Mechanism of NETosis

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1		Deepak Shukla	--	2122	2023-05-17 23:27:02	\|
2	layout & references	Sirius Huang	Meta information modification	2122	2023-05-18 11:11:41	\|

This entry is adapted from the peer-reviewed paper 10.3390/pathogens12020209

Neutrophil extracellular traps (NETs) are net-like structures released from neutrophils. NETs predominantly contain cell-free deoxyribonucleic acid (DNA) decorated with histones and neutrophil granule proteins. Numerous extrinsic and intrinsic stimuli can induce the formation of NETs such as pathogens, cytokines, immune complexes, microcrystals, antibodies, and other physiological stimuli. The mechanism of NETosis induction can either be ROS-dependent or independent based on the catalase producing activity of the pathogen. NADPH is the source of ROS production, which in turn depends on the upregulation of Ca2+ production in the cytoplasm. ROS-independent induction of NETosis is regulated through toll-like receptors (TLRs). Besides capturing and eliminating pathogens, NETs also aggravate the inflammatory response and thus act as a double-edged sword.

NETosis herpes ocular infection survival NETosis lytic NETosis

1. Introduction

The word “osis” in the term NETosis depicts death that implies the loss of the pathogen entrapped in the NET, but it remains debatable whether the NET release is an active and explicit biological outcome of the host response or simply a result of cellular burst due to accumulation of membrane permeable toxins or stress molecules due to an infection. Further highlighting the complex nature of this phenomenon, experts of cell death pathways are unsure about how the active NET release is related to other known programmed cell death pathways such as apoptosis, necroptosis, and pyroptosis. NETosis was initially defined as a suicidal gizmo to trap and kill bacteria extracellularly, but new reports show that NET release can be triggered by numerous other pathogenic (fungi, viruses, and parasites) and non-pathogenic (PMA, Ionomycin, LPS) stimulants ^[1]. Reportedly, NETosis is also involved in the progression of immune-facilitated disorders. Thus, understanding different mechanisms of NETosis is indispensable to comprehend neutrophil-driven infection and/or inflammatory diseases.

2. Suicidal/Lytic NETosis

The formation of NETs is a lesser explored type of cell death that necessitates nuclear envelope disintegration and chromatin decondensation. Upon induction of NETosis, the cell membrane ruptures, and decondensed chromatin releases its granular matter into the extracellular space, leading to the dissolution of plasmatic membrane, ultimately causing neutrophil death. Subsequently, these NETs can entangle different pathogens such as bacteria, fungi, protozoa, and viruses. Using imaging experimentation as a major tool, Fuchs et al. assigned NET formation as the final step of active neutrophil death in response to phorbol ester and Staphylococcus aureus ^[2]. Essentially, this form of cell death allows the complete release of chromatin into the extracellular space without any DNA fragmentation. The detailed cellular mechanism is still under research, but the key elements of lytic NETosis are well defined and constitute neutrophil elastase (NE) and myeloperoxidase (MPO), both of which form the part of primary neutrophilic granules. The reported mechanism of lytic NETosis associates reactive oxygen species (ROS) formation to NET release through an NE-mediated process. ROS generated by NADPH oxidase stimulates NE translocation from cytoplasmic granules to the nucleus, where it cuts histones and promotes the chromatin unfolding and degradation of the nuclear membrane. MPO also synergizes with the NE in DNA decondensation and triggers NET independent of its enzymatic activity, suggesting the complex nature of NETosis ^[3].

Ligation of different pathogens or immune crystals triggers the induction of ROS via MEK–extracellular-signal-regulated kinase (ERK) signaling pathway that further stimulates an MPO-NE pathway. Additionally, Wang laboratory described the role of peptidylarginine deiminase 4 (PAD4) in histone citrullination, heterochromatin decondensation, and NET formation and thus its crucial role in innate immunity ^[4]^[5]^[6]. Upon stimulation of divalent calcium ion (Ca²⁺), PAD4 can reduce the positively charged histones, which transform histone arginines to citrullines. After this stimulation, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase advances to ROS generation, causing the catalyzation of superoxide dismutase (SOD) to produce hydrogen peroxide (H₂O₂). The H₂O₂ then interacts with MPO to produce hypochlorous acid (HOCl) that leads to chlorination of histones and loosens the histone–DNA interactions, similar to histone citrullination ^[7]. Furthermore, reports suggest that Raf-1 proto-oncogene serine/threonine kinase (c-Raf), mitogen-activated protein kinase (MEK), protein kinase B (Akt), extracellular signal-regulated kinase (ERK), and PKC pathways are upstream to NADPH oxidase production and involved in lytic NETosis ^[8]^[9]. Interestingly, the whole c-Raf-MEK-ERK pathway completes in 2–4 h. Additionally, PMA, ionomycin, concanavalin A, bacteria, fungi, and cytokines such as IL-6 and Il-8 are strong inducers of NADPH oxidase-mediated NETosis ^[10]^[11]^[12].

2.1. HSV and Suicidal NETosis

HSV evades host immune responses to establish a successful lytic infection. It protects its clearance from the immune system by a number of mechanisms such as inhibition of interferon response; evasion of complement-mediated destruction by expressing glycoprotein C, which binds to the C3b complement component; inhibition of autophagy by neurovirulence protein ICP34.5; and suppression of the cGAS–STING signaling pathway by HSV–1 protein UL41 and VP22 ^[13]. In addition, to ensure a lifelong infection, HSV employs diverse molecular approaches to escape host cell death responses. For instance, the viral UL39-encoded viral protein ICP6 suppresses both caspase-8 and RHIM-dependent RIPK3 activities in host cells ^[14]. Similarly, HSV-1 ICP27 inhibits GSDME-mediated pyroptosis for enhancing viral replication in host cells ^[15]. Ironically, no reports claim any correlation between HSV infection and NETosis or herpes-mediated modulation of NETosis. It is noteworthy that HSV is known to modulate or affect the pathways that find involvement in suicidal NETosis. Thus, herpes-mediated modulation of such pathways puts forward the possibility of the virally mediated modulation of NEtosis shown in Figure 1.

Figure 1. Schematic showing HSV-1 ocular infection, its recognition by the TLRs on corneal epithelial cells, and mechanisms associated with suicidal and vital NETosis. Implication of NETosis during an ocular HSV-1 infection: (A) sagittal view of an eye infected with HSV-1; (B) zoomed out description of host innate immune response against ocular HSV-1 infection; (C) suicidal NETosis showing ROS-dependent lytic NETosis; (D) lytic NETosis showing TLR-dependent non-lytic NETosis. Created in bioRender.

2.2. Reactive Oxygen Species: In Milieu with NETosis and HSV Infection

ROS are considered essential for NETs formation. ROS generation is a consequence of the activation of the NADPH oxidase (NOX) family of enzymes. NOX-dependent NETosis agonists such as PMA and LPS induce the generation of massive amounts of ROS in neutrophils. High concentrations of ROS and antimicrobial peptides render antimicrobial activity to neutrophil-generated phagosomes. The pharmacological inhibition of NADPH oxidase enzyme by diphenylene iodonium abrogates the NET formation, ROS production, and ultimately leads to cell death in neutrophils that were pretreated with inducers of NETosis. Furthermore, patients with chronic granulomatosis, who have a genetically defective NADPH oxidase enzyme, do not produce NETs ^[16]. Thus, the levels of ROS in neutrophils critically governs the cell death, i.e., NETosis ^[2].

Coincidentally, HSV also induces NADPH oxidase-dependent ROS generation in infected cells. In cultured cells, the increase in ROS levels is detected as early as 1 h post infection ^[17]. The maintenance of an ROS-mediated mild oxidative stress is thought to facilitate replication and pathogenicity of herpes viruses. The supplementation of antioxidants leads to a reduction in the viral load, indicating that replication is favored by a state of oxidative stress or ROS production ^[18]. Treatment with low concentrations of oxidative stress inducers, for instance, 4-HNE, aids in viral replication, whereas increase in concentration beyond a specific level inhibits the viral replication ^[17]. Under a productive HSV replication, the levels of ROS generated by HSV infection are known to impair the interferon response by oxidizing Cysteine 147 on murine STING, which is analogous to Cysteine 148 of human STING ^[19]. However, ROS are known to trigger the phenomenon of cell death only at higher levels where the cell’s antioxidant mechanisms fail ^[20]. This might be the possible reason for inhibition of HSV replication upon treatment with higher concentrations of ROS inducers such as 4-HNE. At lower levels, ROS are involved in different signaling pathways ^[21]. Thereby, they are known to aid HSV replication in a productive infection by suppressing host immune responses. Therefore, it seems that the levels of ROS generated in the HSV-infected cells are insufficient for triggering NETosis. While the induction of different forms of cell deaths curb HSV infection, HSV infection-triggered induction of ROS promotes viral replication, possibly via the suppression of different cell death pathways. However, at the peak of infection, the ROS levels required for NETosis may be achieved, a possibility that requires additional scrutiny.

3. Live Cell/Vital NETosis

Initially, NETs formation was reported as an oxidant-dependent event that leads to lysis of neutrophils. Recently, Pilsczek et al. described a non-lytic mechanism of NETs formation, where neutrophils responded uniquely to Staphylococcus aureus infection. In this form of NETosis, the nucleus of neutrophil condenses and becomes round ^[22]. Then, there is partition of the inner and outer nuclear membranes and budding of vesicles that are filled with nuclear DNA. This marks the expulsion of vesicles from the cell, where they burst and release chromatin. Thus, the whole process occurs swiftly and in an oxidant-independent manner in 5–60 min. Unlike lytic NETosis, vital NETosis contains a very little amount of mitochondrial DNA. Lytic NETosis has a limited amount of proteolytic activity but enough to trap and kill S. aureus.

The lytic form of NETosis is established on the phenomenon of neutrophil death; however, it leaves certain questions unaddressed and creates a confusion regarding how the obligatory events of NETosis such as chemotaxis and phagocytosis are carried out by a dead neutrophil. One possibility is that a PMN could initially perform its live cell functions and degrade intracellular pathogens prior to its death and then trap extracellular pathogens after its lytic NETosis. This constitutes a progressive model of live cell functions advanced by suicidal cell functions. Another possibility exists that demarcates the population of neutrophils into two subsets. One set of neutrophils may lead to live cell functions, whereas another set could lead to lytic form of NETosis. This would suggest that traditional known functions of NETosis and lytic NETosis are mutually exclusive events. To date, inadequate evidence is present to encourage this hypothesis. An additional hypothesis persists in the field that particular subsets of neutrophils endure the NETosis process and persist to execute the tasks necessary to identify, seize, and kill pathogens. This is the most accepted idea given that at most 20% to 25% of PMNs release NETs. This view was also supported by Clark et al., as they demonstrated the NET release from an intact neutrophil ^[23]. In addition, Yousefi et al. demonstrated the similar phenomenon in eosinophils as well as neutrophils ^[24].

The fundamental difference between lytic and vital NETosis other than time of NET release is the nature of stimulation. For example, suicidal NETosis has mostly been exhibited by chemical stimulants. In contrast, vital NETosis has been shown after PRRs recognition by the host. For instance, LPS, a Gram-negative bacterial stimulus, promotes quick, non-lytic NET release. This rapid NETosis was TLR4-mediated on platelets that accelerated PMN activation. Stimulation by a Gram-positive bacteria in vivo also leads to vital NETosis via both TLR2 and the complement system ^[25]. Thus, activation of the vital NETosis pathway has been reported against multiple groups of microbial pathogens. For instance, a recent report found that Candida albicans promoted NETosis within 30 min in a fibronectin- and complement-dependent manner ^[26].

Toll-Like Receptors: In Milieu with NETosis and HSV Infection

The invading pathogens are often detected by PRRs for instigating immune responses. Toll-like receptors are known to play vital role in recognition of pathogens and induction of NETosis ^[27]. Human neutrophils are known to possess all TLRs except TLR3, where different TLR is employed to recognize a different pathogen. For instance, TLR2 signaling pathway is employed to induce NET release against Mycoplasma agalactiae ^[28], whereas TLR2 and TLR4 are vital for ROS-dependent NETosis initiation during Fonsecaea pedrosoi infection ^[29]. Similarly, TLR7 and TLR8 recognize human immunodeficiency virus 1 (HIV) nucleic acid and trigger the induction of NETosis ^[11], whereas chikungunya virus (CHIKV) is captured by TLR7-elicited, ROS-dependent NETosis ^[30]. In addition, mitochondrial DNA (mtDNA) is also known to activate neutrophils via the cyclic GMP-AMP synthase (cGAS) and TLR9 pathways to stimulate NETosis.

Different TLRs are also involved in recognition of HSV by the host immune system. HSV is recognized by TLR2 on the cell membrane, probably in conjunction with TLR1. Different reports have claimed that TLR2, TLR3, TLR4, and TLR9 are capable of detecting definite proteins of HSV such as glycoprotein B (gB), glycoprotein H (gH), glycoprotein K (gK), glycoprotein L (gL), and US2 protein in the activation and reactivation of HSV ^[31]. The dual TLR2/9 recognition has been reported as vital to fight against mucosal HSV infection. The dual ablation of TLR2/9 has been reported in high mortality rates as compared with TLR2 or TLR9 deficiency alone, overlapping with aggravated viral load in central nervous system tissues ^[28]. Similarly, TLR3 is required to control HSV in the central nervous system ^[32]. Although there are no published data on TLRs-mediated NETosis for killing HSV-1, it is obvious that TLRs play a crucial role in combatting HSV; thereby, here arises a possibility that TLRs-mediated antiviral response may be partially warranted by TLRs-trigged NETosis, as shown in Figure 1.

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