key: cord-0861913-1gt4tsu6
authors: Al-Kuraishy, Hayder M.; Al-Gareeb, Ali I.; Akeel Al-hussaniy, Hany; Hadi Al-Harcan, Nasser A.; Alexiou, Athanasios; El-Saber Batiha, Gaber
title: Neutrophil Extracellular Traps (NETs) and Covid-19: A new frontiers for therapeutic modality
date: 2022-01-06
journal: Int Immunopharmacol
DOI: 10.1016/j.intimp.2021.108516
sha: e774422bb299ccc617b0522bb7af77fdaeb29225
doc_id: 861913
cord_uid: 1gt4tsu6

Coronavirus disease 2019 (Covid-19) is a worldwide infectious disease caused by severe acute respiratory coronavirus 2 (SARS-CoV-2). In severe SARS-CoV-2 infection, there are severe inflammatory reactions due to neutrophil recruitments and infiltration in the different organs with the formation of neutrophil extracellular traps (NETs), which involved various complications of SARS-CoV-2 infection. Therefore, the objective of the present review was to explore the potential role of NETs in the pathogenesis of SARS-CoV-2 infection and to identify the targeting drugs against NETs in Covid-19 patients. Different enzyme types are involved in the formation of NETs, such as neutrophil elastase (NE), which degrades nuclear protein and release histones, peptidyl arginine deiminase type 4 (PADA4), which releases chromosomal DNA and gasdermin D, which creates pores in the NTs cell membrane that facilitating expulsion of NT contents. Despite of the beneficial effects of NETs in controlling of invading pathogens, sustained formations of NETs during respiratory viral infections are associated with collateral tissue injury. Excessive development of NETs in SARS-CoV-2 infection is linked with the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) due to creation of the NETs-IL-1β loop. Also, aberrant NTs activation alone or through NETs formation may augment SARS-CoV-2-induced cytokine storm (CS) and macrophage activation syndrome (MAS) in patients with severe Covid-19. Furthermore, NETs formation in SARS-CoV-2 infection is associated with immuno-thrombosis and the development of ALI/ARDS. Therefore, anti-NETs therapy of natural or synthetic sources may mitigate SARS-CoV-2 infection-induced exaggerated immune response, hyperinflammation, immuno-thrombosis, and other complications.

Covid-19 is a global pandemic infectious disease caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2), affecting various organ types, principally the respiratory system, and presenting with pulmonary and extra-pulmonary manifestations [1] . Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are the most severe pulmonary manifestations.

However, extra-pulmonary manifestation likes acute cardiac injury, neurological disorders, pancreatic injury, and acute kidney injury (AKI) are evident [1] . This systemic effect of Covid-19 is linked with the wide distribution of angiotensin-converting enzyme 2 (ACE2), which is an entry point for SARS-CoV-2 [2] . It has been shown that ACE2 is highly expressed in various tissues, including lung alveolar cells type II, proximal renal tubules, immune cells, and intestines [3] . Furthermore, the binding of SARS-CoV-2 to the ACE2 is linked with down-regulation of ACE2, intensification in the level of harmful angiotensin II (AngII), reduction of protective Ang1-7, Ang1-9, and release of pro-inflammatory cytokines [4] .

The World Health Organization (WHO) declaration that this disease is a pandemic, and till late March 2021, the total established cases are 123,012,799, with 2,715,472 deaths. In this universal dilemma, diverse efforts and advancing research are built-up to find effective agents against SARS-CoV-2 from recent or old approved drugs as a repurposing drug strategy [5] . Older age groups and comorbidities such as hypertension, diabetes mellitus, and chronic kidney disease are linked with Covid-19 severity [6] . Regarding the clinical presentations of Covid-19 patients, most of them are asymptomatic or have mild symptoms; however, 10%-20% of them developed and experienced severe to critical clinical presentations with progression of ALI due to the development of hypercytokinemia and cytokine storm (CS) [7] . Thus, the present study aimed to explore the possible role of NETs in the SARS-CoV-2 infection and to identify the targeting drugs against NETs in Covid-19.

Neutrophils (NTs) represent approximately 60% of blood leukocytes and are the primary immune cells and first-line guard alongside entering pathogens [216] . Typically, NTs are formed in humans each day; they have a short half-life and high metabolic functions [9] . It has been reported that NTs are often also participating against viral infections; thereby, a large number of NTs are accumulated in the pulmonary circulation in a steady state due to frequent interactions with invading respiratory viruses [10] . However, exaggerated immune function and activity of NTs are associated with the development of ALI due to the generation of free radicals, reactive oxygen species, and discharge of harmful proteolytic enzymes [11] .

In respiratory viral infections, NTs and other phagocytic cells leave circulation and resident infected pulmonary sites in response to the inflammatory cytokines, chemokines, interferon (IFN), and pathogen-associated molecular patterns (PAMPs) [12] . Likewise, NTs are contemporary in the respiratory tract and show an important role in respiratory immunity against influenza A and avian influenza viruses. In addition, higher NTs in the lower respiratory tract during acute influenza A infection are linked with the infection severity [13] .

Furthermore, NTs illustrate a more vigorous and dynamic defense against respiratory viral infection and other type of infections in cooperation with platelets in forming NETs that protect from viral infections [14] [ Figure 1 ].

Besides, NETosis is defined as the formation of NETs during NTs programmed cell death [14] .

In addition, the formation of NETs may also develop without damage to the NT cell membrane with preserving normal phagocytic and chemotactic functions [15, 217] . Both NETs and NETosis control viral severity; Hiroki et al [16] illustrated that NETosis is a natural process that prevents acute Chikungunya viral infection by reducing systemic viral load.

NETs are net-like structures consisting of neutrophil granule proteins, DNA, and chromatins expelled from the NTs to ensnare invading pathogen [218] . Different enzyme types are involved in the formation of NETs, such as neutrophil elastase (NE), which degrades nuclear protein and release histones, peptidyl arginine deiminase type 4 (PADA4) that releases chromosomal DNA and gasdermin D that creates damage in the NTs cell membrane that facilitating expulsion of NT contents [17] . Despite the beneficial effects of NETs in controlling invading pathogens, sustained formations of NETs during respiratory viral infections are associated with a collateral tissue injury [18] . In addition, various studies showed that higher and excessive formations of NETs might activate inflammatory reactions that induce systemic coagulopathy, localized microthrombosis, and MOD [19] (Figure 2 ).

Generation of Neutrophil extracellular traps (adopted from Papayannopoulos 2018 [19] . exhibited that high mobility group box-1 proteins and histones may trigger the release of proinflammatory cytokines and chemokines from other immune cells; however, this process is limited since high NETs aggregates, degrade and metabolize these chemokines and cytokines [29] . Alongside, NETs also can trigger innate immunity through activation release of IFN from dendritic cells and adaptive immunity via activation of T lymphocytes [29] (Figure 3) . 

In patients with Covid-19, the NTs number is increased with significant lymphopenia, so neutrophil-lymphocyte ratio (NLR) is augmented [222] . High NLR is correlated with underlying inflammatory reactions and is an independent risk factor for Covid- 19 (Figure 4 ). 

The NETs are highly toxic to the vascular endothelium and lung epithelial cells due to their contents, which are histones, MPO, defensins, and cathelicidins [37] . For example, histone has the robust cationic property that binds negative charge host cell membrane leading to cell lysis, tissue damage, and induction of inflammations [37] . Therefore, anti-histone antibody and neutrophil esterase blocking antibodies may reduce NETs-induced tissue damage and ALI [38] .

Thereby, SARS-CoV-2-induced ALI and ARDS might be mediated by induction of NETs formation [223] . Yaqinuddin et al. [39] reported that excessive NETs development is related to the progression of ALI and ARDS due to the creation of the NETs-IL-1β loop that is exaggerated and can cause inflammatory-induced lung damage. NETs-IL-1β loop is developed due to activation of IL-1β by the NETs, and also IL-1β stimulates NETs formation [39] . As well, NETs-IL-1β loop is also created due to activation nod-like receptor pyrin 3 (NLRP3) inflammasome of lung macrophages by NETs during acute SARS-CoV-2 infection the macrophages extrude their contents to form macrophage extracellular traps (METs) similar to that of NETs [223] . NETs 

It has been shown that severe SARS-CoV-2 infection is associated with coagulopathy and NETs level is associated with developing venous thrombosis in mice. However, the precise mechanism of NETs-induced thrombosis is ill-defined.

Nevertheless, diverse studies revealed that platelet activation and immune-mediated fibrin formation could be the proposed mechanisms [64] . During inflammatory reactions caused by dissimilar metabolic and infectious disorders, the platelets membrane-bound TLR4 are activated and interact with the NTs in the formation of NETs [64] . In this way, the NETs also activate the platelet via P-selectin, leading to platelet aggregation and thrombosis [65] . Notably, during mild SARS-CoV-2 infection, physiological immuno-thrombosis is developed and controlled by body homeostatic mechanism; however, in severe SARS-CoV-2 infections, uncontrolled physiological immuno-thrombosis may be controlled extend and develop into pathological immuno-thrombosis [66] . Besides, pathological immuno-thrombosis highly targets pulmonary and renal microcirculations, leading to ALI [67] and acute kidney injury [68] On the other hand, Skendros et al.'s [70] case-controlled study illustrated that activation of both NTs and complemented contribute to inducing thrombotic microangiopathy and hyperinflammation in patients with Covid-19. Complement activation drove immune cells, mainly monocytes, and NTs, to the site of lung injury, and in concert with platelets, can induce micro pulmonary thrombosis and systemic immuno-thrombosis [71] . Various studies showed that sera of Covid-19 patients contain a lot of immune complexes and autoantibodies that activate the complement cascade, mainly C3 [72] . Also, SARS-CoV-2 virions such as nucleocapsid protein can activate C3 through lectin dependent pathway [72] . Complement activations trigger tissue factor expression on the NT cell membrane with significant platelet activation leading to NETs formation and immuno-thrombosis [73] . Therefore, C3 inhibitor (AMY-101) inhibits immuno-thrombosis and thrombo-inflammatory response to the SARS-CoV-2-induced microvascular injury [74] .

NETs production with subsequent suppression of micro-vascular thrombosis [75] . Therefore, C3

inhibitors may reduce NETs-induced immuno-thrombosis and the development of ALI and ARDS in patients with Covid-19 [76] . Indeed, NETs contents can directly lead to immunothrombosis and thrombo-inflammatory [76] . Middleton et al. [77] illustrated that the high MPO-DNA complex released from NETs in the plasma of Covid-19 patients is linked with pulmonary micro-thrombosis and ARDS. In addition, the interaction between NETs and platelet phospholipids activates the plasma kallikrein-kinin system leading to platelet aggregation and thrombosis [78] . Similarly, NETs histones activate platelets TLR causing platelet aggregations [79] . Alongside, progressive binding of NTs to the formed NETs contributes to digestion and degradation of tissue factor inhibitor and anti-thrombin III (natural anticoagulant), thereby augmenting the pro-coagulant activity of thrombin in the induction of intravascular thrombosis [80] .

Moreover, NETs deliver extracellular oxidant contents such as nitric oxide synthase, NADPH oxidase, and MPO that serve as a potential source of toxic histones and DNA, which activate the [105] reported that AMI in Covid-19 might be due to activation and upregulation of prothrombotic factors such as NETs, von-Willebrand factor, factor VIII, and Ddimer. These observations shed light on the critical role of NETs as they link the pathogenesis of AMI and ARDS in Covid-19.

Furthermore, obesity, which is considered an inflammatory status due to adipose tissue dysfunction, is commonly associated with cardiovascular complications that increase the risk for Covid-19 [106, 107] . NETs are increased in obesity due to augmentation of immune cell infiltrations into adipose tissue with subsequent chronic adipose tissue-mediated inflammatory reactions [108] . Therefore, NETs inhibitors may attenuate obesity-induced endothelial dysfunctions and coagulation disorders [109] . Surprisingly, platelet activations are reduced during sepsis in obesity with reducing of NETs formation [110] . Furthermore, experimental studies illustrated that a high-fat diet increases the expression of cathelicidin, a specific marker of NETs formation in mice [111] . Therefore, obesity-mediated NETs formation and endothelial injury might raise the risk of coagulopathy in SARS-CoV-2 infection [112] . serum level is increased in Covid-19, and COX-2 inhibitors might be beneficial in the restoration of human immune response [125] . Inhibition of COX-2 is associated with the elevation of AA, an endogenous antiviral substance associated with inhibiting enveloped viruses such as SARS-CoV-2 and human immune deficiency (HIV) [126] . Therefore, there is a controversy about the potential role in the management of SARS-CoV-2-induced NETs formation.

Thrombomodulin is a cofactor protein mainly expressed on the vascular endothelial cells, modulates thrombin activity [127] . Thrombomodulin-thrombin complex stimulates protein kinase C, which is essential for the anticoagulant pathway [127] . It decreased in sepsis and severe viral infection due to its downregulation by inflammatory cytokines [128] . NETs block fibrinolysis and activate aggregation of platelets, erythrocytes, fibrin, and other clotting factors in the induction of immuno-thrombosis [129] . Recombinant human thrombomodulin (rhTM)

inhibits NETosis and NETs formation [129] . Shrestha et al. [130] showed that rhTM inhibits histone-induced NETs release in vitro and in vivo. Also, rhTM attenuates NETs-induced ALI by reducing NETs accumulation and toxic effects of histone [131] . In Covid-19, SARS-CoV-2 infection is related to high inflammatory cytokine angiopoietin-2 (ANGPT2), which inhibits thrombomodulin-thrombin complex with reduction of physiological anticoagulant [132] . It has been hypothesized by Mazzeffi et al. [133] that administration of rhTM in critically Covid-19 patients may reduce ALI/ARDS via inhibition of NETs formation-induced coagulopathy.

Activated protein C (APC), a serine protease enzyme, has anti-inflammatory, cytoprotective, and anticoagulant effects [134] . APC inhibits the release of extracellular histone, activation of clotting factors, and NETs accumulation and formation [134] . Different preclinical studies demonstrated that APC inhibits thrombin generation and excessive inflammation and ischemicreperfusion-induced tissue injury in bacterial pneumonia [135] . APC also has a protective effect against the development of SARS-CoV-2-induced endothelial injury and coagulopathy via suppression of NETs formation [136] . Guglielmetti et al. [137] offered that recombinant APC might be a therapeutic strategy in Covid-19 through inhibiting inflammation and associated coagulopathy. In critically Covid-19 patients, severe inflammation and thrombosis are the primary determinant factors due to deficiency of APC [138] . In a case series of 10 Covid-19 patients' activities of APC and anti-thrombin are reduced with significant elevation of factor VIII and fibrinogen plasma levels, suggesting a state of hypercoagulability in critically severe Covid-19 patients [139] . Therefore, APC-based therapy is recommended in severely Covid-19 patients to prevent NETs-induced inflammation and coagulopathy.

Anti-high mobility group box-1 (HMGB1) is an endogenous protein released from platelets. It has pro-inflammatory action and regulates NTs chemotaxis [140] . HMGB1 induces thrombus formation, NETosis, and NETs formation through cGMP-dependent activation of plateletneutrophil interaction [140] Exposure of NTs to the higher concentration of HMGB1 results in NETs formation in vitro [141] . Therefore, anti-HMGB1 antibodies may inhibit NETs formation with reduction of circulating DNA-histone complexes [142] . Moreover, anti-HMGB1 antibodies inhibit NETs formation and pro-inflammatory induced-ALI [143] . Street [144] observed that HMGB1 activates autophagy which is concerned with SARS-CoV-2 entry and replication [145] .

Also, down-regulation of ACE2 by SARS-CoV-2 elicits activation of the HMGB1 pathway with subsequent activation of cytokine storm-induced-ALI/ARDS [145] . Therefore, HMGB1inhibitors such as hydroxychloroquine, methotrexate, glycyrrhizin, inflachromene, and salicylic acid derivative might reduce Covid-19 severity [145] . Into the bargain, Dinicolantoio et al. [146] showed that melatonin improves the activity of type I immune response through inhibition of the HMGB1 signaling pathway during SARS-CoV-2 infection. Therefore, HMGB1 is regarded as a target for repurposing drugs in the management of Covid-19.

C1 esterase inhibitor (CIE-INH) is an endogenous inhibitor of the C1 protein of the complement system, regulating the kallikrein system and coagulation pathway [147] . CIE-INH is approved for the management of hereditary angioedema and may reduce NETs-mediated ALI [148] . In addition, CIE-INH blocks histone, NETosis, and NETs formation, thereby reducing the risk of ALI/ARDS [149] . Thomson Heparin is a natural glycosaminoglycan used as an anticoagulant in managing ischemic heart disease, stroke, and deep vein thrombosis [152] . Heparin attenuates NETosis and NETs formation with reduction of circulating histone and associated NF-κB activation in different inflammatory diseases [153] . Moreover, non-anticoagulant heparin (parnaparin) could be used to manage histone and NETs-induced inflammatory diseases [154] . In addition, heparin has a beneficial effect in managing Covid-19 through endothelial protection and prevention of coagulopathy [155] . However, resistance to the heparin effect may develop in severe Covid-19 due to auto-antibody against activated factor X [156] . Thus, high doses of heparin or switching to low molecular weight heparin are recommended [156] . Nonetheless, heparin therapy has a potential role in managing severe Covid-19 through anti-inflammatory, anti-NETs, improvement of lung oxygenation, and prevention development of ARDS [157] .

Human DNAase is a selective enzyme that cleaves and hydrolyzes human DNA in the sputum and mucous, promoting sputum clearance and reducing bronchial secretions' viscosity [158] .

Recombinant human DNAase (rhDNAase) has acute anti-inflammatory effects by suppressing NETosis and NETs formation, reducing NTs infiltrations and expression of thrombin [159] .

Wang et al. [160] illustrated that rhDNAase effectively treats severe sepsis by inhibiting ROS and NETs formation. Therefore, rhDNAase effectively reduces the period of mechanical ventilation in critically severe Covid-19 patients [161] .

Antibiotics may have immunomodulating effects through suppression of NETosis and NETs formation and release of pro-inflammatory cytokines [162] . Azithromycin and other macrolides and chloramphenicol have significant anti-inflammatory effects through inhibition of NETs formation [163] . Furthermore, it has been illustrated that azithromycin inhibits cytokine production, mucin secretion, and bronchial cell proliferation by inhibiting mitogen-activated protein kinase (MAPK) and downstream of the NF-κB pathway [164] . These immunomodulating effects of azithromycin make it a potential candidate in the management of Covid-19 [165] .

Furthermore, doxycycline also blocks NETosis and NETs formation with significant immunomodulating effects; thus, it can be used effectively in Covid-19 [2] . Therefore, the pleiotropic effects of antibiotics, mainly anti-inflammatory and immunomodulatory effects and ani-SARS-CoV-2 effect, can subsidize in controlling severe Covid-19 patients [166] .

Aspirin (acetylsalicylic acid) is a non-steroidal anti-inflammatory drug (NSAID, has antiinflammatory and anti-thrombotic effects used in the treatment of various inflammatory disorders [167] . Aspirin inhibits PGs synthesis and causes irreversible acetylation of platelets COX with suppression of thromboxane A2 leading to a noteworthy antiplatelet effect [168] .

Furthermore, different studies showed that aspirin inhibits NETosis and platelet activations and NETs formation [169] . During inflammatory conditions, the platelets are activated through TLR2/TLR4, leading to the expression of P-selectin, which induces NTs for NETs formation. In addition, activated platelets secret HMBG-1 and platelet factor 4 that together induce NTs for NETs formation [170] . Therefore, aspirin and other antiplatelet inhibits NTs-platelets interactions and formation of intravascular NETs during endotoxemia-induced ALI [171] .

Aspirin also has a direct inhibitory effect on NETs formation via suppression of NTs NF-κB pathway [172] . Recently, aspirin improved clinical outcomes in critically hospitalized Covid-19 patients on mechanical ventilation through suppression of NF-κB pathway and SARS-CoV-2induced NETs formation [173] . Taken together, aspirin is an effective drug in the prevention of SARS-CoV-2-induced ALI and MOF through mitigation of inflammatory reactions and NETs formation [174] .

Sivelestat (ONO-5046) is a competitive, reversible, and selective inhibitor of neutrophil elastase (NEase), not affecting other cellular proteases' activity [175] . Different experimental and preclinical studies showed that sivelestat is effective against ARDS by regulating lung vascular permeability, pulmonary pressure, pathogen clearance, and neutrophil-mediated lung epithelial injury [176] . Okayama et al.'s [177] clinical study observed that sivelestat improves pulmonary function, shortens the duration of mechanical ventilation, and oxygen saturation in patients with systemic inflammatory syndrome and ARDS. Furthermore, Miyoshi et al. [178] illustrated that sivelestat combined with recombinant human soluble thrombomodulin effectively mitigates disseminated intravascular coagulopathy-induced ARDS in intensive care unit (ICU) patients, it increases survival and ventilator-free period. Therefore, sivelestat might be a promising therapy in managing Covid-19-induced ALI, ARDS, and coagulopathy through inhibition of NEase and NETs formation [179] . In addition, sivelestat might be an effective preventive therapy against Covid-19-induced ARDS when given in lymphocytopenic patients before developing neutrophilia and NETs formation [180] . Also, it prevents activation of SARS-CoV-2 S protein and binding with ACE2 [180] .

Chloramidine is an inhibitor of protein arginine deiminase (PAD), which is involved in NETs formation and regulation of immune response [181] . Also, chloramidine reduces NETs formation mediated systemic inflammations in the experimental animals [182] . Recently, it has been shown that PAD inhibitors mitigate inflammatory disorders in multiple myeloma [183] . Therefore, chloramidine and other PAD inhibitors reduce NETs formation in different viral infections [184] .

Furthermore, up to date, Du et al. [185] illustrated that PAD inhibitors might prevent ALI via suppression of NETs formation. Therefore, chloramidine and other PAD inhibitors might be of potential therapeutic role in managing Covid-19 through suppression of NETs-induced inflammatory burst and coagulopathy [186] .

Cyclosporine A is an immunosuppressant agent used to manage autoimmune diseases and organ transplants [187] . It binds cellular cyclophilin and inhibits activated T cells' calcineurin pathway nuclear factor with subsequent inhibition of NETs formation [188] . However, inhibiting NETosis by cyclosporine A may impair the immune response to invading pathogens [189] . In the Covid-19 era, cyclosporine A effectively mitigates exaggerated immune response and cytokine storm with impairment of viral pathogenesis [187, 190] .

Diphenyleneiodonium chloride (DPI) is an oral hypoglycemic drug that inhibits gluconeogenesis and oxidative stress by inhibiting NADPH oxidase, xanthine oxidase, nitric oxide synthase, and oxidoreductase [191] . It has been proposed that DPI may inhibit the release of extracellular DNA and block NETs formation [192] . In addition, DPI exerted potential antiviral effects via suppression of Zika virus-induced NETs formation [193] . Therefore, DPI may reduce SARS-CoV-2-mediated NETs formation and link ALI and immunothrombosis [194] .

Metformin is an insulin-sensitizing agent and is regarded as first-line therapy in managing type 2 diabetes mellitus (T2DM) [87] . It acts through the activation of the AMPK pathway that increases cellular glucose uptake (195) . In addition, Metformin inhibits oxidative stress and the release of pro-inflammatory cytokines through the AMPK pathway-dependent suppression of the mTORC1 signaling pathway [196] . In addition, Metformin has immunoregulating effects via inhibition of NETosis and NETs formation [197] . Recently, Metformin reduces SARS-CoV-2 pathogenesis and NETs formation with subsequent reduced risk of ALI/ARDS in Covid-19 [198] .

Hydroxychloroquine is an antimalarial agent with immunosuppressive and immunomodulating effects used to treat parasitic infections and autoimmune disorders [199] . It inhibits NTs phagocytosis, macrophage activity, and the release of pro-inflammatory cytokines [200] .

Hydroxychloroquine suppressed platelets activation and aggregation induced by inflammatory mediators and thrombin [201] and is regarded as an anti-thrombotic agent in anti-phospholipid syndrome [202] . It has been reported that hydroxychloroquine can interfere with NETosis and NETs formation through clocking TLR9 in mice [203] . Boone et al. [204] observed that hydroxychloroquine attenuates hypercoagulability through inhibition of NETs formation. Therefore, hydroxychloroquine may reduce SARS-CoV-2 induced ALI and cytokine storm by inhibiting inflammatory burst and NETs formation [205] . Thus it can be used effectively in the prevention and treatment of Covid-19 [205] . The up-to-date foundation shows that hydroxychloroquine does not affect clinical outcomes and mortality in patients with Covid-19.

Still, it is effective as a potential prophylactic agent in the early stage of SARS-CoV-2 infection [206] .

N-acetylcysteine (NAC) is a mucolytic agent that decreases mucous viscosity used in the management of paracetamol poisoning, COPD, and oxidative stress treatment [207] . In addition, NAC inhibits ROS-induced NETosis and NETs formation [208] and thrombosis. Thus it may block immunothrombosis in different inflammatory disorders [209] . Recently, NAC has been effective against SARS-CoV-2 infection via interruption of viral replica and development of cytokine storm [210] . In addition, NAC mitigates SARS-CoV-2-induced oxidative stress and endothelial injury and associated coagulopathy through regeneration of endogenous glutathione [211] .

Vitamin D is a fat-soluble secosteroid involved in regulating Ca+2 serum level and bone mineralization [212] . Vitamin D reduces COPD, respiratory viral infections, pulmonary tuberculosis, and metabolic disorders [213] . Moreover, Vitamin D has immunomodulating effects; it blocks NETosis and NETs formation and regulates innate immune response with inhibition release of pro-inflammatory cytokines [214] . Thus, Vitamin D supplementation may reduce Covid-19 mortality and severity via suppression of cytokine storm and regulation of innate immunity against SARS-CoV-2 infection [215] .

NETs formation in SARS-CoV-2 infection is linked with critical complications, including ALI and ARDS, due to the development of hyperinflammation, cytokine storm, and immunothrombosis. Therefore, anti-NETs pharmacotherapy might be a promising goal in the management of patients with severe Covid-19. Additional in vitro and in vivo studies and clinical trials and prospective studies are recommended in this regard.

There is no conflict of interest 

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Is ivermectin-Azithromycin combination the next step for COVID-19

Mechanisms of action and clinical application of macrolides as immunomodulatory medications. Clinical microbiology reviews

Azithromycin in COVID-19 patients: pharmacological mechanism, clinical evidence and prescribing guidelines. Drug safety

Pleiotropic Effects of Tetracyclines in the Management of Covid-19: Emerging Perspectives

Clinical outcomes of aspirin interaction with other non-steroidal anti-inflammatory drugs: a systematic review

Reduced variability to aspirin antiplatelet effect by the coadministration of statins in high-risk patients for cardiovascular disease

Aspirin for prevention and treatment of venous thromboembolism. Blood reviews

Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury. The Journal of clinical investigation

Aspirin, but not tirofiban displays protective effects in endotoxin induced lung injury

Regulation of neutrophil extracellular trap formation by anti-inflammatory drugs

Aspirin use is associated with decreased mechanical ventilation, intensive care unit admission, and in-hospital mortality in hospitalized patients with coronavirus disease 2019

SARS-CoV2 may evade innate immune response, causing uncontrolled neutrophil extracellular traps formation and multi-organ failure

Effect of neutrophil elastase inhibitor (sivelestat sodium) in the treatment of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS): a systematic review and meta-analysis. Internal medicine

Neutrophil elastase inhibitor (sivelestat) attenuates subsequent ventilator-induced lung injury in mice. European journal of pharmacology

Clinical effects of a neutrophil elastase inhibitor, sivelestat, in patients with acute respiratory distress syndrome

Combination therapy with sivelestat and recombinant human soluble thrombomodulin for ARDS and DIC patients. Drug design, development and therapy

Neutrophil Elastase Inhibitor (Sivelestat), may be a Promising Therapeutic Option for Management of Acute Lung Injury/Acute Respiratory Distress Syndrome or Disseminated

Neutrophil Elastase Inhibitors: A potential prophylactic treatment option for SARS-CoV-2-induced respiratory complications

Chloramidine/bisindolylmaleimide-I-mediated inhibition of exosome and microvesicle release and enhanced efficacy of cancer chemotherapy. International journal of molecular sciences

Combination of neutrophil to lymphocyte ratio and platelet to lymphocyte ratio as diagnostic biomarker for rheumatoid arthritis

A novel peptidylarginine deiminase 4 (PAD4) inhibitor BMS-P5 blocks formation of neutrophil extracellular traps and delays progression of multiple myeloma

Pegylated arginine deiminase lowers hepatitis C viral titers and inhibits nitric oxide synthesis

Prevents Renal Ischemia-Reperfusion-Induced Remote Lung Injury

Significance of NETs Formation in COVID-19. Cells

Cyclosporine Attenuates Covid-19: Ensnare or Victory

Efficient neutrophil extracellular trap induction requires mobilization of both intracellular and extracellular calcium pools and is modulated by cyclosporine A. PloS one

Cyclosporine A impairs nucleotide binding oligomerization domain (Nod1)-mediated innate antibacterial renal defenses in mice and human transplant recipients

Cyclosporine therapy in cytokine storm due to coronavirus disease 2019 (COVID-19). Rheumatology international

Preferential inhibition of the plasma membrane NADH oxidase (NOX) activity by diphenyleneiodonium chloride with NADPH as donor. Antioxidants and Redox Signaling

Different procedures of diphenyleneiodonium chloride addition affect neutrophil extracellular trap formation. Analytical biochemistry

Zika virus NS1 affects the junctional integrity of human brain microvascular endothelial cells

Nucleocapsid and Spike Proteins of SARS-CoV-2 Drive Neutrophil Extracellular Trap Formation. Immune Network

Differential effect of metformin and/or glyburide on apelin serum levels in patients with type 2 diabetes mellitus: Concepts and clinical practice

Oxidative stress injury and glucolipotoxicity in type 2 diabetes mellitus: The potential role of Metformin and sitagliptin

Neutrophil extracellular trap mitochondrial DNA and its autoantibody in systemic lupus erythematosus and a proof-of-concept trial of Metformin

Hydroxychloroquine in systemic lupus erythematosus (SLE)

Implant delivering hydroxychloroquine attenuates vaginal T lymphocyte activation and inflammation

Hydroxychloroquine reverses platelet activation induced by human IgG anti-phospholipid antibodies

Hydroxychloroquine as an anti-thrombotic in antiphospholipid syndrome

Hydroxychloroquine inhibiting neutrophil extracellular trap formation alleviates hepatic ischemia/reperfusion injury by blocking TLR9 in mice

Chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps

Chloroquine and hydroxychloroquine in the treatment of COVID-19 with or without diabetes: A systematic search and a narrative review with a special reference to India and other developing countries

Chloroquine and hydroxychloroquine in the treatment of COVID-19: the never-ending story

N-Acetylcysteine as an antioxidant and disulphide breaking agent: the reasons why

Critical role of neutrophil extracellular traps (NETs) in patients with Behcet's disease. Annals of the rheumatic diseases

N-acetylcysteine inhibits thrombosis in a murine model of myeloproliferative neoplasm. Blood advances

Therapeutic blockade of inflammation in severe COVID-19 infection with intravenous N-acetylcysteine

COVID-19: The potential role of copper and N-acetylcysteine (NAC) in a combination of candidate antiviral treatments against SARS-CoV-2. in vivo

Vitamin D: nutrient, hormone, and immunomodulator. Nutrients

Vitamin D deficiency 2.0: an update on the current status worldwide. European journal of clinical nutrition

Vitamin D and immunomodulation in early rheumatoid arthritis: A randomized double-blind placebo-controlled study

Vitamin D in prevention and treatment of COVID-19: current perspective and future prospects

Neutrophil extracellular traps in fungal infection

Capsular polysaccharides from Cryptococcus neoformans modulate production of neutrophil extracellular traps (NETs) by human neutrophils. Scientific reports

Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps

CXCR4 and MIF are required for neutrophil extracellular trap release triggered by Plasmodium-infected erythrocytes. PLoS pathogens

Neutrophil killing of Mycobacterium abscessus by intra-and extracellular mechanisms

Hypertension Severity and Inflammatory Burden as Evaluated by Neutrophil-Lymphocyte Ratio: Role of

Neurological Diseases

Niclosamide for Covid-19: bridging the gap

Common NLRP3 inflammasome inhibitors and Covid-19: Divide and Conquer. Scientific African

Highlights  In severe SARS-CoV-2 infection, there are solid inflammatory reactions due to neutrophil recruitments and formation of NETs.  Sustained formations of NETs during respiratory viral infections are associated with collateral tissue injury.  Anti-NETs of natural or synthetic sources may mitigate SARS-CoV-2 infection-induced complications.  NETs can inactivate a wide range of enveloped and non-enveloped viruses.  NETs can trigger innate immunity through activation release of interferon from dendritic cells and adaptive immunity.  NETs are correlated with underlying inflammatory reactions and is an independent risk factor for Covid-19 severity.