-1 en andere coronavirussen, een onderwerp dat sinds 2003 flink bestudeerd wordt (zie "Verspreiding en complicaties van coronavirussen", 7 maart 2020 ). Het neuro-invasieve potentieel van SARS-CoV-2 wordt echter sinds het begin van deze pandemie onderschat. Redelijkerwijs wordt de vraag gesteld of neurologische manifestaties het gevolg zijn van directe neuro-invasie door SARS-CoV-2, of een gevolg van door SARS-CoV-2 geïnduceerde trombo-inflammatoire verschijnselen.

Vanwege een gebrek aan autopsiemonsters kan virale replicatie van SARS-CoV-2 in het centrale zenuwstelsel (CZ) in veel gevallen niet worden bevestigd. Dat is geen reden om het bewustzijn over het risico op invasie van het CZ en perifere zenuwstelsel door SARS-CoV-2 te laten varen. Afgezien van directe neuro-invasie van het CZ, moeten neurologische manifestaties die worden opgewekt door ontstekingen en stollingsstoornissen (trombose en embolie) nauwlettend worden gemonitord. 

In deze bespreking zal ik ingaan op het neuro-invasieve potentieel van coronavirussen en neurologische manifestaties en mechanismen achter CoV-CZ-infectie. Waarom is het belangrijk om onderscheid te maken tussen neurologische manifestaties als gevolg van directe invasie van het CZ of manifestaties van het CZ die worden veroorzaakt door een ontsteking in de periferie (buiten het CZ)? Het belang is erin gelegen om de juiste therapeutische opties en timing van medische interventie te kunnen bepalen. Ik heb een selectie van casus gemaakt door via de database PubMed en de Wiley Library op "SARS-CoV-2" in combinatie met "Encephalopathy", "Encephalitis", "CNS", "Neuroinvasion", "ADEM", "ANE" en "Encephalomyelitis" te zoeken. Ik beschouw alles in de context: als neurologische klachten door onderliggende medische factoren kunnen worden verklaard, dan vermeld ik dat in de bespreking van de casus. Daarbij staat voorop dat infecties van het zenuwstelsel met grote zekerheid kunnen worden aangetoond, door het hersenruggenmergvocht (CSF) op pleiocytose, eiwitten en de aanwezigheid van SARS-CoV-2 te testen.

1. Over het neuro-invasieve potentieel van coronavirussen (2016-2020)
Net als ademhalingsvirussen in het algemeen kunnen SARS-CoV's schade aan het centrale zenuwstelsel veroorzaken. Coronavirussen kunnen encefalitis veroorzaken. De snelle replicatie van SARS-CoV-2 is een risicofactor voor het initiëren van overreactie van het menselijke immuunsysteem; als gevolg hiervan kan onder meer virale meningitis optreden (Human Coronaviruses and other respiratory viruses: Underestimated opportunistic pathogens of the Central Nervous System?, Viruses 2020, 12 (1), 14, gepubliceerd op 20 december 2019). In een jonge patiëntengroep met CoV werd een lichte ophoping van interleukinen IL-6, IL-8 en MCP-1 in de hersenvloeistof waargenomen. MCP-1 is betrokken bij het initiëren van ontstekingsreacties in de hersenen (Coronavirus infections in the Central Nervous System and respiratory tract show distinct features in hospitalized children, Intervirology 2016, vol. 59, no. 3, gepubliceerd in februari 2017).

In deze studie uit 2016 werd onderscheid gemaakt tussen infectie met het coronavirus en het centraal zenuwstelsel (CoV-CNS) en een infectie met de luchtwegen met coronavirussen (CoV-respiratoir). Het serumniveau van Granulocyte macrophage colony-stimulating factor (GM-CSF) was hoger bij CoV-CNS geïnfecteerde patiënten. GM-CSF heeft pro-inflammatoire eigenschappen en speelt een rol bij auto-immuun-encefalopathie (hersenontsteking door een auto-immuunreactie). De niveaus van interleukinen IL-6, IL-8, MCP-1 en GM-CSF waren significant verhoogd in monsters van hersenvocht van de CoV-CNS-patiëntengroep. Het aantal lymfocyten en eosinofielen was verlaagd in de CoV-CNS-groep, terwijl CoV-luchtwegpatiënten een significant lager aantal neutrofielen hadden. Het aantal monocyten in CoV-CNS-patiënten was verhoogd. Het serumgehalte van de granulocytkolonie-stimulerende factor (G-CSF) was significant verhoogd bij zowel CoV-CNS-patiënten als CoV-respiratoire patiënten.

Autopsies uitgevoerd tijdens de SARS-CoV-1-epidemie toonden viraal RNA in het cytoplasma van hypothalamische en cerebrale corticale neuronen, evenals hersenoedeem en focale neuronale degeneratie en neuronale necrose, uitgebreide hyperplasie van de gliacellen en cellulaire infiltraten. Dit betekent dat coronavirussen in het centrale zenuwstelsel binnendringen. Er werd in de autopsierapporten van de SARS-CoV-1-groep geen demyelinisatie, ofwel verlies van het isolerende merg om zenuwvezels, vermeld. Expressie van Interferon-gamma was te zien in glia. Infiltratie van CD68 + macrofagen en CD3 + T-cellen werd waargenomen in hersenneuronen en glia, wat duidt op het binnendringen van immuuncellen in de zenuwcellen (The neurology of COVID-19 revisited: A proposal from the Environmental Neurology Specialty Group of the World Federation of Neurology to implement international neurological registries, Journal of Neurological Sciences 414 (2020) 116884). 

In een test met ACE2-transgene muismodellen werd het falen van de ademhaling toegeschreven aan medullaire infectie (infectie van de hersenstam) met SARS-CoV. De reukzenuw bleek de ingang voor virale infectie van de hersenen te zijn. Een rapport over COVID-19-encefalitis onthult hemorragische laesies in de thalami (bloedingen), mediale temporale kwabben en de insula ("eilandjes", betrokken bij walging als gradatie van smaak). De mogelijkheid van door virussen veroorzaakte vasculitis (vaatontstekingen) via ACE2-receptoren in de hersenen, verhoogt het risico op cerebrovasculaire aandoeningen (COVID-19 and the nervous system, Journal of Neurovirology, 2020 May 23).

2. Neurologische manifestaties van virale infectie
Er zijn ten minste drie categorieën neurologische manifestaties die verband houden met virale infectie. Encefalopathie, inflammatoire encefalitis, hemorragische en ischemische beroerte zijn complicaties van systemische ziekten, zoals multi-orgaanfalen, ontsteking en coagulatie. Directe virale invasie van het centrale zenuwstelsel resulteert in encefalitis, meningitis en endotheliitis geassocieerd met celnecrose en hersendysfunctie. Acute gedissemineerde encefalomyelitis (ADEM), acute necrotiserende encefalopathie (ANE), het Guillain-Barré-syndroom en Kawasaki-achtige manifestaties zijn immuun-gemedieerde post-infectieuze manifestaties, dat wil zeggen dat zij pas ná virale infectie optreden en worden veroorzaakt door een reactie van het immuunsysteem. 

2.1 Neurologische manifestaties bij SARS-CoV-2-patiënten
In een casestudy onder 214 patiënten met SARS-CoV-2 werden neurologische symptomen gezien bij 36,4% van de patiënten. De gemiddelde leeftijd van de patiënten was 52 jaar. Van alle gemelde patiënten met manifestaties van het zenuwstelsel had 24,8% manifestaties van het centrale zenuwstelsel (hierna: CZ-manifestaties), 8,9% had perifere zenuwstelsel-manifestaties en 10,7% had skeletspierletsel. Typische symptomen van het perifere zenuwstelsel zijn anosmie (verlies van reuk) en ageusie (verlies van smaak). Met name 58,9% van de patiënten met CZ-manifestaties had een niet-ernstige infectie. Neurologische manifestaties traden op binnen 1-2 dagen na de infectie. 2 patiënten werden in het ziekenhuis opgenomen met door SARS-CoV-2 geïnduceerde hemiparese (halfzijdige verlamming), van wie geen koorts, hoest of diarree had. Van CZ-manifestaties werden hoofdpijn, verminderd bewustzijn, acute cerebrovasculaire ziekte, coma, delirium, ischemische beroerte en hersenbloeding gemeld.

2.1.1 Encefalopathie
In een onderzoek onder 58 patiënten had 69% agitatie, 67% had diffusieve tekenen van het corticospinale kanaal en 33% had het dysexecutief syndroom bestaande uit aandachtsproblemen, desoriëntatie en slecht georganiseerde responsbewegingen. Bij 8 patiënten werd verbetering in leptomeningeale ruimtes opgemerkt; bij 11 patiënten werd bilaterale frontotemporale hypoperfusie opgemerkt. 1 van de 8 patiënten had diffuse bifrontale vertraging, consistent met encefalopathie; bij 2 patiënten waren oligoklonale banden ("patronen" van antilichamen) aanwezig, eiwit- en immunoglobuline G-spiegels (antistoffen) waren verhoogd bij 1 patiënt. RT-PCR-reeksen van het hersenruggenmergvocht waren negatief voor SARS-CoV-2. Pleiocytose (toename van het aantal cellen) werd in geen van de gevallen waargenomen. Deze studie concludeert dat encefalopathie wordt geassocieerd met ARDS als gevolg van SARS-CoV-2-infectie, met ergernis en verwarring als kenmerkende klachten. Er kunnen geen conclusies worden getrokken met betrekking tot een ontstekingsreactie als oorzaak van de encefalopathie, aangezien er geen cytokinetests zijn uitgevoerd (Neurologic Features in Severe SARS-CoV-2 Infection, NEJM, 4 June 2020).

2.1.2 MRI-bevindingen bij afwezigheid van specifieke hersenruggenmergvocht-bevindingen: niet-specifieke encefalitis
Een studie waarin 50 COVID-patiënten werden onderzocht, meldt dat de meest voorkomende bevinding corticale signaalafwijkingen bij Fluid-Attenuated Inversion Recovery (FLAIR) waren. Bij 3 patiënten waren subcorticale (onder de hersenschors) en signaalstoornissen van de witte stof aanwezig; afwijkingen bij de frontale kwab waren aanwezig bij 4 patiënten; pariëtale lob in 3; achterhoofdskwab in 4; temporaalkwab in 1; insulaire schors bij 3 en cingulate gyrus bij 3 patiënten. Het hersenruggenmergvocht van 5 patiënten toonde een totaalbeeld van verhoogde eiwitwaarden bij 4 patiënten. Het aantal cellen, albumine, glucosespiegels en immunoglobuline G-index lagen binnen het normale bereik, er werden geen oligoklonale banden waargenomen. Acute intracraniële bevindingen bij afwezigheid van afwijkingen in het corticale signaal waren te zien bij 1 patiënt met acute transversale sinus trombose en 1 patiënt met acuut infarct in het rechter middengebied van de hersenslagader. 

Van de 2 gevallen zonder intracraniële bevindingen werd een verhoogd eiwit gevonden in het hersenruggenmergvocht. De belangrijkste differentiële (niet-specifieke) diagnose van deze bevindingen is infectieuze of auto-immuunencefalitis met toevallen, hypoxie en hypoglykemie. De gevallen van bilaterale frontale afwijkingen worden geassocieerd met hypoxie, omdat ARDS en frontotemporale perfusie gerelateerde factoren zijn. Microbloedingen in de hersenschors en afbraak van de bloed-hersenbarrière vergezeld van hypoxie zouden leiden tot het waargenomen beeldvormingspatroon. Over het algemeen kunnen er geen conclusies worden getrokken met betrekking tot virale of immuniteitsgemedieerde encefalitis (Brain MRI Findings in Patients in the ICU with COVID-19 Infection, RSNA Radiology, 8 May 2020).

2.1.3 Ongebruikelijke microbloedingen
MR-beelden van 9 patiënten met ongebruikelijke verschijnselen werden geanalyseerd. Alle patiënten leden aan ernstige hypoxemie secundair aan ARDS, met een vertraagd herstel van het bewustzijn. Geen van de patiënten had diffuse intravasculaire coagulatie (DIC); alle patiënten vertoonden een hyperfibrinemische toestand evenals septische of inflammatoire coagulatie. Microbloedingen werden waargenomen, specifiek bij het corpus callosum (de hersenbalk). Andere locaties van microbloedingen waren de achterste of voorste delen van de interne capsule en de hersensteel. Bij de meerderheid van de patiënten werden subcorticale regio's aangetast. Deze bevindingen worden níet geassocieerd met hypertensie en amyloïde angiopathie (beschadiging van de kleine bloedvaten van de hersenen). De betrokkenheid van de hersenbalk is niet consistent met hypertensie en amyloïde angiopathie; korte episodes van hypertensie kwamen voor bij slechts 2 van de 9 patiënten, die reeds met succes waren behandeld met antihypertensiva. 

Er wordt verondersteld dat directe schade aan het endotheel weefsel en de bloedvaten verklarend zou kunnen zijn, aangezien directe interactie tussen SARS-CoV-2 en de ACE2-receptor zou kunnen leiden tot beschadiging van de bloed-hersenbarrière en bloeding in het hersenweefsel. Van sommige van de op MR waargenomen laesies werd gemeld dat ze een lineaire vorm hadden die leek op een bloedvat, overeenkomend met het beeld dat wordt waargenomen bij hypercoagulatiestoornissen en microthrombi in longvaten van andere patiënten. Diffuus axonaal letsel (schade aan zenuwvezels) of lipidenembolie (afsluiting van een bloedvat door vet) onderzocht als mogelijke verklaringen (Unusual Microbleeds in Brain MRI of COVID-19 Patients, Journal of Neuroimaging, 8 July 2020).

2.1.4 Systemische hyperinflammatoire dysfunctie van het centrale zenuwstelsel: een mogelijke rol voor anakinra en tocilizumab
1 patiënt zonder comorbiditeiten kreeg linkszijdige verlammingen. Hoofdpijn en een verlamming ontwikkelden zich binnen 12 uur tot verwarring en opwinding. Het hersenruggenmergvocht vertoonde lymfatische pleiocytose (abnormale toename van immuuncellen) en verhoogde eiwitten. PCR van SARS-CoV-2 in het hersenvocht was negatief, maar het monster uit de keelneusholte bevestigde een SARS-CoV-2-infectie. Een tweede patiënt werd opgenomen met onrust, moeite bij het benoemen van objecten, desoriëntatie en verwarring. Het hersenvocht vertoonde ook bij deze patiënt lymfatische pleiocytose en verhoogde eiwitten. Bij beide patiënten was IL-6 verhoogd, bij één patiënt was IL-1β significant verhoogd. Bovendien vertoonde het hersenruggenmergvocht bij beide patiënten verhoogde ACE-spiegels. Er wordt verondersteld dat deze gevallen van encefalitis worden veroorzaakt door een cytokinestorm. Behandelingen gericht op IL-6 (tocilizumab) en IL-1 (anakinra) kunnen nuttig zijn om CZ-dysfunctie te behandelen (Increased CSF levels of IL-1β, IL-6 and ACE in SARS-CoV-2-associated encephalitis, Neuroimmunology & Neuroinflammation, 1 July 2020).

2.1.5 Leuko-encefalopathie en microbloedingen

11 patiënten kwamen in aanmerking voor hersen-MRI vanwege een ongebruikelijke, aanhoudend depressieve mentale toestand. Hersenstamreflexen waren normaal, maar de respons in extremiteiten nam af. Geen van de patiënten had duidelijke gedissemineerde intravasculaire coagulatie. Hersenruggenmergvocht was beschikbaar bij slechts 1 patiënt en was negatief voor meningitis en encefalitis en negatief voor SARS-CoV-2. Van 11 patiënten vertoonden 4 patiënten diffuse leuko-encefalopathie, 1 met microbloedingen en 6 met zowel microbloedingen als leuko-encefalopathie. Patiënten met leuko-encefalopathie hadden symmetrische hyperintensiteiten in beide zijdelingse diepe en subcorticale witte stof. Er werden afwijkingen waargenomen die zich uitstrekten van de precentrale gyrus tot aan het centrum semiovale en corona radiata. Bij de temporale en occipitale hoorns was de posterieure cerebrale witte stof bij alle patiënten afwijkend. De grijze kernen waren gespaard gebleven van leuko-encefalopathie. Afgezien van de pre-centrale gyrus en achterhoofdskwabben, bleef ook de juxtacorticale witte stof gespaard van leuko-encefalopathie. 

Deze bevindingen komen overeen met Delayed Posthypopoxic Leukoencephalopathy (DPHL), die 10-14 dagen na hypoxie optreedt en vergelijkbaar is met het ziektebeeld van slachtoffers van koolmonoxidevergiftiging. Acute hypoxische ischemie is uitgesloten, omdat de bij dat ziektebeeld aangedane hersengebieden in dit geval juist onaangedaan zijn. DPHL is waarschijnlijk gerelateerd aan celdood en daaropvolgende demyelinisatie. Waargenomen milde beperkte diffusie in dit onderzoek kan verband houden met acute demyelinisatie. Mogelijke etiologieën voor de waargenomen leuko-encefalopathie zijn: sepsis-geassocieerde, post-infectieuze demyelinisatie of hemorragische encefalitis en posterieur reversibel encefalopathiesyndroom.

In de juxtacorticale witte stof en het corpus callosum (de hersenbalk), met name het splenium, werden puntbloedingen waargenomen. Soortgelijke bloedingen zijn gemeld in gevallen van hoogteziekte en verwonding van de bloed-hersenbarrière. De waargenomen microbloedingen doen denken aan axonaal letsel. Er worden twee verschillende aandoeningen gediagnosticeerd: demyelinisatie en verstoring van de bloed-hersenbarrière. Beide aandoeningen zijn gerelateerd aan hypoxie veroorzaakt door SARS-CoV-2 (COVID-19-associated Diffuse Leukoencephalopathy and Microhemorrhages, RSNA Radiology, 21 May 2020).

2.1.6 Acute necrotiserende encefalopathie (ANE)
Een eerste studie naar vermoedelijke COVID-19-geassocieerde acute necrotiserende encefalopathie (ANE) gaat over een geval waarbij het hersenvocht niet op SARS-CoV-2 kon worden getest. CT-beelden onthullen symmetrische hypoatuatie binnen de bilaterale mediale thalami; hersen-MRI-beeldvorming onthult hemorragische laesies in de bilaterale thalami, mediale temporale lobben (ter hoogte van de slapen) en subinsulaire regio's. ANE is een zeldzame complicatie van influenza en andere virale infecties, die wordt toegeschreven aan cytokinestormen die leiden tot schade aan de bloed-hersenbarrière, zonder dat sprake is van directe virale invasie van de hersenen of postvirale demyelinisatie (COVID-19-associated Acute Hemorrhagic Necrotizing Encephalopathy: CT and MRI Features, RSNA Public Health Emergency Collection, Radiology 2020 Mar 31: 201187).  .

Een patiënt had een voorgeschiedenis van aplastische anemie met intermitterende transmissie van rode bloedcellen en bloedplaatjes. De patiënt had recent geen middelen gekregen om het immuunsysteem te onderdrukken. De patiënt vertoonde episodes van staren, spraakstilstand, flexie van beide schouders en een korte tonische clonische aanval. Een CT toonde een vroege zwelling van de hersenstam. Nieuwe lymfopenie werd waargenomen. Trombocytopenie in overeenstemming met aplastische anemie werd behandeld met humane leukocyten-antigeen-bloedplaatjes. De tweede CT-scan toonde zwelling van de hersenstam, nieuwe corticale en subcorticale hypodensiteit in de occipitale kwab en progressie van zwelling van de hersenstam met pontinebloeding en afwijkingen in de grijze stof, bestaande uit symmetrische hypodensiteiten, die ook in beide amygdalae te zijn waren. 

Het hersenruggenmergvocht vertoonde een eiwitverhoging, maar een normaal aantal witte bloedcellen. CSF-PCR voor SARS-CoV-2 was negatief. MRI vertoonde progressie van een uitgebreid abnormaal signaal en bloeding in een symmetrische verdeling binnen de dorsolaterale putamina (gelegen in de basale ganglia), thalamische kernen, subinsulaire regio's, splenium van het corpus callosum, cingulate gyri en perirolandische regio's. Er was een vermindering van ventriculi, basale stortbakken, temporale niet-hernia en matige celebellaire hernia. Ernstige trombocytopenie droeg bij tot de hemorragische componenten van encefalopathie. De patiënt was niet lymfopenisch vóór SARS-CoV-2-infectie. Testen op interleukines was niet mogelijk. Directe invasie (neurotroop) van het zenuwstelsel door SARS-CoV-2 is in dit geval onwaarschijnlijk, omdat de CSF-PCR negatief was. Een immunostorm is een zeer plausibele verklaring voor dit medische beeld van ANE (COVID-19-related acute necrotizing encephalopathy with brain stem involvement in a patient with aplastic anemia, Neurology Neuroimmunology & Neuroinflammation, 26 May 2020). 

2.1.7 Acute necrotiserende hersenontsteking (ANE), succesvol behandeld met steroïden
Een geval van acute necrotiserende encefalopathie bij een 51-jarige COVID-19-patiënt zonder een voorgeschiedenis van neurologische aandoeningen wordt mogelijk teweeggebracht door antilichamen. De patiënt reageerde niet, werd comateus, kreunde en vertoonde ritmische bewegingen van het rechterbovenbeen op dag 21 van de ziekte. Subtiele hyperintensiteiten bij bilaterale thalami werden waargenomen. Bloed en hersenvocht onthulden trombopenie, lymfopenie en ontsteking, CSF-albumine-cytologische dissociatie met verhoogde IgG-antilichamen en veranderde bloed-hersenbarriere. MRI vertoonde progressieve laesies met diffuse hyperintensiteit in de thalami, cerebellum, hersenstam en supratentoriale grijze en witte stof. Gebieden van het vezelnetwerk, als ventrale hippocampus commissuur, brachium van de colliculus en stria medullaris leken sterk gekleurd. Bilaterale distributie van afwijkingen aan de hersenstam, thalami, kleine hersenen en witte stof, hyperintensiteiten en schade aan bilaterale thalami is duidelijk te onderscheiden in ANE. Dit is anders bij klinische beelden van ADEM, die worden gedefinieerd door asymmetrische laesies met slecht afgetekende marges, voornamelijk te zien in periventriculaire gebieden en basale ganglia. 

Vroege behandeling met steroïden en polyvalente immunoglobuline was in deze casus zeer effectief. De vermoedelijke oorzaak is de reactie van de gastheer op SARS-CoV-2, geassocieerd met IgG gericht op een neuronaal antigeen. Celvernietiging en de afgifte van grote hoeveelheden auto-antigenen stimuleren zelf-reactieve cellen en kunnen leiden tot zelf-reactieve antilichamen. Ontsteking draagt ​​bij aan de productie van IgG en aantasting van de bloed-hersenbarrière (COVID-19-associated Acute Necrotizing Encephalopathy successfully treated with steroids and polyvalent immunoglobulin with unusual IgG targeting the cerebral fibre network, Postscript Letter, Journal of Neurology, Neurosurgery & Psychiatry, 10 July 2020).

2.1.8 Meningitis / encefalitis geassocieerd met SARS-CoV-2-infectie van het centrale zenuwstelsel
Een rapport uit februari 2020 beschrijft een eerste geval van een 24-jarige bewusteloze patiënt met vermoedelijke meningitis / encefalitis geassocieerd met SARS-CoV-2-infectie. Nekstijfheid en toevallen werden waargenomen. MRI vertoonde abnormale bevindingen van mediale temporale kwab, inclusief hippocampus, wat wijst op encefalitis. Daarnaast werd significante paranasale sinusitis waargenomen, een bevinding die het verdient aandacht te schenken aan retrograde synaptische transmissie van SARS-CoV-2 (transmissie van de neus naar de hersenen, zonder het ademhalingsstelsel). Hoewel SARS-CoV-2 niet werd gedetecteerd in het nasofaryngeale monster, was het hersenvocht positief voor SARS-CoV-2. Het aantal lymfocyten in het hersenruggenmergvocht was licht verhoogd (pleiocytose). Bloedlymfocyten waren verlaagd, het aantal witte bloedcellen was toegenomen, neutrofielen waren dominant en C-reactief proteïne was verhoogd. FLAIR-beeldvorming wees op rechter laterale ventriculitis en encefalitis op de rechter mesiale kwab en hippocampus (A first case of meningitis/encephalitis associated with SARS-CoV-2, International Journal of Infectious Diseases, Vol. 94, May 2020). In het geval van een 41-jarige patiënte met meningoencefalitis is géén RT-PCR van het ruggenmergvocht afgenomen, maar toediening van Hydroxychloroquine (HCQ) heeft opmerkelijke verbetering gebracht (Meningoencephalitis without respiratory failure in a young female with COVID-19 infection in Downtown LA, early April 2020, Brain, Behavior and Immunity, 2020 Jul; 87: 33).

2.1.9 ADEM, acute encefalomyelitis, verbeterd door methylprednisolon
Acute encefalomyelitis (ADEM) is een auto-immuunziekte van het zenuwstelsel, een zeldzame ziekte die voorkomt na virale infecties en die vooral kinderen treft. Er worden echter gevallen van ADEM gemeld die volwassenen treffen na een SARS-CoV-2-infectie. In één geval waarbij een 64-jarige betrokken was, meldde de patiënt een verlies van geur en smaak, dat zich snel ontwikkelde naar totale anosmie en ageusie, gevolgd door bilaterale slechtziendheid en gevoelsverlies aan het rechterbeen. Relatief afferent pupil defect werd gedetecteerd en gezichtsveldtesten vertoonden aan weerszijden defecten. Verder werden prikkelbaarheid, hoofdpijn, aangedaan sensorisch niveau van de rechter buik en linker hyperreflexie van de onderste ledematen gedetecteerd. MRI van de hersenen en de wervelkolom weergaf hersenletsel en één laesie op de achtste thoracale wervel (T8) en bilaterale oogzenuwversterking. Het hersenvocht vertoonde lymfotische pleiocytose, voornamelijk bestaand uit CD3 + CD4 + -T-cellen en milde proteïnorrachie (verhoogde eiwitten in CSF). Identieke immunoglobuline G oligoklonale banden waren aanwezig in CSF en serum. SARS-CoV-2 werd gedetecteerd in het hersenvocht. Serum onthulde anti-SARS-CoV-2 IgG. Het serum was negatief voor antiaquaporine-4 (AQ4) -antilichaam en antimyeline-oligodendrocytglycoproteïne (MOG), waardoor een neuromyelitis optica-spectrumsyndroom minder waarschijnlijk is. Behandeling met methylprednisolon verbeterde geleidelijk de symptomen geassocieerd met ADEM  (Acute disseminated encephalomyelitis after SARS-CoV-2 infection, Neuroimmunology & Neuroinflammation, 1 June 2020).

Een 54-jarige kreeg een episode van bewustzijnsverlies en langdurige reuk- en smaakstoornissen. RT-PCR voor SARS-CoV-2 was positief. Röntgen toonde interstitiële longontsteking. Bloedonderzoek toonde matige lymfocytopenie en lichte verhoging van inflammatoire indices (witte bloedcellen, C-reactief proteïne, fibrinogeen). Bij plotselinge verslechtering werd een bloedgastest uitgevoerd om ernstige hypoxie aan het licht te brengen. Een EEG toonde aan dat er twee aanvallen waren gestart vanuit het rechter frontotemporale gebied, diffuus in homologe contralaterale hemisfeer. Bij MRI werden meerdere hyperintense laesies van de periventriculaire witte stof waargenomen, zonder beperking of diffusie of verbetering. Soortgelijke laesies werden gevonden in de bulbo-medullaire overgang, het cervicale merg en het ruggenmerg. CSF RT-PCR voor SARS-CoV-2 keerde negatief terug. In dit geval wordt aangenomen dat de demyelinisatie wordt veroorzaakt door een vertraagde immuunrespons na de viremie. 

Systemische ontstekingsreactiesyndromen (SIRS) of SIRS-achtige syndromen bij SARS-CoV-2-patiënten kunnen optreden als gevolg van een cytokinestorm, waarbij IL-1, IL-6 en tumornecrosefactor-alfa (TNF-α) verantwoordelijk kunnen zijn voor overactivering van gliacellen, met daaropvolgende demyelinisatie. Er wordt verondersteld dat longontsteking met hypoxie leidt tot een verhoogd anaëroob metabolisme dat neurologische schade veroorzaakt. Het virus kan als alternatief de aanmaak van antilichamen tegen gliacellen veroorzaken, na infectie, die wordt geassocieerd met Guillain-Barré. Toediening van Dexamethason maakte een progressief herstel van de longfunctiestoornis mogelijk (SARS-CoV-2 can induce brain and spine demyelating lesions, Acta Neurochirurgica 162, 1491-1494 (2020), 4 May 2020).

Een niet-typische ADEM werd gezien bij een 71-jarige man, van wie de autopsie milde zwelling van de hersenen en hemorragische laesies verspreid door de witte stof van de hersenhelft aan het licht bracht. Een dag voordat de bevestiging van SARS-CoV-2 binnenkwam, ontwikkelde de patiënt acuut nierletsel, waarschijnlijk secundair aan shocktoestand en ademhalingsfalen in combinatie met een reactie op contrasttoediening. Niveaus van C-reactief proteïne, ferritine en IL-6 waren significant verhoogd. De hersenletsels bestonden uit brandpunten van intraparenchymaal bloed die de witte stof verstoorden, met macrofagen aan de rand van de laesies. Gegeneraliseerde reactieve gliosis was wijdverbreid in de witte stof. Beschadigde axonen markeerden de randen van de bloedingen. Er werd een verlies van myeline vastgesteld, met macrofagen en gefragmenteerde axonale processen binnen de laesies en oligodendrocyt-apoptose rond de laesies. 

Nabij hemorragische laesies was het weefsel relatief bespaard gebleven. Subtiele gebieden van subcorticale witte stof werden gezien met een variabele perivenulaire verdeling. Perivasculaire infiltraten bleken macrofagen te zijn en bijbehorend myeline-verlies was zichtbaar. De laesies vertoonden een bereik van axonaal letsel, tot matig axonaal letsel. Extra laesies in de witte stof vertoonden destructieve laesies met centraal fibrine geassocieerd met geforceerde rode bloedcellen, wat het verlies van myeline rond vaten uitstraalde. Axonaal letsel was gemarkeerd bij deze microscopisch geïdentificeerde laesies. Microscopische corticale infarcten werden geïdentificeerd met astrogliose rond de infarcten. Necrotiseerde neuronen waren verspreid in de neocortex, CA1 hippocampale regio en Purkinje-cellen in het cerebellum, wat wijst op hypoxisch-ischemisch letsel. Er werden weinig perivasculaire T-cellen gezien, maar er werd geen activering van B-cellen waargenomen. Immunokleuring van de hersenstam benadrukte perivasculaire macrofagen geassocieerd met aderverkalking. De laesies van de witte stof zijn hypothetisch vasculair. Microscopische neocorticale infarcten worden waarschijnlijk geassocieerd met microtrombo-embolie als gevolg van SARS-CoV-2-infectie, met een rol voor de ACE2-receptor als een mechanisme voor het letsel (Neuropathology of COVID-19: a spectrum of vascular and acute disseminated encephalomyelitis (ADEM)-like pathology, Acta Neuropathologica (2020): 140:1-6).

2.2 Markers voor ernstige infectie
Patiënten met ernstige infectie hadden een hoger aantal witte bloedcellen, een neutrofielentelling, een lager aantal bloedplaatjes, een lager aantal lymfocyten, een verhoogd C-reactief proteïne-gehalte, een hoog D-dimeer-gehalte, MOF, een verhoogd lactaatdehydrogenase-, alanine-aminotransferase- en aspartaat-aminotransferase-gehalte, verhoogd ureumstikstof, verhoogde creatininespiegels en verhoogde creatininekinasespiegels. Patiënten met skeletspierletsel hadden een verlaagd aantal lymfocyten en ernstig leverletsel en nierletsel (Neurologic Manifestions of Hospitalized Patients with Coronavirus Disease 2019 in Wuhan, China, JAMA Neurology, April 10, 2020). Neurotrope infectie van het centrale zenuwstelsel (directe virusinvasie van het zenuwstelsel) moet worden bepaald met hersenruggenmergvocht-PCR. Postinfectieuze immuun-gemedieerde neurologische aandoeningen (zoals ADEM en Guillain-Barré) kunnen worden onderscheiden van encefalitis door de afwezigheid van hersenvocht-pleiocytose en verhoogd hersenvocht-eiwit.

3. Mechanismen

3.1 Penetratie of vermijding van de Blood Brain Barrier en Blood Nerve Barrier
De ACE2-receptor (angiotensine-converterend enzym 2-receptor) is de gastheerreceptor voor SARS-CoV-2. De binding van SARS-CoV-2 omvat S-glycoproteïne-splitsing door Furin en priming door transmembraanprotease serine 2, TMPRSS2, gelokaliseerd in het celmembraan. ACE2 heeft ten minste twee verschillende functies. In de RAS, het renine-angiotensine-aldosteronsysteem dat de bloeddruk reguleert, klieft het angiotensine I tot ang 1-9 en angiotensine II tot ang 1-7, dat functioneert als een vaatverwijdend peptide. ACE2 reguleert ook de opname van aminonzuren in het endotheel van de darm. ACE2 wordt aangetroffen in het subfornische orgaan (SFO) in de hersenen, een gebied dat het cardiovasculaire systeem reguleert door middel van vochtbalans en hormoonsecretie. Het subfornische orgaan mist de bescherming van de bloed-hersenbarriere, dat maakt het orgaan gevoelig voor invloed van circulerende peptiden, de bouwstenen van eiwitten.

De bloed-hersenbarriere ​​beschermt het centrale zenuwstelsel (hersenen en ruggenmerg) tegen directe interactie met de bloedsomloop. De laag bestaat uit endotheelcellen, een basaalmembraan, astrocyten en gladde cellen (Vascular Smooth Muscle Cells, VSMC). Twee belangrijke immuuncellen die interactie hebben met het zenuwstelsel, zijn perivasculaire macrofagen en microgliacellen. Door bloed overgedragen voorlopers steken de BBB over (The Blood-Brain Barrier, Cold Spring Harbor Perspectives in Biology, 2015 Jan; 7(1)). Het aanvallen van immuuncellen zorgt voor immunologische ontstekingsmechanismen die neurologische manifestaties veroorzaken. Virale replicatie en de afgifte van cytokinen verhogen de doorlaatbaarheid van de BBB (Potential of SARS-CoV-2 to cause CNS infection: Biologic Fundamental and Clinical Experience, Frontiers in Neurology, 18 June 2020).

SARS-CoV's zijn voornamelijk betrokken bij endotheliitis. Dit wordt in verband gebracht met het vermogen van SARS-CoV om het centrale zenuwstelsel rechtstreeks te infecteren door de bloed-hersenbarriere te ontregelen door infectie van het endotheelweefsel van de vaten. De belangrijkste route is de ACE2-receptor die tot expressie wordt gebracht op endotheelcellen. Het vermijden van de bloed-hersenbarriere of de Blood Nerve Barrier (BNB) om toegang te krijgen tot het centrale zenuwstelsel is ook mogelijk, wanneer een virus de dorsale wortelganglia en autonome ganglia binnendringt (The neurology of COVID-19 revisited: A proposal from the Environmental Neurology Specialty Group of the World Federation of Neurology to implement international neurological registries, Journal of Neurological Sciences 414 (2020) 116884).

3.2 Verspreiding via het neurale reuknetwerk
Er wordt verondersteld dat de invasie van het zenuwstelsel door SARS-CoV-2 de ademhalingsfunctie beïnvloedt door beschadiging van het medullaire cardiorespiratoire centrum (gelegen in de hersenstam). In knaagdiermodellen bleek SARS-CoV zich via de reukweg te verspreiden naar subcorticale en corticale hersengebieden. De door SARS-CoV geïnfecteerde hersenstam werpt licht op de mogelijkheid dat de aanvoerende hersenzenuwen bij de invasie betrokken zijn. Aangezien er geen SARS-CoV is gedetecteerd buiten de neuronen van geïnfecteerde hersenweefsels, lijkt het waarschijnlijk dat de infectie uit verdergelegen (perifere) zenuwen wordt ontvangen, door synaptische transmissie tussen de zenuwcellen. SARS-CoV-2 zou de reukweg kunnen binnendringen door epitheelcellen in het slijmvlies te infecteren. Olfactorische axonen, de uitlopers van reukreceptoren, passeren het zeefbeen om te eindigen in de hersenen. Dit mechanisme, waarbij een virus neuronen in de periferie infecteert om axonaal transport te gebruiken om het zenuwstelsel binnen te dringen, wordt retrograde (axonaal) transport genoemd. Opmerkelijk is de bevinding dat grote hoeveelheden ACE2 en TMPRSS2 tot expressie komen in de steuncellen van het reukweefsel, wat verklaart dat het virus zich met succes aan dit weefsel bindt.
 
Bulbus olfactorius-hypothalamus-hippocampus-thalamus-corticale-PAG-amygdala
De reukbol verspreidt zich naar de reukkern, amygdala, piriforme cortex, peri-amygdaloïde cortex, insula en het voorste uiteinde van de parahippocampale gyrus (Anatomy of the olfactory nerves, Nerves and nerve injuries, Chapter 18, Vol. 1: 2015, p. 273-276). De entorhinale cortex van het reukcomplex heeft uitlopers over de gehele lengte van de hippocampus. Het reuksysteem stuurt vezels rechtstreeks naar corticale gebieden, zoals de frontale kwab (Cranial Nerve I: Olfactory Nerve, Textbook of Clinical Neurology, 2007, p. 99-112). De amygdala maakt deel uit van een breed hersencomplex dat de hypothalamus, hippocampus, orbitofrontale cortex en periaqueductale grijze stof (PAG) omvat. De amygdala heeft wederzijdse verbindingen met de hypothalamus via de fornix en stria terminalis en met de PAG. Het ontvangt projecties van de prefrontale cortex (Amygdala and hypothalamus: historical overview with focus on aggression, Neurosurgery Vol. 85, Issue 1, July 2019).

De corticomediale (midden van de cortex gelegen) amygdala is verbonden met de anterieure preoptische en ventromediale kern van de hypothalamus (opmerking: de POA bevat PGD2 (prostaglandine) synthase tot expressie brengende neuronen). De centrale kern van de amygdala is verbonden met de laterale hypothalamus (zie Grey's Anatomy 2e editie, 2008 en 2010). De piriforme cortex bevat feedbackcircuits binnen de cortex en centrifugale axonen naar de ipsilaterale (zelfde zijde) reukbol, evenals verbindingen met de contralaterale (tegenoverliggende) reukweg (Olfaction and Taste, Architecture of the Olfactory Bulb, in: The Senses: a comprehensive reference vol. 4, 2008). Met name ACE2 blijkt sterk tot expressie te komen in de piriforme cortex, evenals in hypothalamische kernen, ventrikels, substantia nigra, amygdala, hippocampi en de frontale cortex
("Angiotensin-converting enzyme 2 in the brain: properties and future directions", Journal of Neurochemistry, 2008 Dec; 107(6)). 

Schematische weergave van de medulla (hersenstam)

Nervus trigeminus en de neurale weg naar het autonome systeem
De grootste hersenzenuw is de nervus trigeminus (nervus trigeminus, V (= vijfde)), die onderkaak-, maxillaire en oftalmische vertakkingen heeft. Het dient als sensorische en motorische zenuw. De trigeminuskernen bevinden zich in de hersenstam. Trigeminusneuronen ontvangen sensorische informatie van het gezichtsslijmvlies. De afferente sensorische vezels van de trigeminuszenuw zijn afkomstig van cellichamen in het ganglion van Gasser. De sensorische primaire afferenten synapsen bij het Trigeminus Brainstem Nuclear Complex (VBNC) dat door de hersenstam loopt. De vezels eindigen in de ventrale posteromediale kern (VPM) van de thalamus.

Het solitaire kanaal, gelegen in de medulla, wordt gevormd door het ganglion van de nervus vagus, glossofaryngeale en geniculaire zenuw. De solitaire kanaalkern (NST) wordt beschreven als de viscerale kern van de hersenstam. De zenuwvezels vertrokken van de NST-synaps in de laterale en paraventriculaire hypothalamische kernen om naar de insula te projecteren. De nucleus ambiguus (NA) is betrokken bij de motorische functies van slikken en spreken. Zowel de NST als de VBNC bevinden zich dicht bij het cardiovasculaire centrum in de medulla oblongata, die de hartslag reguleert door middel van zenuw- en endocriene controle.

Ligging van de drielingzenuw (nervus trigeminus)

Conclusie
Evenals SARS-CoV-2 (2003) heeft SARS-CoV-2 neuroinvasieve capaciteiten. Het bewijs van de invasie van het centrale zenuwstelsel is aanwezig, maar er zal meer materiaal uit autopsies en biopten (afname weefsel), van beeldvormende technieken (CT-scans en MRI) moeten worden geanalyseerd om de aangerichte schade in het zenuwstelsel specifiek te beoordelen. In een aantal gevallen is er bewijs voor directe invasie van het huidige coronavirus in het zenuwstelsel; dat wordt bevestigd door detectie van SARS-CoV-2 in het hersenruggenmergvocht met RT-PCR (CSF of liquor cerebrospinalis). Het gaat bij directe CNS-invasie door het coronavirus om virale encefalitis, virale meningitis en virale endothelialitis (ontsteking endotheel weefsel, resulterend in onder meer microbloedingen in de hersenen). In andere, gevallen worden door  systemische ontstekingsmechanismen of een "cytokinestorm" in reactie op SARS-CoV-2-infectie, encefalopathie, encefalitis en Kawasaki-achtige manifestaties veroorzaakt. Inflammatoire encefalitis, een hersenontsteking die wordt veroorzaakt door de ontstekingsreactie van het lichaam op SARS-CoV-2, wordt bevestigd door het hersenruggenmergvocht op pleiocytose en verhoging van eiwitten te testen. Manifestaties van het zenuwstelsel die ná infectie optreden, zijn ADEM (Acute encefalmyelitis, met verlies van de witte stof), ANE (Acute Necrotiserende Encefalopathie, hersenontsteking met necrose (wegrotten) van het hersenweefsel) en Guillain-Barré. 

Waarom het onderscheid tussen directe neuroinvasie van SARS-CoV-2 of neurologische manifestaties veroorzaakt door inflammatoire mechanismen en immunologische mechanismen in reactie op SARS-CoV-2? Directe neuroinvasie, bijvoorbeeld virale meningitis, moet worden behandeld met antivirale therapie. In één geval van SARS-CoV-2-meningoencefalitis werd een patiënte succesvol behandeld met hydroxychloroquine (HCQ). Als neurologische manifestaties worden veroorzaakt door immunologische reacties, kan bijvoorbeeld Tocilizumab (IL-6-blokker) of Anakinra verbetering geven.