Review Article - revised version
Heidi N du Preez
Glycosaminoglycans (GAGs) are complex linear, negatively charged polysaccharides, expressed at the cell surface, and in the extracellular environment, where they interact with various molecules to regulate many cellular processes implicated in health and disease. Subversion of GAGs is a pathogenic strategy shared by a wide variety of microbial pathogens, including viruses.
The degree of GAG sulfation plays an important role in the host defence system and immunomodulation. Anti-inflammatory drugs are dependent on sulfation for its metabolism. The sulfation of these drugs will deplete the endogenous sulfate levels, affecting the degree of sulfation of the GAGs, therefore impacting on the defence system and the immune response in the lung glycocalyx, predisposing to COVID-19 in SARS-CoV-2 infected individuals.
Keywords: COVID-19, SARS-CoV-2, non-steroidal anti-inflammatory drugs, glycosaminoglycans, sulfation
In the midst of a COVID-19 world pandemic, we can easily lose sight of reality. One coronavirus (SARS-CoV-2) affecting millions of people across the globe. Of these, around 80% of infected people show mild or no symptoms. Approximately 20% of those infected with SARS-CoV-2 experience acute symptoms, life-threatening Acute Respiratory Distress Syndrome (ARDS) or death. The variable factor is the host defence system against the virus. What makes some people more vulnerable to the virus than others?
The French Health Minister, Olivier Veran, warned consumers against the use of anti-inflammatory drugs to minimise the symptoms of COVID-19, such as fever and headache. It was implicated that drugs such as Ibuprofen and cortisone could be ‘aggravating factors’, making the symptoms of the illness even worse. Popular non-steroidal anti-inflammatory drugs (NSAIDs) include aspirin, Ibuprofen (Nurofen, Advil) and naproxen. Veran and health officials around the globe are suggesting the use of acetaminophen (Paracetamol, Tylenol, Panado) instead, which is not a NSAID.
In pharmacovigilance surveys carried out in France, it was found that there is a risk of serious infectious complications when adults and children take NSAIDs, such as Ibuprofen and ketoprofen. These infectious complications (mainly with Streptococcus or Pneumacoccus) were observed after treatment of only 2 to 3 days, especially when NSAIDs were combined with antibiotic treatment. It is also known that NSAIDs can cause serious skin complications (necrotizing fasciitis) when they are used during chickenpox1,2.
In this review article we’ll explore the possible effects of NSAIDs on the outer defence system of epithelial cells, the glycocalyx.
Glycocalyx as the Outermost Cell Region
The glycocalyx, also known as the pericellular matrix or extracellular environment, is a glycoprotein and glycolipid covering that surrounds the cell membranes of microorganisms, epithelia, and other cells. This glycocalyx or cell coat, a constituent of the plasma membrane, is particularly pronounced on all epithelial cells, lining the cell surfaces. It is conspicuous especially on the microvilli of intestinal epithelium and in the lungs, but also the endothelial cell surface3,4.
The glycocalyx consists of proteoglycans (PGs), which are glycoconjugates with a protein core covalently attached to linear polysaccharides consisting of recurrent disaccharide units called glycosaminoglycans (GAGs). GAGs are complex linear anionic and hydrophilic polysaccharides5. The five types of GAGs, classified according to their sugar composition, are heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), and hyaluronic acid (HA)3,6.
Membrane proteoglycans tend to contain mostly either HS or both HS and CS. HS is the most widespread and physiologically relevant GAG3,5. GAG formation is a complex biosynthesis: after the polymerization of the sugar backbone, the chains are modified in different interdependent reactions, including N-deacetylation/N-sulfation, epimerization, and various O-sulfations of hydroxyl groups. The high degree of sulfation of these components provides the glycocalyx with a net negative charge. Proteoglycans with HS moieties (HSPGs) can be classified according to their location: on the cell surface, where two families are found, the transmembrane syndecans and the glycosyl phosphatidylinositol-anchored glypicans; in the extracellular matrix (ECM) where there are three types of HSPGs: agrin, perlecan and type XVIII collagen; and inside intracellular vesicles, where serglycin is located. HSPGs have multiple functions, some of them dependant on the core proteins, but most are related with the GAG chains because of their characteristic epimerization and sulfation pattern. GAG polymerization and modification varies with cell type and tissue source. This structural diversity allows HSPGs to play a key role in many processes including immune response, cell adhesion and migration, organization of ECM, regulation of proliferation, differentiation and morphogenesis, cytoskeleton organisation, tissue repair and inflammation. They are clearly the most information-dense biological molecules. HSPGs can bind several ligands such as cytokines, chemokines, growth factors, and morphogens, protecting them against proteolysis and controlling their gradients. HSPGs co-operate with different molecules to define basement membrane structure and to mediate in cell-ECM attachment, cell–cell interactions, and cell motility. Shedding of membrane bound HSPG ectodomains can be carried out by enzymatic cleavage and is an important factor in host response to tissue injury and inflammation in pathophysiological processes3,4,5.
Damage to and loss of the glycocalyx have been observed in many diseases, including diabetes, ischemia, myocardial infarction, chronic infections, atherosclerosis and tumour metastases, among others. Furthermore, misregulation of GAG sulfation (undersulfation) is associated with several diseases7.
The sulfation of PGs is an important dynamic and complex posttranslational modification that has a regulatory role in cell growth, motility, and metabolism5. The heavily sulfated glycosaminoglycan chains has a net negative charge, due to the sulfate (S04 2-) molecule. In a mammalian cell, the sulfation process begins with uptake of inorganic sulfate from the extracellular milieu7. The universal sulfate donor, 3′-phosphoadenosine 5′-phosphosulfate (PAPS), is synthesised by PAPS synthetase in the cytoplasm. The process of sulfation is strictly governed by various enzymes, such as glycosyltransferases, sulfotransferases, and epimerase5. Different sulfation patterns have been identified for the same organs and cells during their development7.
Owing to its high negative charge, HS interacts with hundreds of proteins and regulates multiple signalling pathways, including fibroblast growth factor (FGF), hepatocyte growth factor (HGF), vascular endothelial growth factor, Wnt, and BMP signalling pathways. The most well-studied functions of HS include its effects on protein conformation, enhancement of protein–protein interactions, role as a co-receptor for growth factors, protection of proteins from degradation, sequestration of protein ligands, and mediation of protein internalisation5,7.
The predominant disaccharide unit in HS is N-sulfoglucosamine/2-O-sulfated IdoA, but sulfation can also occur at the C6 position and, more rarely, at the C3 position of GlcNS, making HP the bio-macromolecule with the highest negative charge, with 2.7 sulfates per disaccharide unit on average. A significant body of evidence shows that sulfated GAGs exert their influence by interacting with other ECM components, mainly proteins, and that the degree of GAGs sulfation—specifically, their negative charge—is the main driver of these interactions. Sulfation can influence GAG cross talk with other bio-entities in the physiological environment either indirectly, such as by regulating protein folding via steric hindrance, exclusion, or recruitment, or directly, through electrostatic interactions that are often sequence specific. GAGs therefore act as a physical and biochemical barrier, creating specific microenvironments around cells. They build size-selective barriers that are permeable only by small entities, such as Ca2+ and Na+, that can freely diffuse and promote extracellular cation homeostasis7.
GAGs as defence mechanism
Infection begins with microbial colonisation of host tissues, but colonisation by most microbes does not lead to infection. In fact, many microbes are supportive of host well-being and survival. Infection occurs when several pathogenic microbes breach the protective barriers of the host, entering the cells, multiply, damage cells, and disrupt normal tissue functions. Fortunately, even after pathogens penetrate the protective borders, disease usually happens in only a small proportion of infected people because the host rapidly mounts an effective defence mechanism to eradicate them8.
We’ll explore the hypothesis that it is the degree of sulfation, therefore the net negative charge of the GAGs, which mainly determine the host’s protection against infections.
Several research groups have studied the electrostatic repulsive forces resulting from the surface electronegativity of GAGs and concluded that it may act to keep cells apart, as with circulating cells. Martins & Bairos (2002) observed this electronegative shield on the lung cell surfaces. They noted that the cause of the frequent infections in cystic fibrosis (CF) patients could be the reduction of the surface charge in CF epithelial cells and the subsequent decrease of the repellent electrostatic forces between cells, thereby facilitating the adherence of bacteria to epithelial cells. The airway epithelial cells are therefore able to defend themselves against invasion by external pathogenic agents from the surrounding environment. This ability results from the existence of a complex series of barriers, including the mucus layer, that may trap the exogenous elements and clear them away; the apical cell membrane that is described as having relatively few viral receptors; and the glycocalyx, which, being present on the surface of the outer leaflet of the plasma membrane, may, among other functions, hinder these receptors or bind the vectors, thus preventing their binding to the membrane receptors3,9. The outcome of a microbial infection is largely governed by the ability of pathogens to subvert host components and their activities and it appears that GAGs, especially HS, plays a major role8. Aquino & Park (2016) indicated that studies done in the 1970’s, examining the anti-infective effects of HS, showed that this highly sulfated GAG inhibited the initial attachment of pathogens to host cells, such as Neisseria meningitides and Chlamydia trachomatis.
Most of the research done on how pathogens, viruses, bacteria, parasites and fungi, subvert GAGs, mainly focussed on the GAG structure and the role that proteoglycans play in infection6. For example, it is postulated that many intracellular pathogens use cell surface HS for host cell attachment and invasion. Several extracellular pathogens secrete factors that release GAGs from cell surfaces and ECMs and exploit the ability of these solubilised GAGs to inhibit antimicrobial factors. Some pathogens coat their surfaces with solubilised GAGs to escape immune recognition8,10.
The glycocalyx may modulate adhesion through a variety of nonexclusive mechanisms, including (1) generation of repulsive forces impeding cell-cell or cell-surface approach, (2) masking of cell adhesive sites, or, alternatively, (3) expression of molecular sites specifically bound by membrane receptors of neighbouring cells11. However, the first barrier function would be glycocalyx-mediated repulsion. Removal of cell surface negative charges is considered to facilitate adhesion11. To demonstrate this fact, coronavirus NL63 (CoV-NL63) and the severe acute respiratory syndrome coronavirus (SARS-CoV) use angiotensin-converting enzyme 2 (ACE2) as a primary receptor for their entry. However, ACE2 expression is not sufficient for infection as directed expression or selective cleavage of ACE2 has no effect on virus attachment. Instead, both CoV-NL63 and SARS-CoV initially bind to cell surface HS and virus entry is dependent on this HS interaction8.
S pneumoniae has the ability to induce the ectodomain shedding of syndecan-1 from the cell surface. This shedding downregulates the host defences, leading to increased bacterial virulence and enhances its survival. Circulating GAG levels in CF patients are increased, not only due to shedding, but also because of other bacterial exoenzymes which are produced to inactivate the action of molecules from the host immune system. Released GAGs are also able to interact with immune factors (LL-37) electrostatically, inhibiting its binding with bacteria and in this way disabling its bactericidal action. Shedding of syndecan-1 is stimulated by proinflammatory substances and, as a consequence, levels of circulating syndecan-1 increase, thereby promoting leukocyte adherence. During septic shock there are high levels of some GAGs in plasma and, although there is no correlation with syndecan-1 levels, they are correlated with mortality. Specifically, the quantity of circulating HS and HA increases. Syndecan-1 ectodomains increase pathogenicity and the potential for bacterial survival by interacting with neutrophils, whose antibacterial function is thus inhibited12.
Most viruses have a negative charge in neutral solutions10,13. Yaoa et al (2013) found that with examination of allowed and unallowed substitutions in viral mutations, that the number and location of negatively-charged residues in the charge-rich motif was critical for S protein incorporation into virions. Analysis of these mutants showed that the quality they had in common was the gain of negatively-charged residues and the loss of positively-charged residues in the charge-rich motif14. Could it be said that SARS-CoV-2, whether a mutated or bio-engineered virus, has an exceptional negative charge? This could explain why hosts with an intact defence system would have no infectious symptoms, due to a strong repulsion force preventing the virus from entering the glycocalyx. However, once the virus gains entry, it will have a high affinity to cationic proteins and other substances in the ECM, enhancing its aggressiveness in creating a cytokine storm. Also, the high ionic force of the virus would enhance covalent bonding to surfaces that would enable its fast and aggressive spreading.
The pathogenesis of viral infection is complex and there are many contradictory theories10,15. The role of GAGs in infections is mostly derived from cell-based experiments performed in vitro, and their physiological significance, relevance, and function in infectious diseases has yet to be determined. Furthermore, as stated previously, most of the research focuses on the proteoglycan structure and shedding, but omits the degree of sulfation and therefore the negative repulsion force or electrostatic potential – relationship between pH and electron charge on the cell surface. Viruses can only gain entry into the glycocalyx and bind to HSPG if they are undersulfated16. Jinno & Park (2015) confirms that the unique and complex sulfation patterns of GAGs enable them to bind specifically to many biomolecules and regulate diverse biological processes6.
The glycocalyx remains poorly studied due to difficulties in preserving its structure upon tissue fixation and the limited number of experimental tools for its visualisation. Therefore, there is no confidently charted time course of damage and disintegration for the glycocalyx, which is quite vulnerable and tends to disintegrate under the influence of various stressors, such as endotoxins, ischemia/hypoxia/reperfusion and oxidative stress, among others.
Immune response and sulfation
Inflammation is a defence mechanism of the body to harmful stimuli. This protective action primarily involves the recruitment of immune cells from the bloodstream into the site of injury or infection17. There are two mechanisms for immunomodulation - immune stimulation and immunosuppression. It is a complicated process that controls the pathogenesis and pathophysiology of different illnesses influencing the immune system. The disintegration of the glycocalyx predisposes to leukocyte adhesion, migration and tissue infiltration by leukocytes, monocyte, macrophages and lymphocytes. Immune cell aggregation favour immune response development. This aggregation is enhanced by glycocalyx degradation, and this degradation was found to occur on lymphocyte activation. Thus, lowering the anti-adhesive barrier by the host, may be a general means of enhancing immune responses11.
The degree of sulfation plays a major role in the inflammatory process. Wang et al (2005) demonstrated this in the endothelial glycocalyx18. During inflammation, leukocytes attach to activated endothelial cells and migrate from the circulation into areas of tissue damage. This process involves multiple adhesion events mediated by different sets of receptors and ligands. P- and E-selectin expressed by activated endothelial cells and L-selectin constitutively expressed by leukocytes, engage carbohydrate ligands and facilitate the initial attachment and rolling of leukocytes under shear flow17. Chemokines, bound to the lumenal surface of the endothelium, activate seven transmembrane-spanning G protein–coupled receptors on leukocytes, resulting in integrin activation and firm adhesion of rolling cells. Subsequently, leukocytes change shape and extravagate across the endothelium into the underlying tissue, where they initiate various protective responses and effect tissue repair. The carbohydrate ligands for the selectins are complex and vary in structure according to the location of the endothelial cells and the subtype of leukocytes being studied. L-selectin recognises sulfated, sialylated, fucosylated mucinous ligands such as CD34, podocalyxin and endoglycan on high endothelial venules in lymph nodes and mediates lymphocyte trafficking through these organs. In the microvasculature, L-selectin works in concert with P- and E-selectin to facilitate neutrophil migration during inflammatory responses. Most of the evidence indicating involvement of HS in inflammation is based on biochemical studies done with isolated cells, purified carbohydrates and recombinant proteins and receptors. To examine its function in vivo, Wang et al (2005) used the Cre-loxP system to inactivate an essential gene required for the addition of sulfate to the HS chains in endothelial cells and leukocytes18. Decreased sulfation of HS, specifically in endothelial cells, resulted in reduced leukocyte extravasation in several inflammatory models. Notably, altering HS in this way reduced L-selectin-mediated adhesion, binding of chemokines to endothelial cells and transcytosis of chemokines across endothelial cells, thus defining three functions for HS in leukocyte recruitment. In contrast, altering HS expression in leukocytes did not affect inflammatory responses in these models. Decreased sulfation of the chains in the endothelium reduced the binding of L-selectin, which led to an increase in neutrophil rolling velocity. Decreased sulfation also decreased firm adhesion of leukocytes, due to decreased chemokine binding and transport across endothelial cells (transcytosis). The reduced interaction of L-selectin with undersulfated endothelial cell HS might explain the reduction in leukocyte infiltration noted in various models of inflammation. Nearly all chemokines bind to HS by way of clustered positively charged amino acid residues. Chemokines with alterations in these domains fail to associate with HS proteoglycans expressed on the endothelial surface. These and related observations suggest that the function of the proteoglycans is to immobilize chemokines near sites of tissue injury, so that rolling leukocytes receive the appropriate spatial signals to activate firm adhesion and eventual diapedesis at sites of injury18,19,20. Decreased sulfation will therefore expedite the cytokine storm. Bode et al (2005) also indicated that HS depletion ampliﬁes TNF-induced protein leakage in an in vitro model of protein-losing enteropathy21. Yi et al (2015) demonstrated that in the H292 cell line, fully sulfated heparan both prevented activation of the ERK1/2, p38, and NF-kB p65 signalling pathways and significantly reduced LPS-induced COX-2 gene and protein expression, while desulfated heparan did not only suppress, but actually over time enhanced LPS-induced COX-2 gene and protein expression. In the HBE1 normal bronchial cell, only fully-sulfated heparan’s effects on ERK signalling were of significance and again, desulfated heparan had little effect upon LPS induced ERK signalling19. The degree of sulfation therefore plays a vital role in both the host defence system and immune response.
Respiratory tract invasion
The airway epithelium, as a result of morphogenesis, forms a continuum beginning in the trachea and ending in the alveoli. The glycocalyx covers the outer leaflet of the airway epithelial and alveolar epithelial cell membranes20. The glycosylation of the airway epithelial cell surface molecules is altered in CF4. This could partially explain the excessive bacterial colonization and infection in the lungs of these patients. Yi et al (2015) demonstrated the in vivo physiological significance of HS in the airway and suggests the potential utility of HS in dampening the inflammatory response to exogenous pathogenic stimuli4,19.
The control of influenza viral infections depends on the ability of virus-specific T cells that are activated in the lymphoid compartment to migrate into the infected lungs where viral replication occurs in epithelial cells. Factors implicated in the high morbidity and mortality of influenza virus infection include robust cytokine production (cytokine storm), excessive inflammatory infiltrates, and virus-induced lung tissue destruction. Although pulmonary inflammatory responses may facilitate virus clearance, they often cause severe lung injury. Interactions of the virus and host factors are crucial for virus replication22.
Around 81% of critically ill COVID-19 patients progress to develop febrile respiratory illness or ARDS. Over-activated neutrophils are the major effector cells in ARDS. Extracellular pathogens triggering TH17-like innate immunity with neutrophil activation, might account for the aetiology of ARDS23.
Effect of anti-inflammatory drugs on GAGs
Sulfation, catalysed by the cytosolic sulfotransferases (STs), plays a critical role in the detoxification of xenobiotics, including drugs, food additives and certain environmental pollutants. Conjugation with sulfate, derived from the active donor PAPS, generally results in a reduction in biological activity and an increase in water solubility, thereby facilitating excretion of the compound in the urine and / or bile24.
NSAIDs are a drug class that groups together drugs that provide analgesic (pain-killing) and antipyretic (fever-reducing) effects, and, in superior doses, anti-inflammatory effects. Sulfotransferases in the small intestine can sulfate orally administered drugs and xenobiotics for which the primary route of conjugation is sulfation (e.g. isoproterenol, albuterol, corticosteroids, opioids25, α-methyl dopa, aspirin and fenoldopam)26. Sulfation and glucuronidation are major pathways for the metabolism of acetaminophen and NSAIDs, such as Ibuprofen, diclofenac, celecoxib, meloxicam and etoricoxib.
The sulfation of these drugs will deplete the endogenous sulfate levels, affecting the degree of sulfation of the glycocalyx, therefore impacting on the defence system and the immune response in the lung glycocalyx, predisposing to COVID-19 in SARS-CoV-2 infected individuals.
Asthma exacerbation seems to be the main adverse event of NSAIDs used in children27 and from the literature we know that the risk of upper gastrointestinal tract bleeding or perforation, due to desulfation of the glycocalyx, increases around twofold with use of oral steroids or low dose aspirin, and increases around fourfold with use of non-aspirin NSAIDs. Acetaminophen, at daily doses of 2000mg and higher, has also been associated with an increased risk. Overall, the risk is dose dependent and is greater when more than one anti-inflammatory drug are simultaneously administered28.
Taking several drugs at the same time will not only further deplete the sulfate pool, but enhance the chances of drug toxicity. The extensive presystemic first-pass sulfation of phenolic drugs, for example, can lead to increased bioavailability of other drugs by competing for the available sulfate pool, resulting in the possibility of drug toxicity. Concurrent oral administration of acetaminophen with ethinyl estradiol resulted in a 48% increase in ethinyl estradiol blood levels. Ascorbic acid, which also requires sulfation for metabolism, increases the bioavailability of concurrently administered ethinyl estradiol. Treating COVID-19 with high dosages of intravenous ascorbic acid (vitamin C), is therefore not recommended. Not only will it deplete the sulfate pool, but will increase the bioavailability and possible toxicity of other drugs29. Drug toxicity can result from a single toxic dose or repeated cumulative dosages, such as in chronic use of NSAIDs.
The current focus of the media, on the use of Ibuprofen during COVID-19, is most likely due to Ibuprofen being one of the most popular NSAIDs, as well as the cautionary warning against its use by the French minister of health. Not only will Ibuprofen deplete sulfate for its metabolism, it is also a potent inhibitor of human liver sulfotransferases, thus further inhibiting sulfation24,30,31. Ibuprofen also inhibits prostaglandin synthesis by blocking the conversion of arachidonic acid to prostaglandins. Prostaglandins plays an important role in the synthesis of sulfated GAGs32. Leukocyte motility and phagocytosis are partially suppressed by Ibuprofen and sulfate uptake in human cartilage tissue culture, is inhibited33. Le Bourgeois et al (2016) observed that even low concentrations of Ibuprofen, such as those obtained during antipyretic use, may have a proinflammatory action that promotes the recruitment and influx of neutrophils. Activated charcoal adsorbs Ibuprofen and should be repeated, since Ibuprofen undergoes enterohepatic recirculation. Forced diuresis, dialysis or hemoperfusion does not enhance Ibuprofen elimination34.
Several researchers35,36,37 found that salicylic acid (aspirin) inhibited phenol sulfotransferase of the human colon mucosa and platelets, and causes salicylate-induced diuresis with a twofold increased excretion of inorganic sulfate, shortly after drug treatment.
Meuwesen (2015) found that most NSAIDs are prescribed by general practitioners in dosages greater than the recommended dosages and many of these drugs are available over the counter, with a great risk of overdosing. NSAIDs are nephrotoxic, but frequently prescribed to patients who have reduced renal function.
NSAIDs use during acute viral infection is associated with an increased risk of empyema in children34. It was observed that NSAIDs may have an inhibitory action of leukocyte adhesion, phagocytosis, and bactericidal activity in vitro, which is in line with the observation of reduced sulfation of HS. NSIADs might temporarily reduce pain and inflammation, but they will eventually accelerate conditions such as osteo-arthritis and similar connective tissue trauma, joint degeneration or related disorders by promoting cartilage destruction and interfering with cartilage repair38, processes that are dependent on sulfation.
The use of NSAIDs also reflects delayed effective treatment, because NSAIDs might mask the onset of viral disease by decreasing the inflammatory response to infection. Associations between bacterial infections and NSAIDs were reported for necrotizing fasciitis (NF) during primary varicella or for invasive group A streptococcal infection. The occurrence of new symptoms or complications were slightly more frequent in children receiving Ibuprofen than in those advised to take acetaminophen during respiratory tract infections34. NF has been linked with cleavage of proteoglycans from the plasma membrane, increasing the susceptibility to bacterial infection2.
Pulmonary eosinophilia (PE) can be found in very diverse pathological processes. Several medications have also been associated with this entity. Acetaminophen is commonly used in multiple drug formulations, many of which are available without a prescription. It has, however, been associated with PE (eosinophilic pneumonia) in a few cases in Japan. Saint-Pierre & Moran-Mendoza (2016) describe the case of a 68-year-old Caucasian female who presented with new persistent dry cough and dyspnoea on exertion after she started using up to 4 grams of acetaminophen on a daily basis40. More studies provide further evidence that use of acetaminophen or paracetamol is associated with an increased risk of asthma and chronic obstructive pulmonary disease (COPD), and with decreased lung function and injury41,42,43.
Melatonin is recommended by many practitioners as treatment for COVID-19 for its immunomodulatory actions, but being a hormone, melatonin also requires sulfation for its metabolism44. Tain et al (2015) and Sutherland et al (2002) found that melatonin may act as a pro-inflammatory agent in asthma leading to bronchial constriction. Those with nocturnal asthma demonstrated the largest cytokine response45,46. This could be explained by the fact that the enzyme cysteine dioxygenase, which converts L-cysteine to cysteine sulfinate, a precursor of sulfate, has a nocturnal effect. When melatonin is prescribed, it is important to add a sulfur-donor supplement.
Any factor or drug, which interferes with the sulfation process, has the potential to disrupt these important events and processes, thereby providing a fundamental mechanism for the pathogenesis of different clinical conditions, and predisposing to the disastrous effects seen in COVID-19. Undersulfation would specifically be pronounced in old age, due to the overuse of chronic prescription medication and decreased alimentary intake of inorganic dietary sulfate29.
Anyone taking prescription NSAIDs for chronic conditions should first consult with their healthcare practitioner before discontinuation. However, over the counter use of NSAIDs, aspirin and acetaminophen for pain and fever, are not recommended for SARS-CoV-2 positive patients.
What are the alternatives?
STS is an approved chemical for human use, and has previously shown to have anti-inflammatory properties in vitro and in vivo47,48,49,50. Sodium thiosulfate (STS) is a known antidote approved for treatment of certain medical conditions, such as cyanide and acrylonitrile poisoning, and cutaneous infections caused by bacteria and fungi. Recent studies demonstrated that STS attenuates neurotoxic effect of lipopolysaccharide (LPS) activated microglia and astrocytes in vitro in glia-neuron co-cultures and protects against ischemic brain injury48 as well as acute lung injury51. Intraventricular STS has been safely administered in the intensive care unit setting48,52, and this route of administration may be an important alternative for treating COVID-19 induced ARDS. Sakaguchi et al (2014) also found that STS inhibited lipopolysaccharide-induced production of cytokines, lung permeability, histological lung injury, and nuclear factor-κB activation in the lung. STS also prevented upregulation of Interleukin-6 in the mouse lung, subjected to caecal ligation and puncture. In endothelial cells, STS increased intracellular levels of sulfate, inhibited LPS or TNFα-induced production of cytokines and reactive oxygen species53. Furthermore, intraperitoneal administration of STS improved survival after endoxemia and acute liver failure. STS is a potent antioxidant and anti-inflammatory and has been used for the treatment of cyanide poisoning and calciphylaxis with a remarkably safe track record54,55,56. Taken together, these observations suggest a therapeutic potential of STS against acute lung injury57.
Other sulfur donors
Sulfation of drugs depends on the availability of PAPS, which requires inorganic sulfate for its synthesis. Therefore, decreased alimentary intake of inorganic sulfate or its precursors, methionine and cysteine, may compromise sulfation of xenobiotics and the glycocalyx29. A balanced wholefood diet can be supplemented with the following sulfur donors:
NAC has been used successfully in COPD patients to reduce symptoms, exacerbations, and accelerated lung function decline58. NAC, and ascorbic acid has been shown to be more effective than any of the two agents alone, in preventing acetaminophen-induced hepatotoxicity in animal studies59. However, Kaplowitz & DeLeve (2013) indicated that prolonged exposure to NAC can be detrimental to liver recovery60 and induced lung adenocarcinoma in mice61. Although NAC is an important sulfur donor, there are necessary cofactors for it to be oxidised to sulfate, such as molybdenum. Long-term use or high dosages of NAC can therefore deplete molybdenum and inhibit sulfation. The same holds true for Methylsulfonylmethane (MSM), although a safer sulfur donor than taking high levels of the sulfur-amino acids methionine and NAC long-term.
MSM, also known by several other names (DMSO, methyl sulfone and dimethyl sulfone), is a naturally occurring source of sulfate available in different fresh fruits and vegetables, grains and animal tissues and is considered to provide health beneﬁts when used to supplement the diet.
Supplemental sulfur may be necessary when the diet does not provide adequate amounts of sulfur-amino acids, particularly for strict vegans and vegetarians, and when the sulfur requirement is higher, such as during disease states, endurance exercise, pregnancy and foetal development. Various studies provide evidence for the ready conversion of sulfur in dietary supplements into inorganic sulfate18,62. Alternative sources of sulfate can spare cysteine for other purposes, such as synthesis of glutathione. MSM has been proven to be an effective anti-oxidant and anti-inflammatory agent63.
Various sulfated glycans, such as glucosamine sulfate, can be used to downregulate the inflammation process12,17,64. Glucosamine sulfate increased serum sulfate concentration more than sodium sulfate – the bioavailability of the sulfur compounds are thus important. Stargrove et al (2008) and Nimni et al (2007) emphasise that it is the sulfate component in glucosamine sulfate that has the functional role in the body38,62.
Heparan derivatives and heparan-mimicking molecules, called heparinoids, have been used in many therapeutic applications as inhibitors of HS-protein interactions. Fucoidans are heparinoids obtained from marine brown algae. These molecules also have different anti-infective activities, including antiviral activity against a wide range of viruses via receptor entry blocking or interference with replicative processes65,67.
It is important to also consider all the precursors of sulfate and the co-factors involved in the trans-sulfuration and sulfation pathways. Vitamin A deficiency will affect sulfation69, magnesium is an important cofactor for PAPS and molybdenum for sulfite oxidases. The various B vitamins, iron, selenium and zinc will play an important role too.
Susceptibility to COVID-19
People susceptible to COVID-19 are those with blood disorders, chronic kidney disease66, chronic liver disease, severely weakened immune response / suppression, diabetes, metabolic and mitochondrial disorders, heart disease, lung disease and neurological disease68. Sulfation plays an important role in all of these conditions7,70,71.
Older people are much more reliant on chronic medication. Although Brinkkoetter (2004) demonstrated that ACE inhibition for blood pressure control preserved HSPG72, Hicks et al (2018) indicated that the use of ACEIs causes an accumulation of bradykinin in the lung, which has been reported to stimulate growth of lung cancer72. Upon inflammation due to viral infection, bradykinins are rapidly generated and can induce IL-1 gene expression through activation of NF73. ACEIs can thus potentially contribute to the cytokine storm seen in COVID-19.
Cellular senescence is irreversible growth arrest. Jung et al (2016) found that HS prevents cellular senescence by fine-tuning of the fibroblast growth factor receptor (FGFR) signalling pathway. They indicated that the HS undersulfation induces augmentation of fibroblast growth factor receptor 1 (FGFR1) activation, ultimately resulting in premature senescence through the p53-p21 signalling pathway5. In old age both factors of, low protein dietary intake62 and the use of chronic medication, will result in undersulfation of HS29,74. Herd et al (1991) found that specific enzyme activity is not age related, but that there is less sulfate in the pool75. The combination of poor dietary intake of sulfur-rich food, use of chronic medication and infections in old age, would predispose this population group to COVID-19 once infected.
From the substantial body of evidence, it is clear that the degree of sulfation, contributing to the net negative charge of the GAGs, does not only affect the host’s protection against infections, but also modulates the immune response76 and viral replication77. Accumulating evidence reveals that severely ill COVID-19 patients have an elevated cytokine profile resembling a cytokine storm and that the use of anti-inflammatory drugs, such as glucocorticoids, has no effective outcome78.
A new COVID-19 HOPE Clinical Trial looks at combining heparin with NAC79 to improve pulmonary function, but fails to recognise that it is the degree of sulfation of the proteoglycans that determines its functionality. Do we need more FDA approved drugs through which someone will capitalise, when we have sulfur-donors freely available? In the acute COVID-19 clinical setting intravenous sodium thiosulfate or NAC should be considered for both preventing and treating the cytokine storm in the lungs. NAC should be administered as per the intravenous protocol for paracetamol overdose. When anti-inflammatory drugs such as glucocorticoids are used, the addition of a sulfur-donor supplement might have a synergistic affect and should ameliorate the adverse effects seen to date when these anti-inflammatory drugs were used without supporting sulfation. In milder COVID-19 cases sulfur donors such as MSM and fucoidans should be considered. These suflur-donors should also be used in those susceptible to COVID-19 as a preventative measure.
Even though we might see the emergence of more aggressive strains of viruses, whether mutated or bio-engineered; it is the weakening of our host defences against these pathogens that warrants global attention.
As a result of poor diet and lifestyle choices, pollution and the over-use of prescription drugs, we are defence-less against these pathogens. With Asia Pacific being the fastest growing market for NSAIDs80, we need to ask ourselves whether this pandemic was self-inflicted? It is by no means implied that NSAIDs are a direct cause of COVID-19, but rather that it could potentially play a huge role in causing an infectious disease state.
Heidi N du Preez, M.Sc., Pr.Sci.Nat.
Heidi N. du Preez is an independent medical researcher in private practice. She holds a Master degree in Science and is registered as a Professional Natural Scientist.
No vested interests other than potentially saving more lives. This research was self-funded by many man-hours.
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