Neutrophil autophagy and NETosis in COVID-19: perspectives

ABSTRACT

The COVID-19 pandemic has caused substantial losses worldwide in people’s lives, health, and property. Currently, COVID-19 is still prominent worldwide without any specific drug treatment. The SARS-CoV-2 pathogen is the cause of various systemic diseases, mainly acute pneumonia. Within the pathological process, neutrophils are recruited to infected sites, especially in the lungs, for the first stage of removing invading SARS-CoV-2 through a range of mechanisms. Macroautophagy/autophagy, a conserved autodegradation process in neutrophils, plays a crucial role in the neutrophil phagocytosis of pathogens. NETosis refers to neutrophil cell death, while auto-inflammatory factors and antigens release NETs. This review summarizes the latest research progress and provides an in-depth explanation of the underlying mechanisms of autophagy and NETosis in COVID-19. Furthermore, after exploring the relationship between autophagy and NETosis, we discuss potential targets and treatment options. This review keeps up with the latest research on COVID-19 from neutrophil autophagy and NETosis with a new perspective, which can guide the urgent development of antiviral drugs and provide guidance for the clinical treatment of COVID-19.

Abbreviations: AKT1: AKT serine/threonine kinase 1; AMPK: AMP-activated protein kinase; AP: autophagosome; ARDS: acute respiratory distress syndrome; ATG: autophagy related; BECN1: beclin 1; cfDNA: cell-free DNA; COVID-19: coronavirus disease 2019; CQ: chloroquine; DMVs: double-membrane vesicles; ELANE/NE: elastase, neutrophil expressed; F3: coagulation factor III, tissue factor; HCQ: hydroxychloroquine; MAP1LC3/LC3: microtubule associated protein 1 light chain of 3; MPO: myeloperoxidase; MTORC1: mechanistic target of rapamycin kinase complex 1; NETs: neutrophil traps; NSP: nonstructural protein; PI3K: class I phosphoinositide 3-kinase; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; ROS: reactive oxygen species; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SKP2: S-phase kinase associated protein 2; TCC: terminal complement complex; ULK1: unc-51 like

KEYWORDS:

• Autophagy
• biomarkers
• clinical treatments
• COVID-19
• neutrophil
• NETosis

Introduction

The widespread coronavirus 2 of the severe acute respiratory syndrome (SARS-CoV-2) has led to the coronavirus disease 2019 (COVID-19) epidemic, which has had a significant health and economic impact worldwide. Globally, 510 million people have been diagnosed with COVID-19, including 6 million deaths before May 2022 [1]. Initial patients with COVID-19 in Wuhan described symptoms that included fever, cough, difficulty breathing, headache, tachycardia, progressive hypoxemia, and other signs and symptoms [2]. In addition, acute respiratory distress syndrome (ARDS), heart damage, kidney damage, symptoms of multiple organ failure, and central nervous system damage are severe complications of COVID-19 that can lead to death [3–6]. Currently, no effective targeted drugs have been developed, only symptomatic treatment for patients with COVID-19. Although vaccines have been developed, it is still urgent to find effective therapeutic targets and drugs. Exploring the mechanism of SARS-CoV-2 invading cells can help researchers find potentially effective therapeutic targets.

Neutrophils are essential for eliminating pathogens and have specific mechanisms against viruses [7]. Neutrophils are the most abundant and effective defense against external pathogens, constituting the first line of cell defense [8]. Neutrophils are recruited into the lungs in the first stage of COVID-19 and eliminate invading SARS-CoV-2 through numerous pathways. However, abnormal neutrophil activation causes a severe cytokine storm and host response in COVID-19 patients [9]. Several clinical studies have shown that neutrophil levels in the blood are associated with a worsening of oxygenation in COVID-19 [10,11]. Neutrophils are the possible persistent inflammation and respiratory system damage factors in COVID-19 [11].

The circulating platelet-neutrophil complexes in all patients with COVID-19 are elevated compared to the healthy individuals [12]. COVID-19 infection induces immunothrombosis, in which activates neutrophil cells to interact with platelets and induce a coagulation cascade, resulting in the formation of intravascular clots in blood vessels [13]. Neutrophils interact with endothelial cells and usually occur in neutrophil recruitment [14]. Through endothelial cells, neutrophils translocate into the pulmonary microvasculature with activated platelets leading to several injuries such as epithelial layer damage, alveolar fibrin deposition, and further resulting in microthrombi formation [15]. Emerging evidence indicates that endothelial dysfunction is involved in COVID-19 by changing the integrity of the vessel barrier, mediating neutrophilic infiltration and inflammation [16], and contributing to end-organ damage [17]. Therefore, these observations powerfully demonstrate that neutrophils could be considered one of the main therapeutic targets in COVID-19.

Macroautophagy/autophagy is a conserved autodegradation process in neutrophils activated under conditions that include pathogen infection, cell starvation, and endoplasmic reticulum stress [18]. Autophagy is the only pathway that can produce random or targeted degradation of entire organelles and invade pathogens [19]. First described by Mitroulis et al., autophagy is involved in phagocytosis-dependent and phagocytosis-independent initiation manners in human neutrophils [20]. Autophagy is also essential for neutrophil differentiation [21]. NETosis is a form of cell death caused primarily by neutrophils and refers to the formation of extracellular neutrophil traps (NETs) during this process [22]. NETs consist of DNA fibers or nuclear chromatins that envelope cytosolic granule proteins and histones in a depolymerized reticulated form to trap and eliminate pathogens [7,23]. These two crucial cell death pathways play a significant role in the COVID-19 process; exploring these two neutrophil pathways may help researchers find suitable targets for treating COVID-19.

In this review, we discuss neutrophil autophagy and NETosis, their connection, and their contribution to the COVID-19 pathogenesis. Furthermore, this review also focuses on potential therapeutic targets and biomarkers of COVID-19 on autophagy and NETosis of neutrophils to guide the clinical development of anti-SARS-CoV-2 virus drugs to improve the prognosis and provide effective indicators to judge the severity of COVID-19.

Neutrophil autophagy in COVID-19

Autophagy of neutrophils in COVID-19: mechanisms

Autophagy plays a vital role in neutrophil formation, differentiation, and function. In addition, it plays a critical role in presenting pathogenic antigens, the secretion of related cytokines, degranulation, bactericidal activity, and inflammation control [24,25]. During pathogen invasion, neutrophils are activated and migrate to the lesion to phagocytose pathogens and produce effector molecules that kill pathogenic microorganisms, such as reactive oxygen species (ROS), proteinases, and NETs. All of these effector molecules can activate neutrophil autophagy [8]. Through autophagy, harmful, redundant, and damaged cellular components are degraded. This process also provides neutrophils with essential lipids, amino acids, carbohydrates, and sufficient energy. Therefore, cell adaptability is improved under pathogen invasion and various pathophysiological conditions [19].

During SARS-CoV-2 invasion, autophagy is activated and plays a vital role in the pathological process of COVID-19. The neutrophil autophagy mechanism for COVID-19 is described in Figure 1. Neutrophil cell structures, such as damaged organelles caused by SARS-CoV-2 and cytoplasmic protein aggregates, are engulfed in phagophores that mature into double-membrane vesicles (DMVs) termed autophagosomes (APs) that are signs of autophagy under the control of proteins encoded by ATG (autophagy related) [26]. These ATGs regulate autophagy in the following four processes: In the initial stage, neutrophils release ROS, proteases, and NET to eliminate the invading SARS-CoV-2, which can cause stress on cellular energy and increase the level of cytoplasmic AMP. Then, increasing AMP leads to the activation of AMP-activated protein kinase (AMPK), phosphorylation, and inactivation of the MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) [27]. The next stage is nucleation controlled by the ULK1/Atg1 (unc-51 like autophagy activating kinase 1) complex downstream of MTORC1 [28]. The ULK1/Atg1 complex activates the class III phosphatidylinositol 3-kinase (PtdIns3K) complex and produces an area rich in phosphatidylinositol-3-phosphate (PtdIns3P) on the surface of omegasomes. WIPI (WD repeat domain, phosphoinositide interacting) proteins recruit the ATG16L1 complex to promote the conversion of MAP1LC3/LC3 (microtubule associated protein 1 light chain 3)-I to the phosphatidylethanolamine-conjugated form, LC3-II [29]. Then, in the expansion stage, autophagy is advanced by the two ubiquitin-like conjugation systems (ATG12–ATG5 and LC3/GABARAP/Atg8), as well as the PtdIns3K complex including ATG14-BECN1 (beclin 1)-PIK3C3/VPS34-PIK3R4/VPS15-NRBF2 [29,]. Subsequently, BECN1 is degraded by SKP2 (S-phase kinase associated protein 2) and AKT1 (AKT serine/threonine kinase 1), a primary degradation pathway. The final stage is the maturation/degradation stage, in which APs and lysosomes fuse to form autolysosomes and the cargo sequestered within APs is degraded.

Figure 1. Autophagy and its mechanism in COVID-19. The autophagy process can be divided into four stages: (1) initiation, (2) nucleation, (3) expansion, and (4) maturation/degradation. In the initial stage, bioenergy stress in the host cell increases after SARS-CoV-2 invasion, thus increasing the level of cytoplasmic AMP: ATP, leading to activation of AMPK and phosphorylation/deactivation of MTORC1. Then in the nucleation stage, the ULK1 complex is activated, thus activating the PtdIns3K complex and producing a PtdIns3P-rich area (PtdIns3P) on the surface of omegasomes. PtdIns3P then recruits the WIPI and ZFYVE1 proteins that bind to the omegasome. In the expansion stage, ATG7 and ATG3 synergistically prime MAP1LC3/LC3, WIPI proteins recruit conjugation factors, including the ATG16L1 complex, leading to the conjugation of MAP1LC3/LC3 to phosphatidylethanolamine (generating LC3-II) to form APs. The last stage is maturation/decomposition, APs and lysosomes fuse to form autolysosomes. Substances wrapped in APs are degraded.

Abbreviations: AMP: adenosine monophosphate; AMPK: AMP-activated protein kinase; ATP: adenosine triphosphate; ATG: autophagy related; ATG16L1: autophagy related 16 like 1; ATG101: autophagy related 101; AP: autophagosome; BECN1: beclin 1; ZFYVE1: zinc finger FYVE domain containing 1; RB1CC1: RB1 inducible coiled-coil 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NRBF2: nuclear receptor binding factor 2; PE: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; PIK3C3: phosphatidylinositol 3‐kinase catalytic subunit type 3; PIK3R4: phosphatidylinositol-3‐kinase regulatory subunit 4; Ptdlns3K, phosphatidylinositol 3‐kinase; ULK1: unc-51 like autophagy activating kinase 1; WIPI: WD repeat domain, phosphoinositide interacting.

Coronavirus and neutrophil autophagy: a double-edged sword

Previous studies have shown that autophagy could be a double-edged sword during coronavirus infection [30]. Autophagy can directly degrade coronavirus particles, promote the response to inflammatory cytokines [31], and act as a negative inflammation regulator in cells such as neutrophils [25]. Furthermore, autophagy promotes the presentation of antigens to induce immunity to resist coronavirus invasion [32]. However, DMVs corresponding to APs (the morphological sign of autophagy) are beneficial in isolating the virus from the external immune response and thus become a site of virus replication and transcription [31]. Previous studies have shown that several pathogens (e.g., influenza A virus, vesicular stomatitis virus, hepatitis B virus, hepatitis C virus, flaviviruses, coronaviruses) replicate in APs [30,33–35] and are capable of arresting autophagy by avoiding detection by the immune system. Nonstructural proteins (NSPs) are coronavirus replicase proteins. Among them, NSP3, NSP4 and NSP6 of SARS-CoV [36], NSP2 and NSP3 of the equine arteritis virus [37], and NSP3 and NSP4 of the middle eastern respiratory syndrome coronavirus [38] induce rearrangement of the endoplasmic reticulum membrane to form DMVs and restrict AP expansion, producing small APs [35].

Furthermore, SARS-CoV NSP6 colocalizes with LC3-positive DMVs [39], indicating that autophagy is closely related to virus invasion. Coronaviruses also inhibit BECN1, which prevents the fusion of APs and lysosomes and interferon production [40]. From this mechanism, coronaviruses control autophagy to facilitate its survival and replication in host cells.

The latest studies showed that SARS-CoV-2 inhibits complete autophagy and hinders AP maturation of APs to block autophagic flux in various ways. Mohamud and colleagues (2021) reported that the overexpression of papain-like protease of SARS-CoV-2 cleaves ULK1 and disrupts the formation of the ULK1-ATG13 complex to block complete host autophagy [41]. Gassen and colleagues (2021) found that SARS-CoV-2 can restrict autophagy signal transduction by activating autophagy inhibitors (SKP2 and AKT1) and preventing AP-lysosome fusion [42]. Network analysis indicated that SARS-CoV-2 could block autophagy flow by negatively regulating autophagy genes, SNAP29 (synaptosome associated protein 29), and lysosomal acidification genes in infected human nasopharyngeal samples [43]. Therefore, the degradation of virus particles is reduced [43]. Miao and colleagues (2021) showed that ORF3a of SARS-CoV-2 can isolate homotypic fusion and protein classification components, thus inhibiting AP and lysosome fusion of APs and lysosomes [44]. ORF3a of SARS-CoV cannot interact with homotypic fusion and protein sorting parts to block autophagy, indicating that this is a newly discovered strategy and a unique characteristic of SARS-CoV-2 to inhibit autophagy [44,45].

In summary, the above studies suggest that SARS-CoV-2 can inhibit complete autophagy. However, Bouhaddou and colleagues (2020) found that the AMPK-MAPK14/p38 pathway is activated and the AKT pathway is inhibited, which can induce the autophagy in COVID-19 [46]. Although that finding seems to contradict the above findings, incomplete autophagy is caused by the virus in DMVs interfering with the fusion of the APs with lysosomes in this circumstance, thus facilitating virus replication [46].

NETosis of neutrophils in COVID-19

NETosis of neutrophils in COVID-19: mechanisms

As a powerful defense method for the elimination and microbial destruction of pathogens, NETosis refers to neutrophil cell death of neutrophils, accompanied by the release of NETs, autoinflammatory factors, and antigens. NETs consist mainly of DNA fibers or nuclear chromatins that envelope cytosolic granule proteins and histones in a reticulated depolymerized form to trap and eliminate pathogens [7,23]. Neutrophils release ELANE (elastase, neutrophil expressed) and MPO (myeloperoxidase) from digestive granules, which can dissolve invading pathogens such as coronavirus, but the process can also cause tissue damage [47]. Although the formation of NETs helps the host resist pathogens, excessive NET formation can trigger a series of inflammatory reactions, destroy surrounding tissues, and promote microthrombus formation [9]. Excess NETs will stimulate the occurrence and development of many diseases and can damage many organs [48].

The mechanism of neutrophil NETosis in COVID-19 is described in Figure 2. There are two forms of NETosis, suicidal and vital [49,50]. Suicidal NETosis is a programmed cell death that ruptures the plasma membrane and releases NETs into the extracellular space. Under stressful conditions such as SARS-CoV-2 invasion, the NADPH oxidase complex is activated to generate large amounts of ROS [51]. The azurosomes then dissociate under ROS, allowing ELANE and MPO to release the particles into the cytosol [52]. Next, ELANE and MPO enter the nucleus to decompose laminin and histones, leading to condensation, destruction of the nuclear shell, and depolymerization of chromatin. PADI4 (peptidyl arginine deiminase 4) citrullinates histones and mediates chromatin decondensation. Decondensed deoxyribonucleic acid, released into the cytoplasm, binds to cytoplasmic proteins, extracellular histone H3, and granular proteins to create NET, which is eventually released extracellularly [53]. In vital NETosis, NET is released without ROS production, and neutrophils retain their viability with normal function [54]. Instead, lipopolysaccharides activate neutrophils and platelets through TLR4 (toll like receptor 4) [50]. Furthermore, given that NETs are abundant in alveolar capillaries [55], cell-free DNA (cfDNA) and NET histones can promote the release of pro-inflammatory cytokines and other comprehensive factors. Once NETs are released heavily in COVID-19, lung tissue is damaged by alveolar microcirculation disorders, and serious consequences can occur, such as ARDS [56,57].

Figure 2. NETosis and its mechanism in COVID-19. NETosis is a process of death with release of NET accompanied by auto-inflammatory factors and antigens. Calcium ionophore activates the CRAC channel of the cytoplasmic membrane and mobilizes Ca²⁺ in the endoplasmic reticulum, increasing the concentration of Ca²⁺ in the cytoplasm. Subsequently, Ca²⁺ overload and oxidative stress in the mitochondrial matrix lead to nonselective mitochondrial pores and the formation of MTROS. The release of MTROS into the cytoplasm activates PRKC and NADPH oxidase and induces the formation of NETs independently of MTROS and MPTP. Under stress conditions such as invasion of SARS-CoV-2, the NADPH oxidase complex is activated, subsequently producing large amounts of ROS. The azurosome is a protein complex that contains the ELANE and MPO proteins. Under ROS, the azurosome releases ELANE and MPO into the cytoplasm. In addition, the azurosomes dissociate in the presence of ROS, causing ELANE and MPO to enter the nucleus to decompose laminin and histones. PADI4 citrullinates histones, resulting in nucleocapsid agglomeration and chromatin depolymerization. DNA fibers or nuclear chromatin, released into the cytoplasm, combined with cytoplasmic proteins, granular proteins, histones, and F3, encapsulate the cytoplasm and finally in the extracellular form of NETs. cDNA and histone promote the release of pro-inflammatory cytokines and TCC. Neutrophils also release ELANE and MPO, which dissolve invasive pathogens such as SARS-CoV-2, digest particles, and induce tissue and bystander damage.

Abbreviations: cfDNA: cell-free DNA; DNA: deoxyribonucleic acid; ELANE/NE: elastase, neutrophil expressed; ER: endoplasmic reticulum; F3: coagulation factor III, tissue factor; IP3: inositol triphosphate; MPO: myeloperoxidase; MTROS: mitochondrial reactive oxygen species; MPTP: mitochondrial permeability transition pore; NADPH: nicotinamide adenine dinucleotide phosphate; NETs: neutrophil extracellular traps; PADI4: peptidylarginine deiminase 4; PRKC: protein kinase C; ROS: reactive oxygen species; TCC: terminal complement complex.

NETosis in COVID-19: clinical studies

SARS-CoV-2 has been shown to activate human neutrophil NETosis and is associated with increased intracellular ROS in neutrophils [58]. There is evidence that low-density neutrophils, particularly prone to NETosis, are closely associated with blood vessel blockage and COVID-19-related ARDS [57]. NET production increases in patients with acute respiratory failure and ARDS [59]. Neutrophils and NETs were present in the airways and lung parenchyma alveoli in 40% of COVID-19 patients, indicating that the increase in NETosis is related to increased leukocytosis and neutrophils [60]. Neutrophils increase in patients with COVID-19, suggesting the possibility of cytokine storms, while the reactivity of dendritic cells and monocytes is altered [61].

NETosis is involved in developing many disease phenotypes (NET-mediated diseases) in COVID-19, including thrombosis, autoimmune diseases, sepsis, and multiorgan diseases such as lung diseases [50,62]. In thrombotic disorders, NETs are lined with F3 (coagulation factor III, tissue factor), the main initiator of blood coagulation [63]. Clinical studies found that NET levels, F3 activity, and the terminal complement complex increased in serum from COVID-19 patients [64]. Neutrophils from COVID-19 patients expressed high F3 and released NET with active F3 [64]. The increased concentration of NETosis markers (cell-free DNA, MPO-DNA complexes, and histone H3) is correlated with neutrophil content and D-dimer (thrombosis marker) [65,66]. The NET formation can be stimulated by activated complement proteins that can interact with coagulation systems. NETs, in turn, can serve as a platform for complement activation and a scaffold for thrombus formation during coagulation [67,68]. Demonstration of autophagy-dependent extracellular delivery of F3 in NET further illustrates the role of neutrophils in thrombosis [68].

Furthermore, components of NET, such as granule proteins and histones, can stimulate the production of antibodies and the development of autoimmune diseases [69]. NET histones are the main contributors to cytotoxic effects toward endothelium injury and multiple organ dysfunction, further supporting the relevance of NET formation in sepsis [62]. Inflammatory cytokines (such as IL1B/IL-1β) released by excessive NETosis are involved in ulcerative colitis and the development of cytokine storms, which are also the leading indicators of the severe course of COVID-19 [9].

Autophagy and NETosis: relationship and potential targets in COVID-19

The relationship between autophagy and NETosis

Studies have shown that autophagy positively affects NET formation [70]. Activation of autophagy (e.g., inhibition of MTOR through rapamycin) can enhance NET production [64], while its inhibition (e.g., the use of a PtdIns3K inhibitor) can reduce NET release [71], and decreased autophagy can decrease NET production [72]. Furthermore, the class I phosphoinositide 3-kinase (PI3K)-AKT-MTOR pathway plays a significant role in both mechanisms, connecting autophagy and NET [70]. Data from phorbol myristate acetate stimulation suggest that autophagy induction is an essential step for the initial stages of NETosis, but not for the cell death process itself [73]. The formation of NETosis requires autophagy, which was investigated by removing WDFY3 to regulate ROS by NET reduction [74]. Lack of either may result in the inability to form NETs and lead to cell apoptosis [71]. In addition, studies have shown that ROS mediates the autophagy process [75]. ROS can cause a rapid increase in the pH value of primary vesicles and induce autophagy [76]. Although ROS is not essential for autophagy, ROS is indispensable for NETosis [73]. Autophagy and superoxide generation is necessary but insufficient to mediate NETosis [73].

Furthermore, several neutrophil-derived proteins, such as F3 and IL1B, are enclosed in the AP and, through these structures, are exposed to NETs [68,77]. Demonstration of autophagy-dependent extracellular delivery of F3 in NET further illustrates the role of neutrophils in thrombosis [68]. Autophagy IL1B maturation and release of IL1B-bearing NETs are closely related to autoinflammatory disorders such as ulcerative colitis and Behçet’s disease [77]. These observations suggest a strong link between autophagy NETs and disease phenotypes. Understanding the relationship between neutrophil autophagy and NETosis is critical for identifying potential targets in these two mechanisms.

Potential therapeutic targets for autophagy and NETosis in COVID-19

Although there is currently no specific drug treatment for COVID-19, regulation of autophagy and NETosis holds promise for developing drugs related to COVID-19. Potential therapeutic targets for autophagy and NETosis in COVID-19 are described in Table 1 and Table 2.

Table 1. Potential therapeutic targets of autophagy in COVID-19.

Potential therapeutic targets	Treatment pathway and mechanism	Potential drugs	References
Lysosomal nutrients	Alkalize lysosomes to inhibit autophagosome and lysosome fusion.	CQ, HCQ, azithromycin, NH4Cl, and bafilomycin A1	[28,90,91]
SKP2-AKT1 pathway	Inhibitors of the SKP2-AKT1 pathway to inhibit BECN1 degradation.	MK-2206	[42]
	Inhibitors of the SKP2 E3 ligase to stabilize BECN1.	Valinomycin	[40]
AMPK pathway	Inhibitors of AMPK inhibit the initial stage of autophagy.	Gilteritinib and ralimetinib	[46]
	Inducers of the AMPK pathway promote autophagy by phosphorylation of autophagy-related proteins in ULK1, MTORC1, and PIK3C3.	Metformin, A-769662, resveratrol, compound C/dorsomorphin, GSK621, and PT1	[102]
Inhibitors of the clathrin-mediated endocytic pathway	Inhibit the entry of SARS-CoV-2 into host cells.	Azithromycin, NH4Cl, bafilomycin A1, chlorpromazine, ouabain, and bufalin	[28,90–92]
MTOR	Inhibitors of MTOR stimulate autophagy by blocking MTORC1.	Rapamycin, sirolimus, and everolimus	[103–105]
PI3K pathway	Inhibitors of the PI3K pathway to upregulate BECN1, ATG12–ATG5, LC3-II-mediated autophagy.	3-Methyladenine	[106,107]
ULK1-ULK2 complex	Inhibitors of the ULK1-ULK2 complex inhibit nucleation by blocking autophagy.	SBI-0206965, MRT67307, and MRT68921	[108,109]
ATG4 and ATG7	Inhibitors of ATG4 and ATG7 cause a block in the processing and lipidation of MAP1LC3/LC3.	Tioconazole and pyrazolopyrimidine sulfamates	[102,110]
Polyamine pathway	Inducers of the polyamine pathway to stabilize BECN1.	Polyamine and niclosamide	[42]
AP-1 signal pathway.	Inducers of the AP-1 signal pathway to improve the immune response to COVID-19.	Oxyphenisatin, clioquinol, and promethazine	[111]
NFKB pathway.	Inducers of the NFKB pathway to transactivate BECN1.	lincRNA-COX2	[112,113]
Vitamin D	Promote BECN 1 and PtdIns3K to induce autophagy.	25-hydroxyvitamin D (25-OHD) and 1,25-(OH)2D	[114]

Abbreviations: AKT1: AKT serine/threonine kinase 1; AMPK: AMP-activated protein kinase; AP-1: activator protein 1; ATG: autophagy related; BECN1: beclin 1; COVID-19: coronavirus disease 2019; CQ: chloroquine; HCQ: hydroxychloroquine; MTORC1: mechanistic target of rapamycin kinase complex 1; NFKB: nuclear factor kappa B; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; SKP2: S-phase kinase associated protein 2; ULK1: unc-51 like autophagy activating kinase 1.

Table 2. Potential therapeutic targets for NETosis in COVID-19.

Potential therapeutic targets	Treatment pathway and mechanism	Potential drugs	References
Immunomodulation.	Essential directions of clinical treatment in COVID-19.	Anakinra, eculizumab, heparin, hyperbaric oxygen, remdesivir, and sirolimus.	[49]
cfDNA for SARS-CoV-2-induced sepsis.	Reduce neutrophil activities and cfDNA levels. A potential therapeutic intervention for COVID-19.	Exogenous administration of long-acting nanoparticle deoxyribonuclease-1.	[94]
NETosis related to sepsis.	Alleviate NETosis. Inhibit neutrophil activity and cytokine storm.Prevent the further progress of COVID-19.	DNase-I-coated melanin-like nanospheres	[53]
They block C-type lectins.	Inhibit the formation of NETs. Reduce intravascular coagulopathy and SARS-CoV-2-induced NETosis.	– –	[98,99]

Abbreviations: cfDNA: cell-free DNA; COVID-19: coronavirus disease 2019; NET: neutrophil extracellular trap; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.

Autophagy features resistance to SARS-CoV and SARS-CoV-2, immune evasion, and replication promotion, which is beneficial for the existence of the coronavirus. However, this practical regulation window is so narrow that both autophagy inhibitors and autophagy inducers can have an antiviral effect on COVID-19 [78]. Although autophagy induction can enhance virus clearance and limit SARS-CoV-2 proliferation [42], the promotion of NETosis is not conducive to treatment and can hinder recovery. Autophagy inhibitors could act indirectly to combat the spread of SARS-CoV-2. Due to the crosstalk between autophagy and apoptosis, the accumulation of APs could activate the host apoptosis mechanism, causing the death of host cells and thus stopping virus replication [29].

Furthermore, inhibition of autophagy can inhibit NET expression, thus reducing inflammation and ARDS and other complications such as sepsis, mediterranean fever, and ulcerative colitis [79–82]. Therefore, we do not recommend autophagy inducers as potential therapeutic drugs since autophagy-mediated NETs induce inflammation and exacerbate tissue damage [79]. In contrast, we recommend autophagy inhibitors, as they also inhibit NETosis.

Among autophagy inhibitors, 3-methyladenine and wortmannin can block autophagy and inhibit virus replication [83]. Chloroquine (CQ) and hydroxychloroquine (HCQ) are lysosomal nutrients that can alkalize lysosomes, inhibit AP and lysosome fusion, block protease activity, and inhibit virus entry. With broad-spectrum antiviral and immunomodulatory properties, these autophagy inhibitors are promising drugs to combat COVID-19 [78,84]. However, its use is controversial given its high cytotoxicity and side effects. Nitazoxanide can inhibit late-stage autophagy and has been shown to inhibit SARS-CoV-2 infection in vitro. Azithromycin is another potential drug for COVID-19, as it can hinder lysosomal acidification, thus inhibiting AP clearance and degradation [85]. Furthermore, azithromycin can extensively block autophagy combined with CQ or HCQ to enhance the curative effect on COVID-19 [86]. Finally, cepharanthine is the other primary drug that can inhibit autophagy with antiviral activity and may be a promising therapeutic option for COVID-19 [87].

Multiple studies have confirmed that the endocytic pathway in autophagy is the primary pathway through which SARS-CoV-2 enters host cells. Therefore, the endocytic pathway has been widely studied as a target for treating COVID-19 [28]. Chlorpromazine, an inhibitor of clathrin-dependent endocytosis and an antischizophrenia drug, has been shown to significantly inhibit the entry of coronavirus into host cells [88]. As an FDA-approved anticoronavirus drug, it can be used alongside other medications to treat COVID-19, but its effectiveness has not been clinically evaluated [89]. Furthermore, NH₄Cl [90], CQ [28], and bafilomycin A₁ [91] are lysosomal nutrients that can inhibit virus entry and prevent virus infection. Ouabain and bufalin [92] are drugs that can hinder the clathrin-mediated endocytic pathway to avoid the access of coronaviruses.

For NETosis, the MPO-DNA complex is a well-defined marker of NETosis [93]. cfDNA, extracellular histone H3, growth arrest-specific 6, and ELANE, which increased significantly in patients with COVID-19, can be used as biomarkers for NETosis [56,65,94]. NET expressions are positively correlated with thrombosis. Thus, inhibition of NET decreases platelet-mediated NET-driven thrombogenicity, which is beneficial for the recovery of COVID-19 [64]. Accurate hematological markers of systemic inflammation, systemic inflammation index, and high neutrophil to lymphocyte ratio were closely related to the severity and mortality of COVID-19 patients [95,96]. Furthermore, circulating nucleosomes and citrulline levels can be used clinically as quick and effective measures of the severity of COVID-19 [97].

From the theory of NETosis, cfDNA may be a potential therapeutic target for treating SARS-CoV-2-induced sepsis, and exogenous administration of long-acting nanoparticle deoxyribonuclease-1 may effectively reduce neutrophil activity and cfDNA levels as a therapeutic intervention for COVID-19 [94]. Furthermore, as with other pathogens, SARS-CoV-2 activates platelets to release extracellular vesicles and activates the C-type lectin receptor that enhances NET formation [98]. Therefore, blocking C-type lectins could be a promising therapeutic strategy to reduce intravascular coagulopathy and SARS-CoV-2-induced NETosis [99]. Furthermore, immunomodulatory treatment is a future direction of clinical treatment of COVID-19, such as the treatment with anakinra, eculizumab, heparin, hyperbaric oxygen, remdesivir, and sirolimus [49].

Furthermore, DNase treatment significantly reduces citrullination of histone H3 levels and degrades NET [100]. Clinical studies showed that DNase-I-coated melanin-like nanospheres can reduce neutrophil counts and mitigate sepsis-associated NETosis dysregulation, thus alleviating systemic inflammation and attenuating mortality [53]. Recent data based on a clinical study in the process (ClinicalTrials.gov Identifier: NCT05279391) to treat COVID-19 suggested that the treatment consisted of inhaled DNase-I to dissolve thrombogenic NETs and agents against cytokine-mediated hyperinflammation, was associated with lower in-hospital mortality and intubation rate, shorter duration of hospitalization, and prolonged overall survival [101]. In addition, the treatment also has an inhibitory effect on the Thrombin axis in fibroblasts [101]. Taken together, inhaled DNase-I has emerged as a potential therapeutic option and has potential applications in the treatment of COVID-19.

Concluding remarks

Autophagy and NETosis are two primary defense mechanisms in neutrophils with different functions during the SARS-CoV-2 invasion process. This review provides an in-depth explanation of the underlying mechanisms of autophagy and NETosis in COVID-19 and summarizes the latest research progress. After exploring the relationship between autophagy and NETosis, we also discussed potential targets and treatment options for COVID-19. A comprehensive understanding of neutrophil responses of patients with COVID-19 is required to understand the heterogeneity of the severity of the disease, define the effectiveness of treatment, and predict the prognosis. In this respect, a single-cell multi-omics approach would contribute to achieving this goal.

Statements and Declarations

The authors declare that they have no conflict of interest.

Data sharing statement

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Acknowledgments

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Disclosure statement

The authors declare that they have no conflict of interest.

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

Junmin Wang was supported by the Zhengzhou University Education and Teaching Reform Research and Practice project (2021ZZUJGLX219; Education and Teaching Reform Research and Practice project of Zhengzhou University.