Genetic Predisposition and Inflammatory Inhibitors in COVID-19: Where Do We Stand?

Severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) and the resulting coronavirus disease-19 (COVID-19) have led to a global pandemic associated with high fatality rates. COVID-19 primarily manifests in the respiratory system as an acute respiratory distress syndrome following viral entry through the angiotensin-converting enzyme-2 (ACE2) that is present in pulmonary epithelial cells. Central in COVID-19 is the burst of cytokines, known as a “cytokine storm”, and the subsequent widespread endothelial activation, leading to cardiovascular complications such as myocarditis, arrhythmias, and adverse vascular events, among others. Genetic alterations may play an additive, detrimental role in the clinical course of patients with COVID-19, since gene alterations concerning ACE2, major histocompatibility complex class I, and toll-like receptors may predispose patients to a worse clinical outcome. Since the role of inflammation is quintessential in COVID-19, pharmacologic inhibition of various signaling pathways such as the interleukin-1 and -6, tumor necrosis factor-alpha, interferon gamma, Janus kinase-signal transducer and activator of transcription, and granulocyte–macrophage colony-stimulating factor may ameliorate the prognosis following timely administration. Finally, frequently used, non-specific anti-inflammatory agents such as corticosteroids, statins, colchicine, and macrolides represent additional therapeutic considerations.


Introduction
Coronaviruses (CoVs) are RNA viruses with a single strand that belong to the Coronaviridae family while four CoVs categories have thus far been identified: α, β, γ, δ. SARS-CoV2 penetrates human cells by attaching to the angiotensin-converting enzyme 2 (ACE2), abundant in alveolar lung cells, vascular endothelium, cardiac myocytes, and other cells [1]. The novel coronavirus disease 2019 (COVID-19) emerged as a severe acute respiratory illness and was proclaimed a pandemic on 30 January 2020, affecting primarily the residents of Wuhan, Hubei Province in China [2,3].
The course of the disease is mild in the large proportion of patients, while severe cases with hospitalization and high mortality rates also occur. Physicians have tried to classify the disease course by dividing it into four stages [4,5]. In the first stage (Stage I), fever, dry cough, tiredness, and myalgia are the most common symptoms which are, however, not specific to the disease. In the second stage (Stage II), bilateral pulmonary parenchymal ground-glass and consolidative pulmonary opacities are presented on the computed tomography scan in the vast majority of COVID-19 patients with viral pneumonia [4]. A hypercoagulable state has been observed, especially in hospitalized patients in Stage III of

Introduction
Coronaviruses (CoVs) are RNA viruses with a single strand that belong to the Co naviridae family while four CoVs categories have thus far been identified: α, β, γ, δ. SA CoV2 penetrates human cells by attaching to the angiotensin-converting enzym (ACE2), abundant in alveolar lung cells, vascular endothelium, cardiac myocytes, a other cells [1]. The novel coronavirus disease 2019 (COVID-19) emerged as a severe ac respiratory illness and was proclaimed a pandemic on 30 January 2020, affecting prim ily the residents of Wuhan, Hubei Province in China [2,3].
The course of the disease is mild in the large proportion of patients, while sev cases with hospitalization and high mortality rates also occur. Physicians have tried classify the disease course by dividing it into four stages [4,5]. In the first stage (Stage fever, dry cough, tiredness, and myalgia are the most common symptoms which are, ho ever, not specific to the disease. In the second stage (Stage II), bilateral pulmonary par chymal ground-glass and consolidative pulmonary opacities are presented on the co puted tomography scan in the vast majority of COVID-19 patients with viral pneumo  , MCP-1, MIP-1α, G-CSF, and TNF-α have been observed in severe infection with marginal statistical significance [27,33]. Airway and alveolar epithelial cell apoptosis was induced by IFN-αβ and IFN-γ, increasing the inflammatory cell infiltration. Apoptosis of endothelial and epithelial cells affects the pulmonary microvascular and alveolar epithelial cell barriers, causing vascular leakage, alveolar edema, and, eventually, hypoxia [7,25]. The studies mentioned above emphasize that a failure in initial type-I and III IFN responses to SARS-CoV-2 leads to an excessive late immune response and severe form of COVID-19. The pro-inflammatory feed-forward loop of cytokines on innate immune cells results in a cytokine storm, coagulopathy, and acute respiratory distress syndrome (ARDS) [34,35]. On the other hand, in contrast with the widespread hypothesis of failure of the immune system, two other studies presented an interesting and quite different concept of cytokine storm onset, differentiating it into two stages. In the first, a short-term immune-deficient state is considered, followed by a second overactive immune condition which tries to counterbalance the agitated entropy from temporary immune target failure, driving to a cytokine storm [7,36]. As such, further molecular research on patients presented with cytokine storm caused by COVID-19 is required to clarify the phenomenon (Figure 1).

Figure 1.
SARS-CoV-2 invasion and hyper-inflammatory state in close relation with genetic predisposition. The presence of Angiotensin-converting enzyme 2 (ACE2) and Transmembrane protease serine 2 (TMPRSS-2) that may cleave the viral spike is required for SARS-CoV-2's cell invasion. Increased levels of pro-inflammatory cytokines, particularly the soluble interleukin 2-receptor (IL-2R) and interleukin-6 (IL-6) have been found. Soluble IL-2R (sIL-2R) is mostly released by activated T helper lymphocytes, although it may also be secreted by endothelial cells (ECs). The capillary leak is caused by the binding of IL-6 and IL-2 to their receptors. The persistent burdening of the endothelium results in increased release of inflammatory cytokines and immune system overreaction, resulting in the so-called "cytokine storm". The above mentioned hyper-inflammatory state is in close relation with the individual genetic profile which can potentially govern the course of the disease. Abbreviations: SARS CoV-2 = Severe acute respiratory syndrome Coronavirus-2, ACE2= Angiotensin-converting enzyme 2, TMPRSS = Transmembrane protease serine 2, IL = Interleukin, ΡΑΙ-1 = Plasminogen activator inhibitor-1, TNF = Tumor Necrosis Factor, ICAM = Intercellular Adhesion Molecule 1, MCP-1 = monocyte chemoattractant protein-1, G-CSF = Granulocyte colony-stimulating factor, IP-10 = Interferon gamma-induced protein 10, MIP-1 = Macrophage inflammatory protein-1, IFN = Interferon.

Genetic Predisposition
Many researchers support the position that cytokine storm in COVID-19 is related to individual genetic predisposition and vulnerability. This theory was strengthened by the evidence of genetic vulnerability in patients marked by primary HLH or cytokine storm in Still disease [14]. Failure of perforin and the activation of NK and cytotoxic T lymphocytes have been demonstrated as the main components of these pathologic entities.
Several studies have reported associations between human genes and COVID-19. ABO blood groups have been assessed in susceptibility to SARS-CoV-2, revealing a higher risk of infection for blood group A than non-A and a lower risk of infection for blood group O compared to non-O [37]. It is assumed that the formation of neutralizing antibodies against protein-linked N-glycans or indirect effects such as the stability of the von Willebrand factor have a partial impact on differing susceptibility [38][39][40]. Although the O blood type group seems to have an advantage concerning the susceptibility to SARS-CoV-2, there was no association between the ABO blood group and the severity of COVID-19 disease or mortality rate according to a recent meta-analysis [41]. As discussed above, invasion of SARS-CoV-2 is dependent on ACE2 and the transmembrane serine protease (TMPRSS2) [42]. Polymorphisms on the gene of ACE2 have been associated with The presence of Angiotensin-converting enzyme 2 (ACE2) and Transmembrane protease serine 2 (TMPRSS-2) that may cleave the viral spike is required for SARS-CoV-2's cell invasion. Increased levels of pro-inflammatory cytokines, particularly the soluble interleukin 2-receptor (IL-2R) and interleukin-6 (IL-6) have been found. Soluble IL-2R (sIL-2R) is mostly released by activated T helper lymphocytes, although it may also be secreted by endothelial cells (ECs). The capillary leak is caused by the binding of IL-6 and IL-2 to their receptors. The persistent burdening of the endothelium results in increased release of inflammatory cytokines and immune system overreaction, resulting in the so-called "cytokine storm". The above mentioned hyper-inflammatory state is in close relation with the individual genetic profile which can potentially govern the course of the disease. Abbreviations: SARS CoV-2 = Severe acute respiratory syndrome Coronavirus-2, ACE2 = Angiotensin-converting enzyme 2, TMPRSS = Transmembrane protease serine 2, IL = Interleukin, PAI-1 = Plasminogen activator inhibitor-1, TNF = Tumor Necrosis Factor, ICAM = Intercellular Adhesion Molecule 1, MCP-1 = monocyte chemoattractant protein-1, G-CSF = Granulocyte colony-stimulating factor, IP-10 = Interferon gamma-induced protein 10, MIP-1 = Macrophage inflammatory protein-1, IFN = Interferon.

Genetic Predisposition
Many researchers support the position that cytokine storm in COVID-19 is related to individual genetic predisposition and vulnerability. This theory was strengthened by the evidence of genetic vulnerability in patients marked by primary HLH or cytokine storm in Still disease [14]. Failure of perforin and the activation of NK and cytotoxic T lymphocytes have been demonstrated as the main components of these pathologic entities.
Several studies have reported associations between human genes and COVID-19. ABO blood groups have been assessed in susceptibility to SARS-CoV-2, revealing a higher risk of infection for blood group A than non-A and a lower risk of infection for blood group O compared to non-O [37]. It is assumed that the formation of neutralizing antibodies against protein-linked N-glycans or indirect effects such as the stability of the von Willebrand factor have a partial impact on differing susceptibility [38][39][40]. Although the O blood type group seems to have an advantage concerning the susceptibility to SARS-CoV-2, there was no association between the ABO blood group and the severity of COVID-19 disease or mortality rate according to a recent meta-analysis [41]. As discussed above, invasion of SARS-CoV-2 is dependent on ACE2 and the transmembrane serine protease (TMPRSS2) [42]. Polymorphisms on the gene of ACE2 have been associated with adverse cardiovascular and pulmonary conditions in severe COVID-19 patients due to alterations to angiotensinogen. Furthermore, the localization of the ACE2 gene on the X chromosome may contribute to the generally higher burden observed in males as compared to in females [42]. On the other hand, a recent study illustrated that several ACE2 variants including K31R, N33I, H34R, E35K, E37K, D38V, Y50F, N51S, M62V, K68E, F72V, Y83H, G326E, G352V, D355N, Q388L, and D509Y have less affinity to bind SARS-CoV-2 [43]. Although large cohort studies have not been completed yet and these variants are rare in the general population, this observation could be a cornerstone in the management of the disease [43]. In different populations, no polymorphisms or mutations associated with S binding protein have been documented [43]. Another analysis suggested that polymorphisms including rs233574, rs2074192, and rs4646188 would change COVID-19 binding to ACE2 expressing a protective profile [44]. Of interest is the fact that three well-known polymorphisms of ACE2 (p.(Asn720Asp), p.(Lys26Arg), and p.(Gly211Arg), as well as two rare variants p.(Leu351Val) and p.(Pro389His) have been identified and associated with a better course of the disease [45]. TMPRSS2 enzyme activity is important for coronavirus spread and pathogenesis in the infected host. The polymorphism p.Val160Met (rs12329760) seems to be related to increased susceptibility to SARS-CoV-2 while the oncogenic role of TMPRSS2 may be linked to poor disease outcomes [46].
The apolipoprotein E (ApoE) e4e4 homozygous genotype has been observed to enhance the risk of severe COVID-19, regardless of prior dementia, cardiovascular illness, or type 2 [47][48][49]. ApoE e4 rules the macrophage pro-/anti-inflammatory phenotypes, and it is expressed in type II alveolar cells in the lungs where the ACE2, which SARS-CoV-2 employs for cell entrance, is abundantly co-expressed [48,49]. Association of major histocompatibility complex (MHC) class I genes (human leukocyte antigen [HLA] A, B, and C) and the susceptibility to SARS-CoV-2 have been observed. More specifically, assessing the binding affinity across HLA phenotypes and viral peptides, it is shown that harbors of HLA-B*46:01, HLA-A*11:01, -B*51:01, -C*14:02, HLA-DRB1*15:01, -DQB1*06:02, and -B*27:07 alleles are more vulnerable to SARS-CoV-2, and these mutations predispose patients to a worse disease course [50]. Loss-of-function variants of the X chromosomal Toll-Like Receptor 7 (TLR7) gene have been described in individuals. The primary pathophysiologic mechanism predisposing patients to severe COVID-19 disease is the impaired type I and II IFN response and the retarded immune system reaction [50]. A wide genetic analysis of blood samples of 332 COVID-19 patients in China revealed that the most significant gene loci related to disease severity were the Transmembrane protein 189 and Ubiquitin Conjugating Enzyme E2 V1 (TMEM189-UBE2V1) which play an orchestrating role in the IL-1 signaling pathway [51]. As far as the complement protein system is concerned, polymorphisms such as C3 FF, C3 FS, and C3 SS have been recognized as potentially deleterious for the susceptibility to SARS-CoV-2 and the course of the disease. These preliminary data should be confirmed by the large ongoing SOLID-C19 trial [52]. Finally, the Solute Carrier Family 6 Member 20 (SLC6A20), Leucine zipper transcription factor like 1 (LZTFL1), C-C chemokine receptor type 9 (CCR9), FYVE and Coiled-Coil Domain Autophagy Adaptor 1 (FYCO1), C-X-C Motif Chemokine Receptor 6 (CXCR6), and X-C Motif Chemokine Receptor 1 (XCR1) are genes highly expressed in human lung cells, and polymorphisms have been related to the increased risk of respiratory failure and ARDS in severe COVID-19 cases [53,54] (Table 1). Better cardiovascular and pulmonary course of the disease, less susceptibility to SARS-CoV-2 [42][43][44][45].
Severe course of the disease and potentially higher odds for ARDS [53,54].
TMEM189-UBE2V1 rs6020298-A Severe course of the disease [51].  Several pro-inflammatory cytokines have been studied to find therapeutic options in severe COVID-19 cases. IL-1 is a pro-inflammatory cytokine comprised of two ligands, IL-1α and IL-1β. IL-1 promotes inflammation by recruiting immune cells and inducing secondary cytokine production, culminating in acute phase responses. Anakinra is a modified form of the human IL-1 inhibitor [55], which targets the IL-1 family, and studies showed decreased CRP and IL-6 levels, markers related to a substantial increase in mortality in severe COVID-19 illness [56,57]. In a cohort trial, patients with COVID-19 and ARDS who received Anakinra lived without the requirement for non-invasive ventilation outside of the ICU, and high-dose therapy was safe and linked with clinical improvement in 72% of patients [58]. Observational research discovered that taking Anakinra in addition to methylprednisolone decreased mortality in individuals with hyperinflammation, respira-tory dysfunction, or who were on mechanical ventilation [59]. Finally, Canakinumab could be a beneficial IL-1 antagonist with research showing improvement in oxygen-support requirements and overall mortality rates [60]. Canakinumab's effectiveness for COVID-19 needs additional investigation in randomized controlled trials.

Inhibition of IL-6 Signaling
Infection and tissue injury provoke the secretion of IL-6, which promotes the differentiation of B and T cells, the production of acute-phase proteins, and the regulation of hepcidin levels. As such, IL-6 is a principal pro-inflammatory component that regulates the native immune response. Tocilizumab, an IL-6 receptor antagonist that inhibits signal transduction by binding soluble IL-6R and membrane-bound IL-6R and can be used in patients with bilateral pneumonia caused by SARS-CoV19, is one of the IL-6 options [61]. It was formally included in the National Health Commission of China's COVID-19 diagnosis and treatment program and was recently approved by the Infectious Diseases Society of America. Clinical data demonstrated that most patients' clinical course, hypoxygenemia, and computed tomography opacity alterations improved rapidly following Tocilizumab therapy [62]. The medication was associated with better overall recovery but more extended hospital stays due to unfavorable metabolic, respiratory, and infectious complications [61][62][63][64]. In a study that included 1351 patients, the use of Tocilizumab showed a significant decrease in the risk of mechanical ventilation or death. Of interest is the fact that the agent was associated with a 55% increase in survival rate in patients with mechanical ventilation needs [65]. Finally, Sarilumab, another anti-human IL-6 receptor monoclonal antibody previously used to treat rheumatoid arthritis, was found to be effective in patients with COVID-19 and hospitalization. A preliminary analysis of a trial in the United States found that Sarilumab was helpful in severely ill COVID-19 patients (those who needed mechanical breathing or high-flow oxygenation or who needed to be treated in an intensive care unit) [66].

Inhibition of TNF-α Signaling
During the acute inflammatory state, macrophages, as well as monocytes, B cells, and other tissues, can produce TNF-α. Activation of TNF-α induces the secretion of IL-1 and IL-6. As such, it stands to reason that drugs targeting this cytokine would be of great benefit throughout the disease [67]. Although increased levels of TNF-α have been revealed in various types of cytokine storms, no significant benefit was observed considering the treatment of the phenomenon. Etanercept, Adalimumab, and Infliximab have to be administered as soon as possible from the onset of symptoms since delayed therapy may reduce the efficacy of these agents [68,69]. Although no studies have been completed, preliminary data show that it could be used to treat mild COVID-19 pneumonia to reduce IL-1 and IL-6 levels as possible endpoints [68,69].

Inhibition of IFN-γ Signaling
IFN-γ has been recognized as a critical pro-inflammatory component in various cytokine storms such as primary HLH [70]. Its ability to stimulate the inflammatory response has made it an exciting research topic, particularly during the COVID-19 era. Since its FDA approval in 2018, Emapalumab, an anti-IFN-monoclonal antibody, has been used to treat primary HLH. Trials showed a significant reduction of C-X-C motif chemokine ligand 9 (CXCL9) levels in the bloodstream with clinical improvement. Trials on COVID-19 patients using Emapalumab are ongoing with hopes that it could be a valid treatment option [71].

Inhibition of JAK Pathway
The Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway is commonly involved in various cytokine activation processes [72]. JAK is an intracellular tyrosine kinase that mediates cytokines, hormones, and growth factor signals. The use of JAK inhibition in COVID-19 patients will potentially reduce the inflammatory burden, inhibiting the secretion of common inflammatory cytokines (IL-1, IL-6, TNF-a, etc.), and impeding the entry and proliferation of SARS-CoV-2 [73]. The latter can be explained by the ability of JAK inhibitors to reduce the action of the AP2-associated protein kinase-1 regulator of SARS-CoV-2 invasion in alveolar epithelial cells. Studies on COVID-19 patients have illustrated rapid clinical improvement with Ruxolitinib or Fedratinib treatment while Baricitinib reduced the rate of intensive care unit admission and fatality and increased discharge rates [72][73][74].

Inhibition of Granulocyte-Macrophage Colony-Stimulating Factor Signaling
Alveolar epithelial cells secrete GM-CSF, which can act as a growth factor and proinflammatory cytokine. GM-CSF regulates the native immune response in the lungs driving pulmonary host defense function against pathogens [75]. In severe COVID-19 patients, particularly during hyper-inflammation state and cytokine storm, GM-CSF levels have been found to be unexpectedly high. As such, and considering the upregulation of inflammatory cytokines and chemokines levels via GM-CSF's action, downregulation of this ligand would be potentially beneficial [76,77]. Multiple trials are trying to assess the efficiency of monoclonal antibodies neutralizing GM-CSF as possible treatments for preventing and curing ARDS, such as Gimsilumab, Lenzilumab, Namilumab, and TJM2 targeting to decrease cytokine levels and mortality rates [76,77].

Corticosteroids
Corticosteroids are effective cytokine inhibitors, primarily suppressing the nuclear factor kappa-light-chain-enhancer of the activated B cell (NF-B) transcription factor [3]. Furthermore, corticosteroids seem to have anti-fibrotic properties with good patient tolerance [78]. Corticosteroids inhibit the production of pro-inflammatory cytokines, avoiding a protracted cytokine response and hastening recovery from pneumonia. Since it is well known that corticosteroids downregulate the native immune response with wide spectrum immunosuppression, studies recommend their use on a low dose (≤0.5-1 mg/kg/day of methylprednisolone or equivalent) and short duration (≤7 days) for patients with severe cases of COVID-19 or ARDS. It has been observed that dexamethasone treatment helps SpO2 levels approach more than 90% in each case. Furthermore, it reduces hospitalization, intubation, and the number of patients who do not require mechanical ventilation [3,79]. The RECOVERY trial showed a significant reduction of mortality risk in the group of patients on mechanical ventilation (by one-third) receiving oxygen (by one-fifth) when applied to the administration of low-dose dexamethasone (6 mg once daily, orally or intravenously) for ten days. The death rates during hospitalization and one month after discharge were reduced [3,79,80]. Corticosteroids were related to decreased all-cause mortality at 28 days in a recent meta-analysis of 1703 critically ill patients with COVID-19. There was no evidence of an increased risk of side effects, and survival rates were comparable in the dexamethasone and hydrocortisone groups [3]. The tone of physicians' awareness is the higher mineralocorticoid activity of methylprednisolone, which indicates a strict control of fluid and sodium administration. A larger dose of corticosteroids did not provide more advantages than a smaller dose [3].

Statins
COVID-19 inflammatory cytokines and chemokines share many similarities with those seen in the early and late phases of atherosclerotic plaque development [81,82]. Statins' anti-inflammatory properties are supported by their action on the mevalonate pathway, which influences endothelial function, inflammation, and coagulation [83]. Furthermore, statins inhibit the overexpression or underexpression of the myeloid differentiation primary response 88 (MyD88) gene by keeping its level in the bloodstream relatively stable. Alterations in the expression of MyD88 have already been linked with higher mortality rates and vulnerability in COVID-19 disease [84,85]. Finally, statins downregulate the secretion of pro-inflammatory cytokines such as CRP and IL-6 in animal models, while they have become the gold standard therapy for cardiovascular risk reduction and prevention [86].

Colchicine-Macrolides
Colchicine's anti-inflammatory properties are based on two mechanisms: (a) direct inhibition of (NOD)-like receptor protein 3 (NLRP3) inflammasome and, consequently, of IL-1β production, and (b) inhibition of the polymerization of microtubes [87]. Although it was considered a novel therapeutic approach for COVID-19 patients, no established benefit has yet been met [88,89]. On the contrary, dose-dependent inhibition of surfactant by affecting alveolars types II pneumocytes has been observed, consequently burdening ARDS patients with potential multiorgan failure and disseminated intravascular coagulation [90,91].
Macrolides such as azithromycin and clarithromycin are agents with high patient tolerance. Their anti-inflammatory and immunomodulatory effects are used widely in cases of viral pneumonia as adjunctive therapy to prevent bacterial infection [92,93]. In the COVID-19 era, azithromycin is used daily for its anti-inflammatory and bacterial preventive properties, but the exact benefit is not fully understood [92,93]. The use of azithromycin in COVID-19 patients is not well established and is still debated, while adverse effects such as prolongation of the QT interval could potentially increase the risk of sudden cardiac death in non-monitored patients [94] (Table 2, Figure 2).

Conclusions
SARS-CoV-2 and the resulting COVID-19 have led to a global pandemic with catastrophic outcomes due to the induced hyperinflammatory state and systemic endothelial activation, which result in pulmonary and cardiovascular complications. Genetic predisposition, as in other inflammatory diseases, might be responsible for

Conclusions
SARS-CoV-2 and the resulting COVID-19 have led to a global pandemic with catastrophic outcomes due to the induced hyperinflammatory state and systemic endothelial activation, which result in pulmonary and cardiovascular complications. Genetic predisposition, as in other inflammatory diseases, might be responsible for alterations in the clinical course of COVID-19 patients through polymorphisms in crucial genes such as ACE2 and MHC class I. Components of the immune response to the virus appear to be primarily related to disease severity, whereas genes related to the binding of the ACE2 cell surfacethe entry point for SARS-CoV-2-during the early stages of infection appear to be largely responsible for the varying susceptibility to SARS-CoV-2. Inflammatory inhibitors are at the forefront of pharmacological management in COVID-19, although their potential has not been fully elucidated till now. The above mentioned would have a potentially large impact on targeted medicines and, more critically, vaccine development. Moving forward, we must acknowledge that each decade of the twenty-first century has seen a new significant coronavirus epidemic: SARS in the 2000s, MERS in the 2010s, and now COVID-19. As such, evidence-based risk assessment might result in individualized preventive methods and treatment approaches while further SARS-CoV-2 molecular research is needed to develop more sensitive genetic-based detection and treatment methods to overcome the pandemic.