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- 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).
