Chronic Expression of Pro-Inflammatory Cytokines in Long COVID/PASC

Version	Summary	Created by	Modification	Content Size	Created at	Operation
1		Rupert Donald Holms	--	4912	2022-09-26 09:35:44	\|
2	format	Jason Zhu	-3 word(s)	4909	2022-09-27 03:39:30	\|

This entry is adapted from the peer-reviewed paper 10.3390/immuno2030033

Long COVID, also referred to as Post-Acute Sequelae of COVID (PASC), is probably triggered during SARS-CoV-2 infection and acute COVID-19 by SARS-CoV-2 Spike-protein binding and hyper-activating the cell-membrane expressed Receptor for Advance Glycation End-products (mRAGE) and Toll-Like Receptor 4 (TLR4). SARS-CoV-2 infects lung monocytes by Spike binding to mRAGE (not ACE2). During acute COVID-19, high levels of IL-6 hyper-stimulate S100A8/A9 expression and secretion. Although no viral protein nor mRNA can be detected in half of long COVID (PASC) patients, there is a significant elevation of serum levels of IL-1b, IL-6, TNFa, and S100A8/A9. It appears that a pathological pro-inflammatory feedback loop (the TLR4/RAGE-loop) is established during acute COVID-19, which is maintained by S100A8/A9 > RAGE/TLR4 chronic inflammatory signalling, even after SARS-CoV-2 has been cleared from the body. During long COVID/PASC, Ca2+-binding protein S100A8/A9 chronically stimulates TLR4/RAGE-signalling to induce chronic expression of IL-1b, IL-6 and TNFa. Secreted IL-6 binds to its IL-6R receptor on the surface of other cells and signals via STAT3 and C/EBPb for more S100A8/A9 expression. Secreted IL-1b binds to its receptor IL-1R on other cells, and signals via NFkB for more mRAGE and TLR4 expression. New S100A8/A9 can bind and activate cell-surface mRAGE and TLR4 to stimulate expression of more IL-1b, IL-6 and TNFa. This process establishes a pathogenic pro-inflammatory TLR4/RAGE-loop: IL-1b + IL-6 > IL-1R + IL-6R > TLR4/mRAGE + S100A8/A9 > IL-1b + IL-6, which generates multi-organ inflammation that persists in the blood vessels, the brain, the liver, the heart, the kidneys, the gut and the musculo-skeletal system, and is responsible for all the complex pathologies associated with long COVID/PASC. Chronic expression of IL-1, IL-6 and TNFa is critical for the maintenance of the TLR4/RAGE-loop and persistence of long COVID/PASC.

long COVID PASC RAGE TLR4

1. Long COVID and Post-Acute Sequelae of COVID-19 (PASC)

1.1. Prevalence of Long COVID/PASC

Even after SARS-CoV-2 mRNA and proteins cannot be detected in half of long COVID/PASC patients, COVID-19 sequelae related to multi-organ chronic inflammation persist for months. Generally, persistence of symptoms between 4 and 12 weeks after SARS-CoV-2 infection is known as long COVID. However, no SARS-CoV-2 mRNA can be detected in half of symptomatic long COVID patients at a 55-day mean time-point after confirmed infection ^[1].

Persistence of symptoms beyond 90 days is common and is generally referred to as Post-Acute Sequelae of COVID-19 (PASC). At least seventy different organ-specific disorders have been reported to be associated with long COVID/PASC symptoms, but all seem to originate from a common systemic inflammatory process initiated by SARS-CoV-2 infection.

In the UK as of 4 June 2022, the prevalence of ongoing long COVID following SARS-CoV-2 infection was 2 million people (3% of the whole UK population). The number of long COVID cases increased by ten per cent over the prior three months since an earlier report on 5 March 2022. Of the UK population suffering long COVID, 405,000 (21%) had been infected with SARS-CoV-2 less than 12 weeks previously, 1.4 million people (74%) more than 12 weeks previously, 807,000 (41%) more than one year previously and 403,000 (21%) more than two years previously ^[2].

All SARS-CoV-2 variants caused long COVID: 570,000 (29%) first had COVID-19 before the Alpha variant became dominant in November 2020; 237,000 (12%) during the Alpha variant wave, 394,000 (20%) during the Delta variant wave between May and December 2021, and 642,000 (33%) during the subsequent Omicron variant period.

There is no significant difference between the prevalence of long COVID/PASC symptoms between hospitalized and non-hospitalized acute COVID-19 patients. Many patients with only mild acute COVID-19 go on to develop long COVID symptoms. The prevalence of long COVID symptoms in the UK is higher in women compared with men, while the age-group estimated to be most greatly affected by long COVID symptoms is 35–69.

The UK Government estimated that a total of 22.2 million people had been infected with SARS-CoV-2 since the start of the pandemic, suggesting that long COVID/PASC developed in 13.5% of all SARS-Cov-2 infected people ^[3]. Studies from different countries suggest that the UK estimate understates the scale of the long COVID/PASC problem.

1.2. The Frequency of Long COVID/PASC Due to Infection and Re-Infection

A study of the outcomes of SARS-CoV-2 infection versus re-infection was performed using data collected by The Department of Veterans Affairs, USA, to determine the six-month burdens of all-cause mortality, hospitalization, and a set of pre-specified incident outcomes. The study also detected the incidence and prevalence of long COVID/PASC. The cohort comprised of first infection (n = 257,427), re-infection (two or more infections, n = 38,926), and a non-infected control group (n = 5,396,855) ^[4].

The first infection symptom burden per thousand persons was compared to re-infection symptom burden per thousand persons, six months after SARS-CoV-2 infection and ranked by burden frequency.

Ranking the data by burden per thousand persons six months after estimated date of infection revealed the frequency of long COVID/PASC in this cohort: of the first infected, 36% had long COVID/PASC at 6 months (at least 1 sequela) and 55% of the re-infected group had long COVID/PASC at 6 months (at least 1 sequela). People with a re-infection also had an increased frequency of hospitalization and death compared to those with first infection.

In people with a first SARS-CoV-2 infection, re-infection contributed additional risks of all-cause mortality, hospitalization, and adverse health outcomes of long COVID/PASC. The descending order of frequency of symptoms and organ pathologies for long COVID/PASC was revealed to be: mental health problems, neurologic, musculo-skeletal, gastro-intestinal, cardio-vascular, diabetes, pulmonary, fatigue, coagulation-haematology and kidneys.

These risks of long COVID/PASC were significant in those individuals who were unvaccinated, who had had 1 vaccine shot, or had had 2 or more vaccine shots, prior to a second SARS-CoV-2 infection. Evidence is accumulating that the re-infection risk is significantly higher with the SARS-CoV-2 B.1.1.529 Omicron and its descendants. This highly infectious variant appeared suddenly in November 2021 in Botswana but there is still no evidence of the expected prior natural ancestor variants. Generally, re-infection with SARS-CoV-2 increases the risk of developing long COVID/PASC.

2. SARS-CoV-2 Infection, Acute COVID-19 and Long COVID

2.1. Markers of Immune Perturbation after Acute COVID-19

SARS-CoV-2 infection causes reduced activity of the interferon system, pathological hyper-activation of inflammatory mechanisms, malfunction of early macrophage mobilisation, alterations in the induction of adaptive immune response due to stimulation of effector T-cells with pro-inflammatory properties, followed by ineffective elimination of SARS-CoV-2 ^[5].

Recovered acute COVID-19 patients have elevated levels of pro-inflammatory cytokines: IL-1b, IL-6, TNFa, INFg, macrophage inflammatory protein-1 (MIP-1) and vascular endothelial growth factor, even six months after infection, compared to uninfected control subjects. long COVID/PASC patients maintain excessively high plasma levels of pro-inflammatory IL-1b, IL-6, and TNFa that are also associated with higher levels of antiplasmin (2AP) leading to hyper-coagulability and formation of fibrinolysis-resistant blood micro-clots. Dysregulation of the immune system in long COVID/PASC patients is also characterized by pathological changes in CD4+ and CD8+ lymphocyte subpopulations, dysregulation of the CD14+/CD16+ monocyte subset, reduced HLA-DR expression and deficits of B-lymphocytes.

2.2. Complexity of Symptoms in long COVID/PASC Patients

A range of symptoms can remain long after SARS-CoV-2 infection and acute COVID-19. However, the relative intensity of symptoms vary between patients, but can be categorized by the relative effects on different organ systems. For example, in the lungs and airways, long COVID patients can suffer chronic sore throat and cough, dyspnoea (breathing difficulty), chest pain, and evidence of chronic inflammation. Inflammation within and around the airways may induce concentric fibrosis around the bronchioles, resulting in airway narrowing or obliteration. This is termed constrictive (or obliterative) bronchiolitis, the development of which may result in persistent dyspnea after resolution of the acute infection ^[6]. In the heart, long COVID patients suffer chest pains, palpitations and myocardial inflammation. In the blood vessels, there is evidence of vessel damage, coagulopathy, microangiopathy, and chronic inflammation ^[7].

In relation to the brain, long COVID/PASC patients report cognitive impairment, concentration problems, sleep disturbances, depression, anxiety, symptoms similar to post-traumatic stress disorder, and evidence of ongoing inflammation. In the gut, long COVID symptoms include nausea, dysbiosis and diarrhoea. In the spleen: depressed T-cell and B-cell counts and evidence of chronic inflammation. In the liver: elevated aspartate-aminotransferase and alanine aminotransferase, and evidence of chronic inflammation. In the kidneys: renal impairment, damage and chronic inflammation. In the pancreas: injury, pancreatitis and evidence of chronic inflammation. In the musculo-skeletal system: fatigue, muscle pain, joint pain, mitochondrial dysfunction and evidence of chronic inflammation.

The origin of this inflammatory process can be traced to the alveoli of the lungs where sustained production of pro-inflammatory cytokines and reactive oxygen species (ROS) is originally released into the surrounding tissue and bloodstream in response to SARS-CoV-2 infection. Endothelial damage caused by the inflammatory response triggers the activation of fibroblasts, which deposit collagen and fibronectin resulting in fibrosis. Endothelial injury, complement activation, platelet activation, and platelet-leukocyte interactions also enhance the release of pro-inflammatory cytokines and disrupt normal coagulant pathways. The hypoxia and hypercoagulable state increases the risk of thrombosis, which affects all organs.

2.3. Long COVID/PASC Compared and Contrasted with ME/CFS

The molecular mechanism that causes sustained symptoms in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) seems to be related to the self-stimulated persistent immune cell perturbation in long COVID/PASC ^[8].

ME/CFS is a disease triggered by an acute viral infection but maintained by a systemic inflammatory cytokine response, chronic immune cell activation and dysregulation. There is no conclusive evidence of any long-term chronic viral infection that maintains ME/CFS, while evidence is accumulating of a chronic self-sustained immune dysfunction.

Similar to long COVID/PASC, early in disease development of ME/CFS, there is a characteristic pattern of pro-inflammatory cytokines in the plasma, in which protein concentrations of IL-1beta, IL-6, IL-8, TNFalpha and INFgamma are elevated, together with other inflammatory cytokines. However, in ME/CFS patients, in contrast to long COVID, the most upregulated gene expression is for the inflammatory cytokine IL-8, (5.6× more mRNA than healthy controls) ^[9].

ME/CFS patients also express 2.4× more mRNA from NFKBIA than healthy controls, which results in the production of the negative regulator NF-kB-Inhibitor-Alpha (also known as IkBa protein), a suppressor of transcription factor NFkB. In addition, ME/CFS patients also express 3.6× more mRNA from TNFAIP3, which results in the production of Tumour-Necrosis-Factor-Alpha-Induced-Protein-3 (also known as A20 protein), which inhibits further TNFa expression, in response to TNFa-induced NF-kB activation. Pro-inflammatory cascades that activate transcription factor NF-kB induce IL-1, IL-6 IL-8 and TNFa, plus a multitude of other cytokine and chemokine receptors required for immune recognition, antigen presentation, and cell adhesion.

In addition, in ME/CFS, there is also evidence of active suppression (compared to healthy controls) of expression of the cytokine-like pro-inflammatory calcium binding proteins S100A8 and S100A9, which trigger inflammatory RAGE and TLR4 signalling. In contrast, S100A8 and S100A9 expression is elevated in long COVID/PASC, and the significance of this shall be discussed later.

Similarly to long COVID/PASC patients, ME/CFS patients suffer brain-centred symptoms of neuroinflammation, including loss of homeostatic control, “brain fog” affecting cognitive ability, impaired circadian clock function, lack of refreshing sleep, and poor response to small stresses. In ME/CFS, it appears that pro-inflammatory cytokines chronically activate microglia in the brain, which further promotes inflammation and neurological dysfunction.

Neuroinflammation activates microglia to release Reactive Oxygen Species (ROS) that have a destructive effect on mitochondria. Neuroinflammation is closely associated with mitochondrial dysfunction, compromised energy production and further oxidative stress. Immortalized lymphocytes from ME/CFS patients have dysregulated mitochondrial function and inefficient production of ATP by Complex-V of the mitochondrial electron transport chain. The interdependence of oxidative stress, neuroinflammation and mitochondrial dysfunction are causative of many of the common symptoms seen in ME/CFS and long COVID/PASC.

3. Chronic Expression of Pro-Inflammatory Cytokines in Long COVID/PASC

3.1. IL-1b, IL-6 and TNFa Expression in Long COVID/PASC

In a study of 121 Post-Acute Sequelae of COVID (PASC) patients, markers of immune activation and inflammation correlated with the persistence of a long COVID state. Inflammatory cytokines were sampled “Early” between 38 and 64 days after infection, and again “Later” between 116 and 136 days, and compared with a Control Group who had recovered completely from acute COVID-19 ^[10].

Comparison between Early and Later sampling of the PASC group showed IL-6 increased over time by up to 50 pg/mL in some PASC patients, and was on average 44% higher than the control group in Later sampling, while IL-6 declined in the Control Group. TNFa was also significantly higher in the PASC group than the Control Group, up to 8 pg/mL higher in some patients, but TNFa decreased over time in both the PASC group and Control Group. There was no significant difference in the serum levels of anti-inflammatory cytokine IL-10 between the PASC group and the Control Group, and in both the Early and Later sampling, there were only low levels of IL-10, approximately 0.8 pg/mL.

IL-6 and TNFa are both proinflammatory cytokines that contribute to leukocyte recruitment, activation, and differentiation, as well as B-cell maturation and the expansion of T-helper cell subsets. The data supports the important roles for chronic expression of IL-6 and TNFa in driving long COVID/PASC. However, IL-1b was not measured.

In DigiHero, a digital epidemiology study in Germany that collected information on consequences of acute COVID-19, 8077 individuals responded to questionnaires and a sub-group was followed-up in a long COVID/PASC-focused survey. Generally, the clinical spectrum of PASC symptoms included fatigue and exercise intolerance, brain fog, shortness of breath, joint pain, fever, sleep and anxiety disorders, as well as gastrointestinal symptoms and palpitations. It was found that after only mild acute COVID-19, post-acute sequelae could persist from 8 months up to 24 months in 60% of patients and the severity ranged from mild to debilitating ^[11].

Using a 21 cytokine panel assay that included IL-1b, IL-4, IL-6, IL-8, IL-13, IL-17a, TNFa, LTA and INFa2, cytokine plasma levels were measured in n = 641 Long COVID /PASC patients, and compared with plasma samples from never-infected control participants and previously SARS-CoV-2 infected/acute COVID-19 recovered participants without PASC. Despite the 8-month interval between acute SARS-CoV-2 infection and blood sampling, participants with prior acute COVID-19 showed patterns of systemic cytokine dysregulation. However, it was found that only chronic elevated expression of IL-1b, IL-6 and TNFa was significantly associated with at least a 10-month persistence of chronic PASC symptoms.

In PASC patients, plasma levels of IL-1b, IL-6 and TNFa remained high and stable, whereas in non-PASC patients they decreased in the post-infection/post-acute COVID-19 phase. The plasma concentrations of these three cytokines also positively correlated with each other in PASC patients, suggesting the expression of IL-1b, IL-6 and TNFa was being driven by a common pro-inflammatory mechanism.

In acute COVID-19, highly elevated levels of plasma IL-6 and high levels of IL-1b and TNFa are associated with over-activated CD14+CD16+ monocytes and macrophages and CD38+HLA-DR+ myeloid (bone-marrow derived) cells in lung tissue and bronchoalveolar fluid (BALF), but not with the activation of peripheral blood monocytes. Single-cell analysis of lung tissue and BALF, compared to the peripheral blood, revealed that the lung monocytes and macrophages are uniquely hyper-activated and dysregulated during acute COVID-19. In addition, BALF-derived macrophages from patients with acute COVID-19 show especially high response scores for IL-1b and TNFa when measuring the respective cytokine receptors IL1R1, IL1R2, TNFRSF1A and TNFRSF1B.

3.2. IL-6 > IL-6R Driven S100A8/A9 Expression in Acute COVID-19

A small clinical study of 12 acute COVID-19 patients compared to 10 healthy controls was performed to assess the efficacy of monoclonal anti-body Tocilizumab, a competitive inhibitor of IL-6 binding to IL-6R.

COVID-19 patients (n = 10) were recruited and compared to age and sex-matched healthy control subjects (n = 13). At various time points Peripheral Blood Mononuclear Cell (PBMC) samples were collected and isolated. Monocytes from patients with progressive acute COVID-19 were expressing high levels of S100A8 and S100A9 proteins, but low levels of HLA-DR. As expected, patients with severe acute COVID-19 had lymphopenia and increased blood levels of inflammatory biomarkers such as C-Reactive Protein (CRP), IL-1b, IL-6, IL-8, and TNFa ^[12].

S100A8, S100A9 and hetero-dimer S100A8/A9 all trigger mRAGE and TLR4 signalling which results in high levels of NFkB mediated IL-6 expression. In addition, secreted IL-6 binds IL-6R and stimulates S100A8 and S100A9 expression in monocytes via activation of STAT3 and C/EBPb. These stimulated monocytes release S100A8/A9 that binds and activates mRAGE and TLR4 to induce much more IL-6. This pro-inflammatory positive-feedback loop between IL-6 expression and S100A8/9 expression is a rapid amplifier of innate pro-inflammatory responses. As expected, anti-IL-6R tocilizumab treatment inhibited IL-6 signalling in the acute COVID-19 patients. However, in addition, tocilizumab treatment also inhibited S100A8 and S100A9 expression and reduced levels of S100A8/A9 in the serum.

4. Other Pro-Inflammatory Factors in Acute COVID-19 (Metabolic Reprogramming, AGEs and HMGB1)

4.1. Virally Induced Metabolic Re-Programming

Recent studies have revealed that SARS-CoV-2 infection distorts and re-programs human metabolic pathways. One hypothesis is that Long COVID/PASC may be due to the chronic pro-inflammatory signalling arising from metabolic re-programming by SARS-CoV-2 proteins, driving high serum concentrations of AGEs and chronic AGE > RAGE signalling (see next section). Virally induced metabolic re-programming inhibits pyruvate and ATP production, while increasing the rate of glycolysis and stimulating synthesis of AGEs, which then amplifies AGE > RAGE pro-inflammatory signalling ^[13].

Hours after SARS-CoV-2 infection of cells, there is inhibition of the production of pyruvate, the end product of glycolysis. This causes a significant amplification of the rate of glycolysis and an accumulation of up-stream metabolic intermediates. The reduced supply of pyruvate to the mitochondria, which is normally catabolized by the Tricarboxylic Acid (TCA) cycle to provide fuel for oxidative phosphorylation, reduces the production of ATP, the basic energy molecule of living systems.

Experimental transfection of plasmids expressing SARS-CoV-2 proteins into lung epithelial BEAS-2B cells demonstrated that SARS-CoV-2 proteins disrupt the terminal step of glycolysis. Normally phosphoenolpyruvate (PEP) is converted into pyruvate, catalysed by isoforms of Pyruvate Kinase Muscle (PKM). PKM has two distinct isoforms: PKM1 and PKM2. PKM1 is continuously expressed and constitutively active, while the enzyme activity of PKM2 is controlled allosterically. PKM2 is only enzymatically active in its tetrameric form, but inactive as a monomer or in dimers.

In lung epithelial BEAS-2B cells, SARS-CoV-2 proteins stimulate increases in the expression of the PKM2 isoform and also stimulate increases in the expression of Polypyrimidine Tract Binding Protein (1PTBP1), which interferes with tyrosine kinases. Virally-induced phosphorylation of PKM2 at Tyrosine-105 inhibits the formation of the active PKM2-tetramer, blocking enzymatic activity. The result is that the conversion of PEP into pyruvate stops and inactive PKM2 dimers accumulate. In this virally perturbed environment, metabolic regulatory feed-back loops cause the rate of glycolysis to increase and increases the concentrations of early intermediates.

Measurement of the mitochondrial Oxygen Consumption Rate (OCR) and the Extracellular Acidification Rate (ECAR) in lung epithelial BEAS-2B cells shows that transfection of SARS-CoV-2 Spike protein also decreases the reaction rate of the TCA cycle and inhibits mitochondrial oxidative phosphorylation.

SARS-CoV-2 induced metabolic re-programming and the elevated concentrations of early intermediates of glycolysis stimulate the non-enzymic chemical synthesis of Advanced Glycation End-products (AGEs). SARS-CoV-2 proteins induce increases in the concentration of AGEs such as N-Carboxymethyl-lysine (CML) and N-carboxy-ethyl-lysine (CEL) in the serum, during the early phase of SARS-CoV-2 infection.

High serum concentrations of CML, CEL and other AGEs trigger mRAGE signalling, causing expression of the pro-inflammatory cytokines IL-1b, IL-6 and TNFa. In addition, the elevated concentrations of AGEs and increased mRAGE-signalling results in higher levels of mRAGE-expression, creating a self-sustaining positive-feedback loop that further amplifies inflammation.

4.2. The Role of AGEs in Acute COVID-19

Advanced Glycation End-products (AGEs) are a heterogeneous group of compounds produced by glycation of amino acids, lipids, and DNA molecules by non-enzymic chemical reactions with glucose and fructose glycolysis intermediates in the presence of inflammation-induced Reactive Oxygen Species (ROS). Serum concentrations of AGEs in SARS-CoV-2 infected patients are significantly higher in those who develop severe acute COVID-19, compared to asymptomatic SARS-CoV-2 infected patients ^[14].

Some AGEs are produced by threonine and lipid peroxidation in reactions that result in the generation of highly reactive alpha dicarbonyl groups. Methylglyoxal (MGO), a by-product in glycolysis during the conversion of dihydroxyacetone phosphate to glyceraldehyde-3, can chemically modify amino acids such as lysine and arginine. AGES, such as N-Carboxymethyl-lysine (CML) and N-carboxy-ethyl-lysine (CEL), are produced in hyperglycaemic disorders such as diabetes. In cardiovascular disease, chronic expression of pro-inflammatory cytokines is implicated in increased ROS production that drives higher serum-AGE concentrations, which favour platelet aggregation, atherosclerotic plaque formation, the entry of inflammatory cells into atherosclerotic plaque lesions and a higher risk of inflammatory thrombocytosis, atherosclerosis and hypertension.

Increased plasma concentrations of AGEs also correlate with age-associated increases in systemic inflammation, sometimes referred to as ‘‘inflammaging’’. AGEs accumulate gradually, and in adults aged 65 and older, serum concentrations of AGEs correlate with an increased risk of mortality due to cardiovascular disease.

4.3. AGE > RAGE-Signalling in Acute COVID-19

Generally, AGEs in the extra-cellular medium trigger pro-inflammatory cytokine expression, particularly in monocytes and macrophages, by binding to cell-membrane Receptor for Advanced Glycation End-products (mRAGE) ^[15]. Clinical studies on acute COVID-19 show that higher cell-surface expression and signalling activity of mRAGE correlates with acute COVID-19 severity. In addition, the comorbidities that are risk factors for severe acute COVID-19 are also associated with hyper-activated RAGE signalling. The COVID-19 co-morbidities include: ageing, diabetes, obesity, atherosclerosis, cancer, and Chronic Obstructive Pulmonary Disease (COPD). RAGE is associated with a broad range of inflammatory, degenerative and hyperproliferative diseases, including sepsis, rheumatoid arthritis, diabetic nephropathy, atherosclerosis, cancer, and neurological disorders ^[16].

Membrane-expressed RAGE (mRAGE) belongs to the immunoglobulin superfamily. It comprises of five domains: an extra-cellular ligand binding Ig-V domain, on top of a Ig-C1 domain, sitting on a Ig-C2 domain that is connected to its Transmembrane domain and Cytoplasmic tail, which binds sub-membrane signalling proteins. Cell-surface mRAGE is expressed in high density on the linings of the airways. In the lungs, RAGE is found on the luminal membrane of type I pneumocytes, as well as monocytes, macrophages and endothelial cells. RAGE signalling also plays a role in the differentiation of monocytes and macrophages in the lungs. mRAGE is also expressed on the surface of many types of cell including epithelial cells, fibroblasts, endothelial cells, monocytes, macrophages, neutrophils, dendritic cells, vascular cells, neuronal cells, cardiomyocytes, adipocytes, podocytes and many others.

mRAGE binds Pathogen-Associated Molecular Patterns (PAMPs) in macromolecules of invading micro-organisms. mRAGE-signalling plays a key role in rapid triggering of innate immune responses to new infections and molecular patterns that indicate host tissue damage. RAGE signalling is also involved in the induction of phagocytotic activity in neutrophils, in controlling the adhesion and transmigration of granulocytes, and in the maturation of dendritic cells. mRAGE is also triggered by “alarmins”, such as High Mobility Group Box-1 protein (HMGB1) and S100 proteins, and other Damage Associated Molecular Pattern molecules (DAMPs) such as β2-Integrin, Macrophage-1-antigen (Mac-1) and CD11b.

RAGE is highly expressed during embryonic development, but generally at low levels in most tissues of healthy adults. The significant exception is that mRAGE remains abundant on type 1 Alveolar Epithelial Cells (AECs), monocytes and macrophages in adult lung tissue. However, RAGE is re-expressed and over-expressed in age-related chronic inflammatory diseases, such as atherosclerosis, cardiovascular disease, liver disease, type 2 diabetes, osteoarthritis, nephropathy, neuropathy, brain diseases and in cancer. RAGE is encoded by the AGER gene that has multiple single-nucleotide polymorphisms. AGER genetic polymorphisms correlate with the differential severity of pathological and age-related inflammatory conditions. AGE > RAGE signalling sustains “inflammaging”, the low-grade chronic inflammation that develops during aging.

RAGE also exists in serum as soluble RAGE (sRAGE), which is the proteolytic product of Matrix-Metallo-Proteinase (MMP) cleavage of mRAGE. Soluble sRAGE in the intra-cellular medium acts as a competitor and regulator of mRAGE signalling by binding and inactivating RAGE ligands, such as AGEs. High levels of RAGE-signalling in severe acute COVID-19 correlates with the detection of high levels of sRAGE in serum ^[17].

4.4. SARS-CoV-2 Spike-Protein Binds mRAGE to Infect Lung Monocytes

SARS-CoV-2 infection induces a severe immune dysregulation in monocytes and macrophages, which determines the severity of acute COVID-19 and the probability of long COVID/PASC. However, circulating primary monocytes and macrophages express neither the ACE2 Receptor nor TMPRSS2, the main cell membrane proteins assumed essential for cell invasion by SARS-CoV-2.

It has been recently established in vitro, in Peripheral Blood Mononuclear Cells (PBMCs), that SARS-CoV-2 infects monocytes as a result of specific binding of the viral Spike protein S1-RBD domain to the Receptor for Advanced Glycation End-products (RAGE). This interaction between SARS-CoV-2 Spike and RAGE was verified in co-immunoprecipitation and anti-body blocking experiments. Spike-binding RAGE induces cell signalling via p38MAPK, activation of transcription factor NFkB and up-regulation of expression of the inflammatory cytokines: IL-1, IL-6 IL-8 and TNFa ^[18].

4.5. Virus Induction of HMGB1 > RAGE Signalling in Acute COVID-19

In healthy individuals, High Mobility Group Box-1 protein (HMGB1) is found in the nucleus where it binds to DNA. However, HMGB1 also acts as a Damage Associated Molecular Pattern molecule (DAMP) in the serum, where it forms complexes with extra-cellular DNA and RNA arising from infection and cell damage. HMGB1-RNA/DNA complexes bind to mRAGE, and Toll-Like Receptors such as TLR2 and TLR4, to initiate pro-inflammatory responses ^[19].

In respiratory epithelial cells, monocytes and macrophages, HMGB1 triggering of mRAGE and TLRs leads to p38MAPK phosphorylation and activation of transcription factor NFkB. This causes expression of pro-inflammatory cytokines IL-1, IL-6, IL-8 and TNFa, together with chemokines and adhesion molecules, such as ICAM-1. In addition, the activation of mRAGE receptors triggers ROS-dependent activation of MEK > ERK > IKKb signalling, which also amplifies NF-κB signalling pathways and pro-inflammatory cytokine expression.

HMGB1 plays a significant role in the disease progression of acute COVID-19. In acute COVID-19 patients, high HMGB1 serum levels from SARS-CoV-2 lysed cells and dying non-immune cells cause hyper-expression of inflammatory cytokines, Acute Lung Injury (ALI), Acute Respiratory Distress Syndrome (ARDS) and mortality. Elevations of serum concentrations of pro-inflammatory cytokines trigger the secretion of even more HMGB1 from monocytes and macrophages. High serum levels of HMGB1 are associated with “late” amplification of inflammation and ALI.

HMGB1-driven pro-inflammatory processes observed in SARS-CoV-2 infection and acute COVID-19 resemble pro-inflammatory processes induced by other viral infections. For example, Respiratory Syncytial Virus (RSV) causes lower respiratory tract infection and acute bronchiolitis in children, which correlates with high concentrations of HMGB1 in the nasopharyngeal samples. Dengue virus induces systemic inflammation and endothelial injury that is caused by the release of HMGB1 from monocytes.

Obesity and kidney disease also increase serum HMGB1 concentrations and are co-morbidities that increase the risk of severe acute COVID-19. In the obese, dying adipocytes release HMGB1 and trigger mRAGE, causing the secretion of inflammatory cytokines and production of reactive-oxygen species (ROS). In kidney disease, HMGB1 stimulates RAGE-mediated production of ROS and sustained nephropathy. Generally, high serum HMGB1 concentrations in the lungs trigger endothelial inflammation, the formation of Neutrophil Extracellular Traps (NETs), the release of Platelet Derived Growth Factor (PDGF) and Transforming Growth Factor (TGF). Activation of platelets leads to coagulopathy, micro-clots and thrombosis. In acute COVID-19, high serum concentrations of HMGB1 predispose patients to high risk of acute ischemic stroke.

4.6. S100A8/A9 > RAGE-Signalling in Acute COVID-19 and Long COVID

The expression of genes for calcium-binding proteins S100A8 and S100A9 are significantly upregulated in severe forms of acute COVID-19 and Acute Respiratory Distress Syndrome (ARDS). In severely ill acute COVID-19 patients, serum levels of S100A8/A9 hetero-dimer protein (also known as calprotectin) are highly elevated. S100A8/A9 acts like a pro-inflammatory cytokine and is associated with the development of severe acute COVID-19 and increased mortality. High serum concentrations of S100A8/A9 trigger both TLR and RAGE signalling, driving hyper-inflammation in severe acute COVID-19.

In acute COVID-19, mature neutrophils containing large amounts of S100A8/A9 migrate into the lungs, where the S100A8/A9 is released. Retro-analysis of clinical studies of acute COVID-19 patients revealed that S100A8/A9 heterodimer levels predicted cases associated with poor clinical outcomes. In acute COVID-19 patients, S100A8 expression was markedly higher compared to healthy subjects, and serum concentrations of S100A8/A9 correlated with the severity of acute COVID-19 ^[20]^[21]^[22]^[23]^[24].

Elevated serum levels of S100A8/A9 at hospital admission of SARS-CoV-2-infected patients that correlate with inferior clinical outcomes with acute COVID-19 are between 5 micro-gram (10⁻⁶g) /mL and 10 micro-gram (10⁻⁶g) /mL. In contrast, the pro-inflammatory cytokines IL-1b and IL-6 can activate cells at serum concentrations of 10 pico-grams (10⁻¹²g) per ml. S100A8/A9 is approximately a million times less potent than IL-1b and IL-6 as a pro-inflammatory effector, but its serum concentrations are huge. In vitro experiments show that 15 μg/mL S100A8/A9 can induce IL-8 release from bronchial epithelial cells, 50 μg/mL S100A8/A9 induces freshly isolated monocytes to express significant levels of TNFa, and 100 μg/mL S100A8/A9 directly activates endothelial cells to induce pro-inflammatory chemokines and adhesion molecules ^[25].

Long-term longitudinal whole-blood RNA sequencing of SARS-CoV-2 infected patients who had suffered acute COVID-19 has revealed specific perturbations of the peripheral immune system, even six months after their confirmed date of SARS-CoV-2 infection. Cell analysis revealed expression of S100A8/A9 and HMGB1 were still strongly up-regulated six months after SARS-CoV-2 infection. Long COVID/PASC also correlates with over-expression of S100A8/A9 heterodimers in CD4+ T-cells. So far, no clinical study on the serum concentrations of S100A8/A9 in long COVID/PASC patients has been published ^[26]^[27].

References

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