Dying to Defend: Neutrophil Death Pathways and their Implications in Immunity

Abstract

Neutrophils, accounting for ≈70% of human peripheral leukocytes, are key cells countering bacterial and fungal infections. Neutrophil homeostasis involves a balance between cell maturation, migration, aging, and eventual death. Neutrophils undergo different death pathways depending on their interactions with microbes and external environmental cues. Neutrophil death has significant physiological implications and leads to distinct immunological outcomes. This review discusses the multifarious neutrophil death pathways, including apoptosis, NETosis, pyroptosis, necroptosis, and ferroptosis, and outlines their effects on immune responses and disease progression. Understanding the multifaceted aspects of neutrophil death, the intersections among signaling pathways and ramifications of immunity will help facilitate the development of novel therapeutic methods.

Keywords: NETosis; ferroptosis; necroptosis; neutrophil apoptosis; pyroptosis.

Figure 1

Neutrophil Homeostasis and Multifaceted Death Pathways in Steady‐State and Inflammatory Conditions. Neutrophils are generated in the bone marrow through granulopoiesis and subsequently enter the circulatory system. Depending on the specific microenvironment, neutrophils undergo various mechanisms of cell death.^{[ 7 ]} These mechanisms encompass both non‐lytic apoptosis and lytic death modalities, including necroptosis, pyroptosis, ferroptosis, and NETosis. These lytic processes are accompanied by the release of cytotoxic cellular proteases, cell‐free DNA, and chromatin into the microenvironment. Each death pathway operates through distinct molecular mechanisms and regulatory networks, ultimately resulting in either immunosuppressive or pro‐inflammatory outcomes.^{[ 10 ]} Defects in the clearance of apoptotic neutrophils and the accumulation of cellular remnants contribute to the onset of inflammatory diseases and autoimmune disorders. Figure created with BioRender.com.

Figure 2

Molecular Mechanisms Underlying Neutrophil Apoptosis. a) Extrinsic Apoptosis: Initiated by cell surface death receptors, such as FAS, TNFR1, and TRAIL receptors, the extrinsic apoptotic pathway begins with the activation of Caspase‐8, which also promotes Mitochondrial Outer Membrane Permeabilization (MOMP), ultimately leading to Caspase‐3 activation, responsible for the execution phase.^{[ 18 ]} Concurrently, the generation of reactive oxygen species (ROS) by NADPH oxidase serves as a complementary factor in this pathway.^{[ 19 ]} b) I ntrinsic Apoptosis: Within the intrinsic apoptosis pathway, pro‐apoptotic dimers from the Bcl‐2 family, Bax and Bak, embed themselves in the mitochondrial outer membrane, inducing MOMP. The release of Cytochrome c into the cytoplasm then initiates the activation of Caspase‐9, eventually leading to Caspase‐3‐mediated apoptosis.^{[ 16 ]} c) Pathogen‐Induced Cell Death (PICD): This specialized process integrates the phagocytic elimination of microbial pathogens with the initiation of apoptosis. Following their antimicrobial actions, neutrophils are targeted for clearance via efferocytosis, typically carried out by macrophages. Effective efferocytosis ensures the timely removal of apoptotic neutrophils, preventing their progression to secondary necrosis. Inefficient clearance of apoptotic neutrophils may lead to the extracellular release of toxic granules and damage‐associated molecular patterns (DAMPs), thus amplifying local inflammatory responses and perpetuating tissue injury.^{[ 29 ]} Figure created with BioRender.com.

Figure 3

Pathways of Neutrophil Extracellular Trap (NET) Formation. a) Lytic NETosis: This form of NET formation is characterized as a cell death pathway. It commences with the disassembly of actin cytoskeletal structures within the neutrophil, followed by nuclear delobulation, which involves the reorganization of nuclear components.^{[ 47 ]} Subsequent histone citrullination facilitates chromatin decondensation, allowing de‐agglutinated chromatin to mix with cytoplasmic granular components. The process culminates in plasma membrane rupture, releasing NETs into the extracellular milieu.^{[ 50 ]} b) Vital NETosis (Non‐Lytic NETosis): In contrast, vital NETosis enables neutrophils to form NETs without accompanying cell death.^{[ 48 ]} During this process, NETs are extruded from neutrophils while maintaining membrane integrity. This non‐lytic mode of NET formation maintains the neutrophil's functional capacity for tasks such as phagocytosis, allowing them to engage in microorganism engulfment and contribute to host defense while simultaneously releasing NETs.^{[ 49 ]} Figure created with BioRender.com.

Figure 4

Regulatory Mechanism of NET Formation and Release. The generation of ROS serves as a cornerstone in modulating NET release. Key kinases such as AKT, PI3K, and PKC are integral to the dynamic regulation of ROS levels.^{[ 63} , ^{64 ]} Elevated intracellular calcium ions are indispensable for NETosis and act as activators for PAD4, which in turn catalyzes histone citrullination and chromatin decondensation.^{[ 52 ]} In synergy with PKC and CDK4/6, PAD4 facilitates the disassembly of nuclear architecture during NETosis.^{[ 70 ]} The azurosome, a specialized organelle, harbors key enzymes like Myeloperoxidase (MPO), Neutrophil Elastase (NE), and Cathepsin G (CG), essential for NET biogenesis. Upon neutrophil activation, these enzymes translocate to the nucleus, furthering chromatin relaxation. The release of NE into the cytosol is ROS‐ and MPO‐mediated and precedes its nuclear translocation, wherein it targets and degrades F‐actin. Both pathogens and Pathogen‐Associated Molecular Patterns are potent NET inducers.^{[ 69 ]} Dysregulated NET formation or defective clearance can result in pathological NET accumulation, exacerbating inflammation and autoimmune diseases. Furthermore, NETs can interact with platelets, presenting potential complications such as vascular or catheter obstructions.^{[ 111 ]} This figure serves as a comprehensive synopsis of the multifaceted processes underpinning NET formation, underscoring its relevance in host defense, inflammatory regulation, and disease etiology. ACPAs, autoantibodies to citrullinated protein antigens; I/R injury, ischemia‐reperfusion injury; RA, rheumatoid arthritis; SLE, Systemic Lupus Erythematosus. Figure created with BioRender.com.

Figure 5

Potential Mechanisms of Neutrophil Pyroptosis. a) Mechanisms Underpinning Resistance to Neutrophil Pyroptosis: Pattern recognition receptors (PRRs), including NLRP3 and NLRC4, enable neutrophils to sense exogenous pathogens, culminating in inflammasome assembly and Caspase‐1 activation.^{[ 126 ]} In addition, this activation cascade results in the mature release of IL‐1β without invoking neutrophil pyroptosis.^{[ 128 ]} Several regulatory pathways contribute to this resistance: (1) neutrophil‐derived GSDMD‐NT targeting azurosomes and autophagosomes, limiting its impact on cell membranes. ^{[ 129 ]} (2) the serine protease inhibitors from the SERPINB family can impede inflammatory Caspases and neutrophil serine protease (NSP) activity, preventing unwarranted neutrophil pyroptosis.^{[ 130 ]} (3) the triggering of membrane repair mechanisms by GSDMD perforation, such as ESCRT‐III, which might support neutrophil resistance to GSDMD‐caused cytoplasmic membrane disruption.^{[ 133 ]} b) Neutrophil Pyroptosis‐Induced NETs Pathway: Intracytoplasmic LPS and bacterial agents activate Caspases, including Caspase‐11 and Caspase‐4/5, resulting in GSDMD‐dependent neutrophil death. These Caspases act in concert with GSDMD to facilitate nuclear membrane penetration and histone degradation, processes integral to NET formation.^{[ 136 ]} Neutrophil granules harbor specialized serine proteases, such as neutrophil elastase (NE) and cathepsin G (CTSG), capable of uniquely cleaving GSDMD, thus contributing to neutrophil pyroptosis.^{[ 138 ]} Figure created with BioRender.com.

Figure 6

Molecular Pathways Regulating Neutrophil Necroptosis. Community‐acquired methicillin‐resistant Staphylococcus aureus (CA‐MRSA) is phagocytosed by neutrophils yet manages to persist intracellularly. This intracellular survival of CA‐MRSA instigates RIPK3‐mediated necroptosis, a form of programmed cell death, independently of MLKL.^{[ 150} , ^{155 ]} In the absence of the XIAP, a crucial member of the IAP family, the inhibition of Caspase‐8 triggers a shift in TNF‐induced neutrophil cell death from apoptosis to RIPK1‐RIPK3‐MLKL‐dependent necroptosis.^{[ 158 ]} This transition represents a critical regulatory juncture in determining cell fate. Additionally, CA‐MRSA stimulates an autocrine production of TNFα in neutrophils, further amplifying the necroptotic cascade. In the presence of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), ligation of adhesion receptors activates the RIPK1‐RIPK3‐MLKL‐p38 MAPK‐PI3K axis, culminating in ROS production via NADPH oxidase.^{[ 147 ]} This condition is another pathway of neutrophil necroptosis. Figure created with BioRender.com.

Figure 7

Implications of Neutrophil Ferroptosis in the Pathogenesis of Systemic Lupus Erythematosus (SLE) and Tumor Progression. In patients with SLE, autoantibodies and IFN‐α augment ferroptosis in neutrophils by intensifying the binding of the transcription suppressor cAMP response element modulator alpha (CREMα) to GPX4 promoters through activation of calcium/calmodulin kinase IV (CaMK IV), thereby reducing GPX4 expression and subsequently amplifying phospholipid‐containing polyunsaturated fatty acid hydroperoxides (PL‐PUFA‐OOH).^{[ 178 ]} Neutrophils significantly contribute to the synthesis of PUFA via the fatty acid transport protein 2 (FATP2)‐mediated uptake of arachidonic acid.^{[ 186 ]} Key enzymatic players, including acyl‐CoA synthetase long‐chain family member 4 (ACSL4), lysophosphatidylcholine acyltransferase 3 (LPCAT3), and arachidonate lipoxygenases (ALOXs), amplify the generation of PL‐PUFA‐OOH, serving as potent inducers of neutrophil ferroptosis. Neutrophil ferroptosis within the tumor microenvironment fosters tumor growth by establishing an immunosuppressive milieu, thereby impeding T‐cell‐mediated antitumor responses.^{[ 187 ]} Neutrophil ferroptosis can lead to the release of Fe²⁺ and PL‐PUFA‐OOH, thereby contributing to a range of immune regulatory mechanisms. Figure created with BioRender.com.