key: cord-0692173-vzqeul71 authors: Henrique Agra Cavalcante-Silva, Luiz; Cristina Madruga Carvalho, Deyse; de Almeida Lima, Éssia; Galvão, José G.F.M.; de França da Silva, Juliane S.; de Sales-Neto, José M.; Rodrigues-Mascarenhas, Sandra title: Neutrophils and COVID-19: The road so far date: 2020-11-30 journal: Int Immunopharmacol DOI: 10.1016/j.intimp.2020.107233 sha: 432b48ff2a9eac584d5185a4bae6b9e2d8d29e90 doc_id: 692173 cord_uid: vzqeul71 The SARS-Cov2 infection triggers a multisystem inflammatory disorder, knowing as COVID-19, a pandemic disease. This disease is characterized by acute respiratory distress syndrome, cytokine-driven hyperinflammation, and leukocytes count changes. The innate immune response has been linked to COVID-19 immunopathogenesis (e.g., dysfunctional IFN response and myeloid inflammation). In this regard, neutrophils have been highlighted as essential effector cells in the development of COVID-19. This review summarized the significant finds about neutrophils and its effector mechanisms (e.g., neutrophils enzymes and cytokines, neutrophil extracellular traps) in COVID-19 so far. The COVID-19 infection starts by exposure to microdroplets present in the exhalations of infected individuals. Then, the SARS-CoV-2 spreads to the bronchioles and alveolar spaces [25] , entrancing into the host cells (e.g., endothelial, epithelial, and smooth muscle cells) by binding the angiotensin-converting enzyme (ACE)-2, a metallopeptidase present on the cell surface [26] [27] [28] [29] . In the lung, SARS-CoV-2 infects the alveolar cells (type I and II pneumocytes and alveolar macrophages) and then starts intracellular replication in pulmonary tissues. Type I and III interferons (IFN) production is an early defense mechanism in the alveolar cells [25] . However, recent researchers have found deficient expression of these cytokines, besides the upregulated expression of chemokines and interleukins [30, 31] . In normal human bronchial epithelial (NHBE) cells culture, the cytokine profile includes the IFNs deficiency and elevated expression of CCL20, CXC-type chemokines, IL-1β, IL-6, and tumor necrosis factor (TNF) [31] . The type I and III IFN absence shows that, although SARS-CoV-2 is sensitive to IFN antiviral effect, the virus can inhibit its induction [31] [32] [33] [34] . This ability may come from, at least, one mechanism of blocking the activation of the IFN signaling pathway at an early step following the nuclear transport of interferon regulatory factors (IRF) [35] . Furthermore, the recruitment of leukocytes, a hallmark of inflammation, is strongly related to the chemokine profile. For example, CCL2 and CCL8 recruit monocytes/macrophages, CXCL16 is a chemoattractant of NK cells, CXCL8 is the principal neutrophil chemoattractant, and CXCL9 and CXCL10 chemoattract T cells. Thus, the chemokine profile may be a driver of the signature pathology of SARS-CoV-2 [36] . The immune features between moderate and severe disease are modified after ten days of infection when severely ill patients remain with high proinflammatory cytokines [37] . Furthermore, deregulated inflammatory response to an infection may result in the cytokine storm syndrome, which is associated with severe COVID-19 [38, 39] . This syndrome is characterized by high levels of interleukins, TNF-α, G-CSF, MCP-1, and MIP-1α, which are higher in intensive care unit patients than non-intensive care unit patients [37, 40, 41] . Additionally, the inflammasome NLRP3, a multiprotein complex crucial to the host defense, is highly activated in COVID-19 patients. Inflammasome-induced cytokines IL-1β and IL-18 also contribute to cytokine storm, and sustained NLRP3 inflammasome activation is directly associated with the disease's severity [42] [43] [44] . The cytokines milieu recruits immune cells and activate T helper type 1 (Th1) response, which is related to the activation of a specific immune response. Moreover, Th1 cells stimulate IL-6 production by inflammatory monocytes in severe COVID-19 and contribute to the cytokine storm [45] . However, Th2 cytokines are also presented in COVID-19 serum patients and may impair the Th1inflammatory response [40] . Thereby, chemokines/cytokines milieu comprises a possible therapeutic target for COVID-19 [46] . Peripheral blood immune cells (PBMCs) of COVID-19 patients present low T cell number and frequency in both CD4 + and CD8 + populations, which are more activated. On the order hand, monocytes are increased, but they present a reduction in HLA-DR expression compared with the control group (non-infected) [37] . Additionally, in severe COVID-19, patients present a reduced number of B cells and natural killer (NK) cells associated with severe T cell depletion, and a high neutrophil population [37, 40, [47] [48] [49] . This neutrophilia occurs after seven days symptoms onset [50] . Neutrophils are the most abundant immune cells in human blood. They account for approximately 50 -70% of all leukocytes. Besides serving as first responders to many infections, neutrophils have critical homeostatic functions being also implicated in chronic inflammatory diseases [51] . These polymorphonuclear cells play a protective role during bacterial or fungal infections; however, their role in viral infections is not fully understood [52, 53] . Although the evidence is limited, it has been suggested that neutrophils enhance antiviral defenses by interaction with other immune cell populations, virus internalization and killing mechanism, cytokines release, degranulation, oxidative burst, and neutrophil extracellular traps (NETs) [53, 54] . Neutrophils are present in many lung diseases associated with ARDS, as reported in infections by influenza virus and SARS-CoV-1 [55] . A bioinformatic study presented data indicating that neutrophil activation and degranulation are highly activated processes in the SARS infection [56] . Recently, the recruitment of this polymorphonuclear (PMN) was observed in the immune response triggered by SARS-CoV-2. Furthermore, neutrophilia has been described as an indicator of severe respiratory symptoms and a poor outcome in patients with COVID-19 [57] [58] [59] . Several studies have reported that neutrophil-to-lymphocyte ratio (NLR), a clinical inflammation biomarker, is increased and predicts severe illness in the early stage of SARS-CoV-2 infection [59] [60] [61] [62] . Higher D-dimer and C-reactive protein (CRP) levels follow NLR's increase in these patients [63, 64] . Also, increased NLR has been considered an independent risk factor for mortality in hospitalized patients [41, 65, 66] , related to some comorbidities (e.g., diabetes and cardiovascular disease) [67] . A study observed that COVID-19 diabetes patients with higher NLR had heavier severity and more extended hospital stay [68] . This fact supports the idea that pre-existing chronic inflammation contributes to COVID-19 severity [65, 69] . In addition to the NLR, neutrophil to CD4 + lymphocyte ratio (NCD4LR) has been associated with the negative conversion time (NCT) of SARS-CoV-2. A study found that high NCD4LR indicates worse immune function and prolonged virus clearance [70] . Another biomarker involving this PMN, the neutrophil count to albumin ratio (NAR), has been described as a new predictor of mortality in COVID-19 patients [71] . Therefore, the NCD4LR and NAR values also could be used as clinical markers for COVID-19 progression in addition to the NLR [41] . Besides, the increase of neutrophils is not reported only in the bloodstream but also in the lungs [72] . PMN infiltration in pulmonary capillaries with extravasation to alveolar space and neutrophilic mucositis was observed in lung autopsies obtained from patients who died from COVID-19, indicating inflammation in the entire lower respiratory tract [73, 74] . Moreover, immature phenotype and/or dysfunctional mature neutrophils have been described in severe COVID-19 patients [75, 76] . These studies indicate that the increased infiltration of immature and/or dysfunctional neutrophil contributes to the imbalance of the lungs' immune response in severe cases. Respiratory epithelium infection by SARS-CoV-2 leads to cell secretion of multiple cytokines, chemokines, and DAMPs, as previously described [31, 77] . Transcriptional analysis of bronchoalveolar lavage fluid (BALF) from COVID-19 patients reported high levels of CXCL-2 and CXCL-8, chemokines that facilitate the PMN recruitment to the site of infection [78] [79] [80] [81] [82] . Although the neutrophils could present a protective role, extensive and prolonged activation of these leukocytes can lead to detrimental effects in the lungs and result in pneumonia and/or ARDS [83, 84] . Wang and colleagues [50] also demonstrated that neutrophilia coincides with lung injury in severe COVID-19 patients. It has been described that neutrophils play a pivotal role in the development of ARDS caused by influenza infection [55] . In COVID-19, neutrophils accumulation generates toxic molecules that might contribute to ARDS's physiopathology [85] . Respiratory burst from activated neutrophils induces ROS release, such as superoxide radicals and H 2 O 2 , leading to oxidative stress that contributes to the cytokine storm and blood clots formation in SARS-CoV-2 infection [86, 87] . Moreover, decreased expression of the antioxidant enzyme superoxide dismutase 3 (SOD3) in the lung tissue of old patients with COVID-19 was also reported [88] . Therefore, excessive oxidative stress induced by PMN infiltration is related to the alveolar damage, thrombosis, and severity in COVID-19 [87] . In addition to ROS formation, neutrophil elastase has been implicated in COVID-19 pathogenesis [89] [90] [91] . This proteolytic enzyme, which is stored in azurophil granules, is secreted to degrade antigens. Nevertheless, an imbalance of the elastase and other proteinases induces damage in the alveolar-capillary barrier, resulting in tissue injury and edema formation [92] . Furthermore, persistently activated neutrophils contribute to maintaining the inflammatory state in the lungs by cytokine release, as observed in MERS and SARS-CoV-1 infections [93] . Similar findings were described in SARS-CoV-2 infection by Parackova and colleagues [76] that reported the neutrophils as drivers of hyperinflammation by enhanced degranulation of primary granules and pro-inflammatory cytokines release. Taken together, these molecules secreted by PMN can cause severe damage in alveolar tissue, independently of the virus cytopathic effect. Additionally, Meizlish and colleagues [94] identified neutrophil activators (IL-8 and G-CSF) and effectors (resistin, lipocalin-2, and hepatocyte growth factor) as early biomarkers of severe COVID-19 patients. The authors also demonstrated a positive association between high levels in immature granulocytes and neutrophil counts with increased mortality [94] . These data highlight the neutrophil role in the severity of COVID-19 disease. Viral infection can also induce the release of neutrophils extracellular traps (NETs) by neutrophils [95] . The NETs mechanism was first described by Brinkmann and colleagues in 2004 [96] . These traps consist of chromatin fibers associated with enzymes such as neutrophil elastase, cathepsin G, and myeloperoxidase [97, 98] . NETs are known to immobilize and degrade bacteria, fungi, viruses, being a critical effector mechanism to contain infections [99] . However, NETs can act as a double-edged sword of immunity [98] , having a pro-or anti-inflammatory effect [100, 101] . Schauer and colleagues [102] reported that an aggregate of NETs can degrade cytokines and chemokines, reducing inflammation. This anti-inflammatory effect has also been demonstrated in the ocular microenvironment [103] . On the other hand, NETs can promote tissue damage, having already been shown that NETs and platelets' interaction can cause endothelial damage in infections by Escherichia coli [104] . NETs can also participate in the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis, where elevated levels of NETs have been seen in serum and synovial fluid, respectively, in patients with these diseases [105, 106] . Studies have been reported an elevated level of NETs in patients with COVID-19 [107] [108] [109] , and an increased plasma NETs is correlated with increased COVID-19 severity [109] , besides contributing to lung injury and microvascular thrombosis [107] . The vascular occlusion caused by NETs is not only reported in lung tissue [110] but also in kidney and liver [111] , which suggests that NETs thrombotic effects may be related to systemic and harmful effects of COVID-19. This relationship between NETs and thrombosis may also be related to complement system activation. Indeed, C3 [112] and C5 [113] inhibition dampen NET release in COVID-19 patients., Since coagulation disorders are a worse prognosis to COVID-19 [114] [115] [116] , and both NETs and complement proteins are associated with these thrombotic events [113] , therapies that focus on this triple complement-NETs-coagulation axis may be a therapeutic opportunity. At the transcriptional level, Wang and collaborators [50] demonstrated activation of several NETs-associated in COVID-19 patients. They hypothesized that some of them could be related to negative regulation of NK and T cell, dampening antiviral response [50] . In severe COVID-19, Veras and colleagues [109] demonstrated that neutrophils, both circulating and lunginfiltrating, release high levels of NETs. The authors also present data that demonstrate a NETs release directly induced by SARS-CoV-2 [117] . This SARS-CoV-2-induced NETs release is PAD-4-dependent [109] . PAD4 is critical to NET formation because it promotes a process of hypercitrulination of histones, resulting in chromatin decondensation [118] . The SARS-CoV-2-activated neutrophils can also induce apoptosis in lung epithelial (A549 cells), reinforcing neutrophil role in COVID-19 immunopathology and other coronavirus infections [109] . The literature related to neutrophil and COVID-19 so far demonstrated a crucial role of these polymorphonuclear cells in the pathogenesis of COVID-19 (Fig. 1) . Despite the immune system modulation needs being tightly controlled to avoid immunosuppression, the different neutrophil mechanisms (e.g., neutrophils enzymes and cytokines, NETs) are potential targets to treat COVID-19, mainly the severe cases. (4) immunothrombosis. These many steps may be a therapeutic target. Several other cells and mediators are involved in COVID-19 immunopathology, but they are suppressed in this figure to highlight the neutrophil role. The figure was created with BioRender.com [119] . The trinity of COVID-19: immunity, inflammation and intervention A novel coronavirus from patients with pneumonia in China The proximal origin of SARS-CoV-2 The COVID-19 epidemic The Origin of COVID-19 and Why It Matters Epidemiology and cause of severe acute respiratory syndrome (SARS) in Guangdong, People's Republic of China A Novel Coronavirus Associated with Severe Acute Respiratory Syndrome The Middle East respiratory syndrome coronavirus Isolation of a Novel Coronavirus from a Man with Pneumonia in Saudi Arabia Clinical characteristics of 24 asymptomatic infections with COVID-19 screened among close contacts in Nanjing Transmission mode associated with coronavirus disease 2019: A review Asymptomatic Carrier Transmission of Coronavirus Disease 2019 (COVID-19) and Multipoint Aerosol Sampling to Assess Risks in the Operating Room During a Pandemic Gastrointestinal and Liver Manifestations of COVID-19 Detection of Novel Coronavirus by RT-PCR in Stool Specimen from Asymptomatic Child, China, Emerg Corona Virus Disease (COVID-19) Fecal-oral transmission: Is it a potential risk for Indians? Fecal-oral transmission of SARS-CoV-2: review of laboratory-confirmed virus in gastrointestinal system Perinatal transmission with SARS-CoV-2 and route of pregnancy termination: a narrative review Possible vertical transmission and antibodies against SARS-CoV-2 among infants born to mothers with COVID-19: A living systematic review Updated understanding of the outbreak of 2019 novel coronavirus (2019-nCoV) in Wuhan An Updated Comparison of COVID-19 and Influenza, SSRN Electron COVID-19: What has been learned and to be learned about the novel coronavirus disease Clinical Characteristics of 138 Hospitalized Patients with 2019 Novel Coronavirus-Infected Pneumonia in Estimating clinical severity of COVID-19 from the transmission dynamics in Wuhan, China COVID-19: immunopathogenesis and Immunotherapeutics Immune Response, Inflammation, and the Clinical Spectrum of COVID-19 A pneumonia outbreak associated with a new coronavirus of probable bat origin Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis Single-cell RNA-seq data analysis on the receptor ACE2 expression reveals the potential risk of different human organs vulnerable to 2019-nCoV infection A new coronavirus associated with human respiratory disease in China Comparative replication and immune activation profiles of SARS-CoV-2 and SARS-CoV in human lungs: an ex vivo study with implications for the pathogenesis of COVID-19 Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19 Coronavirus infections and immune responses Immunopathogenesis of coronavirus infections: Implications for SARS SARS-Coronavirus Open Reading Frame-9b Suppresses Innate Immunity by Targeting Mitochondria and the MAVS/TRAF3/TRAF6 Inhibition of Beta Interferon Induction by Severe Acute Respiratory Syndrome Coronavirus Suggests a Two-Step Model for Activation of Interferon Regulatory Factor 3 Chemokine receptors: Multifaceted therapeutic targets Exuberant elevation of IP-10, MCP-3 and IL-1ra during SARS-CoV-2 infection is associated with disease severity and fatal outcome In the eye of the COVID-19 cytokine storm Clinical features of patients infected with 2019 novel coronavirus in Neutrophil-to-lymphocyte ratio as an independent risk factor for mortality in hospitalized patients with COVID-19 Targeting the NLRP3 Inflammasome in Severe COVID-19 Severe COVID-19: NLRP3 Inflammasome Dysregulated Pathogenic T-cells and inflammatory monocytes incite inflammatory storms in severe COVID-19 patients Coronavirus disease 2019 (COVID-19): Immunological approaches and emerging pharmacologic treatments Dysregulation of Immune Response in Patients With Coronavirus Immunopathological characteristics of coronavirus disease 2019 cases in A single-cell atlas of the peripheral immune response in patients with severe COVID-19 Excessive Neutrophils and Neutrophil Extracellular Traps in COVID-19 Neutrophils as protagonists and targets in chronic inflammation Neutrophils and Neutrophil Extracellular Traps Drive Necroinflammation in COVID-19 Neutrophils in viral infections: Current concepts and caveats Neutrophils in viral infection A role for neutrophils in viral respiratory disease Cytokine storm and leukocyte changes in mild versus severe SARS-CoV-2 infection: Review of 3939 COVID-19 patients in China and emerging pathogenesis and therapy concepts A meta-analysis of SARS-CoV-2 patients identifies the combinatorial significance of D-dimer, C-reactive protein, lymphocyte, and neutrophil values as a predictor of disease severity Continuous tracking of COVID-19 patients' immune status Neutrophil-to-lymphocyte ratio predicts critical illness patients with 2019 coronavirus disease in the early stage The clinical implication of dynamic neutrophil to lymphocyte ratio and D-dimer in COVID-19: A retrospective study in Suzhou China Clinical criteria for COVID-19-associated hyperinflammatory syndrome: a cohort study Biomarkers associated with COVID-19 disease progression Dynamic changes of D-dimer and neutrophil-lymphocyte count ratio as prognostic biomarkers in COVID-19 Neutrophil-to-Lymphocyte Ratio and Outcomes in Louisiana COVID-19 Patients The diagnostic and predictive role of NLR, d-NLR and PLR in COVID-19 patients Average Values and Racial Differences of Neutrophil Lymphocyte Ratio among a Nationally Representative Sample of United States Subjects The role of neutrophil-lymphocyte ratio and lymphocyte-monocyte ratio in the prognosis of type 2 diabetics with COVID-19 Admission fasting plasma glucose is an independent risk factor for 28-day mortality in patients with COVID-19 Neutrophil to CD4+ lymphocyte ratio as a potential biomarker in predicting virus negative conversion time in COVID-19 Neutrophil count to albumin ratio as a new predictor of mortality in patients with COVID-19 ınfection The emerging spectrum of cardiopulmonary pathology of the coronavirus disease 2019 (COVID-19): Report of 3 autopsies from Houston, Texas, and review of autopsy findings from other United States cities Targeting potential drivers of COVID-19: Neutrophil extracellular traps Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment Disharmonic Inflammatory Signatures in COVID-19: Augmented Neutrophils' but Impaired Monocytes' and Dendritic Cells Neutrophil Extracellular Traps (NETs) and Damage-Associated Molecular Patterns (DAMPs): Two Potential Targets for COVID-19 Treatment Transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in COVID-19 patients COVID-19 Hyperinflammation: What about Neutrophils? Heightened Innate Immune Responses in the Respiratory Tract of COVID-19 Patients Compartmental immunophenotyping in COVID-19 ARDS: a case series Immune characteristics distinguish patients with severe disease associated with SARS-CoV-2 Neutrophils and acute lung injury Immunopathogenesis of SARS-CoV-2-induced pneumonia: lessons from influenza virus infection Understanding the role of neutrophils in acute respiratory distress syndrome SARS-CoV-2 infection pathogenesis is related to oxidative stress as a response to aggression Tissue damage from neutrophil-induced oxidative stress in COVID-19 Is Low Alveolar Type II Cell SOD3 in the Lungs of Elderly Linked to the Observed Severity of COVID-19? Neutrophil Elastase Inhibitors: A potential prophylactic treatment option for SARS-CoV-2-induced respiratory complications? Anti-protease treatments targeting plasmin(Ogen) and neutrophil elastase may be beneficial in fighting covid-19 Neutrophil elastase inhibitor (sivelestat) may be a promising therapeutic option for management of acute lung injury/acute respiratory distress syndrome or disseminated intravascular coagulation in COVID-19 Clinical utility of the neutrophil elastase inhibitor sivelestat for the treatment of acute respiratory distress syndrome The pathogenesis and treatment of the 'Cytokine Storm'' in COVID-19 A neutrophil activation signature predicts critical illness and mortality in COVID-19 Neutrophil extracellular traps go viral Neutrophil Extracellular Traps: Double-Edged Swords of Innate Immunity Neutrophil extracellular traps in immunity and disease Neutrophils and neutrophil extracellular traps orchestrate initiation and resolution of inflammation Nanoparticles size-dependently initiate self-limiting NETosis-driven inflammation Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines Frontline Science: Aggregated neutrophil extracellular traps prevent inflammation on the neutrophil-rich ocular surface Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood The role of neutrophils and NETosis in autoimmune and renal diseases Neutrophil extracellular traps (NETs) in autoimmune diseases: A comprehensive review Neutrophil Extracellular Traps (NETs) Contribute to Immunothrombosis in COVID-19 Acute Respiratory Distress Syndrome Neutrophil extracellular traps in COVID-19 SARS-CoV-2-triggered neutrophil extracellular traps mediate COVID-19 pathology Neutrophil extracellular traps infiltrate the lung airway, interstitial, and vascular compartments in severe COVID-19 Vascular occlusion by neutrophil extracellular traps in COVID-19 Complement C3 vs C5 inhibition in severe COVID-19: Early clinical findings reveal differential biological efficacy Complement and tissue factor-enriched neutrophil extracellular traps are key drivers in COVID-19 immunothrombosis Multifactorial pathogenesis of COVID-19-related coagulopathy: Can defibrotide have a role in the early phases of coagulation disorders? Thrombosis and COVID-19 pneumonia: The clot thickens! Clotting disorder in severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 triggered neutrophil extracellular traps (NETs) mediate COVID-19 pathology An emerging role for neutrophil extracellular traps in noninfectious disease Cytokine Storm We are grateful to CAPES (PROCAD) and CNPq ("Conselho Nacional de Desenvolvimento Científico e Tecnológico") to fellowships support (154801/2019-4 and 141304/2017-0). None.