Neutrophil Death-More Than Meets The Eye

Abstract

Neutrophils play an indispensable role in the innate immune system as the body’s first line of defense against pathogens. These highly specialized cells are rapidly recruited to infection sites, where they execute a variety of critical functions essential for pathogen clearance. These functions include phagocytosis, degranulation, the release of antimicrobial peptides and reactive oxygen species (ROS), as well as the formation of neutrophil extracellular traps (NETs), which serve to directly neutralize pathogens or restrict their spread. Despite their abundance—accounting for 40-70% of total white blood cells in human circulation, neutrophils have a relatively short lifespan. To maintain immune homeostasis, approximately 1 billion neutrophils per kilogram of body weight are produced and cleared each day, a highly regulated and energy-intensive process. Neutrophil death is a highly heterogeneous process, with neutrophils undergoing different forms of cell death depending on the stimuli, signaling, and microenvironment. Even during aging or cell death, neutrophils continue to exert significant effects on the immune landscape. In this review, we discuss the dynamics of neutrophil turnover during homeostasis and inflammation, the diversity of mechanisms governing their death, and the multifaceted roles of neutrophils in modulating the immune environment both during and after their demise.

Short-lived but adaptable: the regulation of neutrophil lifespan

Neutrophil half-life under homeostatic conditions

The lifespan of circulating neutrophils is generally believed to be quite short, typically ranging from 7 to 24 hours. Early studies used DFP³² to label blood cells isolated from volunteers¹. DFP³² irreversibly bound intracellular serine proteases, demonstrating relative specificity for neutrophils while binding minimally, if at all, to lymphocytes and monocytes. The labeled blood cells were then reinfused into the volunteers, and by detecting the radioactive decay of DFP³² in white blood cells, researchers calculated the half-life of reinfused neutrophils to be approximately 4 to 9 hours². Subsequent studies using ⁵¹Cr³ and ³HTdr⁴ produced similar findings, with granulocyte half-lives ranging from 5 to 17.5 hours in peripheral blood. While this form of ex vivo labeling provided valuable insights, the method had limitations, including the inadvertent activation of neutrophils⁵ and alterations to their migratory characteristics⁶, which influenced half-life calculations.

To improve accuracy, direct in vivo labeling methods were utilized, such as the intravenous injection of DFP³² into volunteers⁷. Using a mathematical model to analyze the radioactivity in neutrophils isolated from bone marrow and peripheral blood, Cartwright et al., estimated the half-life of neutrophils to be 7.2 hours⁷. In another study, Pillay et al. administered low doses of heavy water to volunteers over nine weeks, measured radioactivity in peripheral blood neutrophils, and estimated a lifespan of 5.4 days in humans and 12.5 hours in mice⁸. Later, Lahoz-Beneytez et al. conducted a similar study using both heavy water and deuterium-labeled glucose, developing a refined mechanistic model applied to their own data as well as Pillay et al.’s data⁹. Due to a difference in the estimated neutrophil precursor-to-mature neutrophil ratio, the human peripheral blood neutrophil half-life was determined to be less than one day⁹.

Mice models are widely used in immunological research, making an understanding of neutrophil dynamics crucial. Through BrdU tracing or in vivo radioactive labeling, researchers have demonstrated that the half-life of circulating neutrophils in mice is shorter, estimated to be 10.7 to 12.5 hours to that of humans^8,10. Ballesteros et al. utilized a parabiosis model to demonstrate that the lifespan of neutrophils in most mice tissues are less than 24 hours¹¹. However, surgical separation in parabiosis experiments might inadvertently extend neutrophil lifespan¹². To address this, they employed a tamoxifen-induced model expressing transient expression of tdTomato specifically in neutrophils, enabling the tracking of individual fluorescent waves. Their findings revealed that neutrophil half-life varied across tissues, with a lifespan of less than 10 hours in blood in contrast to 18.8 hours in the skin¹¹.

Neutrophil life span is altered during inflammation

During inflammation, neutrophils exhibit an extended lifespan, a phenomenon particularly evident in certain inflammatory diseases such as cystic fibrosis, rheumatoid arthritis, or psoriasis^13–15. In these cases, neutrophils demonstrate delayed apoptosis, which is associated with significantly lower levels of Bax, a protein that promotes cell death¹⁶. Furthermore, under the hypoxic conditions often associated with inflammation, neutrophils can extend their half-life through activation of HIF-2α pathways, in addition to the downregulation of MCL-1, an important anti-apoptotic protein, which leads to a prolonged neutrophil lifespan ^17,18. The same extension of neutrophil half-life has been reported during treatment with various inflammatory cytokine stimulations, such as IL-1β, IFN-γ, exposing to bacterial products such as LPS, or streptococci, where activated neutrophils stabilize Mcl-1 thereby inhibiting apoptosis¹⁹. Tumor Necrosis Factor-alpha (TNF-α) which is a potent pro-inflammatory activator of neutrophils has been shown to have a dual effect on neutrophil apoptosis. TNF-α has been shown to accelerated apoptosis via TNFR55 /TNFR75 signaling and caspase induced MCL-1 turnover^20,21. Conversely TNF-α treatment for prolonged periods or with a lower concentration lead to decreased apoptosis levels via IL-8 autocrine signaling or expression of the antiapoptotic molecule, BFL-1^21-23.

There is currently a challenge in precisely calculating neutrophil lifespans under inflammatory conditions due to alterations in systemic factors, granulopoiesis, microenvironmental cues, and apoptotic clearance. Under inflammatory conditions, granulopoiesis is markedly upregulated, resulting in heterogeneous and dynamic neutrophil kinetics. G-CSF, a cytokine that promotes granulopoiesis, has also been shown to strongly upregulate PI3K/Akt pro-survival signal and inhibit the activation of calcium-dependent cysteine proteases, calpains, which act upstream of caspase-3 activation^24,25. Additionally, neutrophil recruitment to inflamed tissues can modify their lifespan through interactions with the local microenvironment, exposure to specific regional cytokine signals, and the efficiency of apoptotic cell clearance mechanisms. This complexity, in addition to varying stages of neutrophil maturation, survival, and function, poses significant challenges for accurate in vivo modeling of neutrophil lifespan during inflammation.

Prolonged neutrophil lifespan in tumors

Neutrophils in the tumor microenvironment have been shown to survive significantly longer than the less-than-24-hour lifespan of circulating neutrophils, suggesting that the tumor provides conditions that extend their longevity to more than 96 hours²⁶. In a recent study, Ng et al. employed BrdU tracing in tumor-bearing mice, demonstrating that the half-life of neutrophils is significantly prolonged not only in the tumor microenvironment but also in the bloodstream²⁷. Specifically, circulating neutrophils in tumor bearing mice exhibited a half-life of 31.4 hours, indicating an increased lifespan compared to previously predicted values for wild-type neutrophils¹¹. Remarkably, neutrophils residing in tumors displayed an even longer half-life of 41.8 hours, with a predicted lifespan of 135 hours (up to 5.625 days). Similarly, in human cancer patients, both peripheral and tumor-infiltrating neutrophils demonstrate reduced apoptosis rates and extended persistence^28,29.

The extended lifespan of neutrophils has been suggested to result from intrinsic signals, such as cytosolic proliferating cell nuclear antigen (PCNA), which inhibits caspase activation²⁹, or from extrinsic pro-survival factors like G-CSF or IFN-β, which are produced by nearby tumors or other immune cells³⁰. Further investigations are required to define the biological significance of altered neutrophil apoptosis and its potential influence on cancer progression and therapeutic responses.

The modes of neutrophil death

Apoptosis

Apoptosis is a fundamental mechanism for maintaining neutrophil homeostasis, which can be initiated through intrinsic or extrinsic pathways^12,31. The intrinsic pathway is triggered by intracellular stress signals, leading to the activation of caspase-9, whereas the extrinsic pathway is mediated by membrane-bound death receptors, which activate caspase-8. Both pathways converge upon the cleavage and activation of caspase-3 which in turn triggers cellular events such as DNA fragmentation, chromatin condensation, cytoskeletal disruption, nuclear envelope breakdown, cell shrinkage, and apoptotic body formation, ensuring programmed cell death occurs with minimal damage to surrounding tissues^32,33. Under physiological conditions and during inflammation resolution, neutrophil turnover predominantly occurs through apoptosis, though variations exist in morphology and regulatory mechanisms^31,34. Neutrophil apoptosis shares key features of classical apoptosis, including cytoplasmic vacuolation and the externalization of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane³⁵.

Many classical pro-survival and pro-apoptotic pathways regulate neutrophil survival and death³⁴. For instance, the pro-survival PtdIns(3,4,5)P₃/Akt signaling pathway also plays a key role in neutrophil death^36,37. In PTEN-deficient neutrophils, enhanced PtdIns(3,4,5)P₃ signaling prolongs survival and improves host outcomes during E. coli-induced pneumonia³⁸. Conversely, disruption of the focal adhesion complex significantly downregulates the PtdIns(3,4,5)P₃/Akt pro-survival pathway, leading to increased neutrophil death³⁹. An extended neutrophil lifespan can contribute to tissue damage, as seen when cigarette smoke extract and nicotine reduce InsP7 levels, thereby increasing Akt activity and promoting lung neutrophil accumulation and damage. Neutrophil death can be restored by overexpressing InsP6K1 to elevate InsP7 levels or by inhibiting Akt activity⁴⁰.

Intracellular reactive oxygen species (ROS) are also key mediators of neutrophil apoptosis which accumulate intracellularly in aging neutrophils³⁶. In apoptotic neutrophils, ROS accumulation disrupts actin-mediated positive feedback loops, acting as a negative regulator of PI3Kγ³⁶. In addition, ROS can directly oxidize and damage granules, causing serine proteases or other enzymes to leak into the cytosol, which results in damage and ultimately leads to cell death, a process known as lysosomal membrane permeabilization (LMP) ^41,42. Proteinase 3 which is typically stored in granules is released into the cytosol during aging through LMP. This release facilitates the cleavage of procaspase-3, thereby inducing caspase activation and apoptosis in a caspase-8/9-independent manner⁴³. The primary anti-apoptotic protein in neutrophils is Mcl-1, in contrast to the more commonly expressed Bcl-2 and Bcl-X⁴⁰. Mcl-1 levels directly correlate with neutrophil survival, declining as neutrophils undergo apoptosis to facilitate the apoptotic process^44,45. Conversely, factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1β, sodium butyrate, and lipopolysaccharide can upregulate Mcl-1 expression, thereby enhancing neutrophil survival⁴⁵.

Unlike most other cell types, neutrophils do not rely on mitochondria for energy production but primarily depend on glycolysis. They have relatively low mitochondrial oxidative metabolism. Throughout neutrophil differentiation, they continuously lose cytochrome c and other mitochondrial proteins, leaving only trace amounts in mature neutrophils ^46,47. Despite this loss, neutrophils still exhibit significant caspase-9 activation during apoptosis, a process attributed to their elevated levels of apoptotic protease activating factor-1 (Apaf-1).^46,47. The extrinsic apoptotic pathway in neutrophils is activated upon engagement of surface death receptors such as FAS, tumor necrosis factor receptor 1 (TNF-R1), and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors. In addition to these classical death receptors, neutrophil apoptosis can also be triggered by interactions with pathogens. The phagocytosis of pathogenic bacteria, including B.cepacia, S.aureus, and S.pyogenes, has been shown to accelerate apoptosis in neutrophils^34,48. Furthermore, a separate study demonstrated that the digestion of E. coli induces apoptosis through an oxygen-dependent mechanism⁴⁹. Additionally, the toxic metabolite pyocyanin, produced by P.aeruginosa, directly promotes neutrophil apoptosis by inducing early lysosomal dysfunction, followed by mitochondrial membrane permeabilization and downstream caspase activation⁵⁰.

Apoptotic neutrophils are rapidly cleared by macrophages through efferocytosis, a process mediated by the recognition of phosphatidylserine (PS) on the surface of dying cells^35,51. This interaction stimulates the release of transforming growth factor beta (TGF-β) and interleukin-10 (IL-10) from macrophages, playing a crucial role in promoting the resolution of acute inflammation by suppressing immune activation^35,51-54.

Pyroptosis

Pyroptosis is a specialized, inflammatory form of programmed cell death mediated by inflammatory caspases, playing a critical role in host defense and the propagation of inflammation. Inflammatory caspases such as CASP1, CASP4, CASP5, and CASP11 can cleave full-length GSDMD into an active, pore-forming N-terminal fragment. This fragment then translocates to the plasma membrane via palmitoylation^55–58, where it forms pores, leading to cell swelling and eventual rupture, thereby facilitating the release of cytokines like IL-1β^59,60.

Neutrophil pyroptosis is generally considered a pro-inflammatory mode of cell death, leading to the release of damage-associated molecular patterns (DAMPs), IL-1β, and neutrophil extracellular traps (NETs). Therefore, the regulation of upstream pyroptotic mediators, including inflammasomes, caspases, and proteases, as well as functional GSDMs, must be tightly controlled to fine-tune the downstream inflammatory response. These regulatory mechanisms were recently reviewed in detail by Dubyak et al.⁶¹. Pyroptosis in neutrophils extends beyond its well-established pro-inflammatory roles^62,63. Ma et al. identified gasdermin E (GSDME) as a key regulator of lytic cell death during spontaneous neutrophil aging in murine models. The controlled cleavage and activation of GSDME are mediated by proteinase-3 and caspase-3, leading to pyroptotic cell death. Interestingly, GSDME deficiency does not affect overall neutrophil lifespan but specifically inhibits the pyroptotic pathway of spontaneous death, shifting the mode of death toward apoptosis. This apoptotic skewing enhances macrophage-mediated efferocytosis, thereby reducing inflammatory responses⁶². Furthermore, Zhu et al. demonstrated that GSDME cleavage in murine neutrophils facilitates peptidyl arginine deiminase 4 (PAD4) activation, promoting the extrusion of neutrophil DNA into the extracellular space⁶³.

Despite extensive research on GSDMD and GSDME in neutrophil pyroptosis, the upstream signaling events that initiate this process remain poorly understood. Specifically, the molecular cues that regulate the translocation of cleaved GSDMD fragments to distinct cellular membranes are unclear. While some stimuli direct these fragments to the plasma membrane, inducing pore formation, lysis, and cytokine release, others promote their trafficking to intracellular granules, preventing pyroptosis. This differential localization suggests the involvement of regulatory mechanisms such as post-translational modifications, adaptor protein interactions, or variations in proteolytic processing. Further investigation is needed to elucidate these upstream pathways and assess the role of other pore-forming proteins, such as NINJ1, in neutrophil pyroptosis. A deeper understanding of these mechanisms could uncover new strategies for modulating inflammation and developing targeted therapies for inflammatory diseases.

Necrosis

Neutrophil necrosis frequently occurs under inflammatory conditions, including hypoxia, nutrient deprivation, elevated temperatures, exposure to toxic compounds, and mechanical stress, all of which can trigger necrotic cell death^64,65. Similar to other cell types, neutrophil necrosis is characterized by organelle swelling, plasma membrane rupture, and eventual cell lysis, leading to the release of intracellular contents into the surrounding tissue and contributing to tissue damage^64,65. Primary necrosis, in contrast to secondary necrosis-the final phase of apoptosis is distinguished by increased cell volume (oncosis), plasma membrane rupture, and the uncontrolled release of intracellular material into the extracellular environment^66,67. Like other forms of lytic cell death, neutrophil necrosis can actively contribute to a pro-inflammatory microenvironment by promoting the secretion of IL-6, IL-8, and TNF-α by neighboring cells through the release of damage-associated molecular patterns (DAMPs) from dying neutrophils⁶⁸. It is worth noting that the definition of necrosis has mostly been based on lytic morphology and may overlap with other, more well-defined lytic forms of cell death.

Necroptosis

Described first in 2005, necroptosis was identified as a TNF-α-triggered, non-apoptotic form of lytic cell death occurring independent of caspase-8 and when apoptotic caspases are blocked⁶⁹. It shares key necrotic features like organelle swelling, plasma membrane rupture, and intracellular leakage top other forms of lytic cell death⁷⁰. Mechanistically, necroptosis is driven by the necrosome, comprising of MLKL, RIPK1, and RIPK3 signaling in an intracellular ROS dependent fasion^71–73. Neutrophils can undergo necroptosis during TNF receptor 1 (TNFR1) stimulation⁷⁴, after GM-CSF priming followed by cell adhesion⁷³, activation by MSU crystals or PMA⁷⁵, or phagocytosis of S. aureus⁷⁶. Neutrophil necroptosis can often lead to DAMP release ^77,78, proinflammatory cytokine production, and formation of NETs^79,80, fueling inflammation. However there have been reports suggesting that the rapid lysis of cells during necroptosis may lead to an earlier resolution of inflammation via early shut down of TNF/TLR-induced inflammatory cytokine production. RIPK3 can also exert anti-inflammatory function independent of necroptosis^81–84.

Ferroptosis

Ferroptosis is a form of lytic cell death characterized by lipid peroxidation and iron accumulation. In systemic lupus erythematosus (SLE), neutrophils are shown to undergo ferroptosis, triggered by autoantibodies (IgE) and interferon-alpha (IFN-α), which suppress the activity of GPX4, highlighting how immune dysregulation can drive ferroptotic cell death in SLE neutrophils⁸⁵. Similarly, in the subcutaneous tumor microenvironment (TME), tumor infiltrating neutrophils (TINs) are prone to ferroptosis, which may contribute to immune suppression and tumor progression⁸⁶. These tumor-associated neutrophils are particularly sensitive to ferroptosis induced by agents like RSL3. Importantly, when ferroptosis is inhibited in TINs, T-cell proliferation is enhanced, suggesting that TIN ferroptosis plays a role in suppressing anti-tumor immunity⁸⁶. TINs can evade ferroptosis through GM-CSF signaling in the mammary TME. GM-CSF upregulates Acod1, leading to increased itaconate production, which inhibits Keap1, a negative regulator of Nrf2⁸⁷. This activation enhances GPX4 expression, suppressing ferroptosis in TINs and ultimately promoting tumor growth and metastasis⁸⁷. In both scenarios, the TME modulates TIN ferroptosis to drive tumor progression, with the key difference lying in the tumor models used and the specific phases of tumor development.

NETosis

Neutrophils possess a broad array of antimicrobial defense mechanisms. In addition to their traditional responses, recent research has highlighted the formation of unique fibrous, web-like chromatin structures known as neutrophil extracellular traps (NETs). These structures were first observed in neutrophils challenged with S.aureus and have since been noted in response to protozoal infections as well^88,89. NETs were later defined as extracellular strands of decondensed neutrophil DNA complexed with histones and granule proteins. Their formation during neutrophil stimulation is dependent on the citrullination of histones by peptidyl arginine deiminase 4 (PAD4), which leads to DNA decondensation^90–93. The classical pathway of NET formation, also known as lytic NETosis (suicidal NETs), is triggered by various stimuli, including bacterial and fungal infections, cytokines, damage-associated molecular patterns (DAMPs), and pathogen-associated molecular patterns (PAMPs). This process is largely dependent on ROS production by NADPH oxidase, which activates PAD4 to citrullinated histone H3 along with CDK4/6 activation leading to chromatin decondensation and release^90–92,94. In contrast, non-lytic NETosis (Vital NETs) allows neutrophils to extrude NETs without compromising their viability, enabling them to continue performing immune functions^95,96. It is worth noting that in some cases the DNA released during vital NETosis is of mitochondrial origin, allowing unimpaired neutrophil function^97,98. The mechanistic regulation between lytic and vital forms of NETs is still not fully understood. NETs have also been associated with other forms of lytic neutrophil death such as pyroptosis and necroptosis ^79,99. Mechanistically, whether NETosis constitutes a distinct form of cell death featuring PAD4-mediated chromatin recondensation or simply represents a natural consequence of neutrophils undergoing lytic cell death remains elusive. It has been reported that neutrophil NETosis can occur independently of PAD4 and histone citrullination in models of K. pneumoniae pneumonia¹⁰⁰ or when neutrophils are stimulated with PMA¹⁰¹.

Metabolic changes in neutrophil death

Neutrophils primarily rely on glycolysis for metabolic energy to carry out their effector functions, despite possessing functional mitochondria and internal glycogen stores^102–104. During apoptosis, their glycolytic activity diminishes due to the downregulation of key glycolytic enzymes or reduced glucose uptake leading to decreased energy production^105,106. To counteract this, glycerol-3-phosphate (G3P) can transfer electrons to complex III of the mitochondrial respiratory chain via ubiquinol, helping maintain mitochondrial membrane potential in turn, preventing cytochrome c release and delay the onset of apoptosis⁴⁶. In addition to serving as a source of ATP and pro-survival functions, glucose can also be diverted into the parallel pentose phosphate pathway (PPP), which plays a critical role in supporting ROS production, NET formation, and death receptor expression, processes that collectively contribute to neutrophil death^103,107. These observations suggest that the routing of glucose into distinct metabolic pathways can influence neutrophil fate.

Glutamine metabolism also plays a dual role in regulating neutrophil lifespan. On one hand, glutamine serves as a precursor for the synthesis of antioxidants such as glutathione, which protects against oxidative damage. Exogenous glutathione supplementation has been shown to delay neutrophil death and can also act as a metabolic fuel under inflammatory conditions^108,109. On the other hand, glutamine can be converted to malate through glutaminolysis, generating NADPH to support ROS production and NET formation which can contribute to neutrophil death^110,111.

Fatty acid oxidation (FAO) is a key metabolic pathway that generates acetyl-CoA through the breakdown of fatty acids. During differentiation, neutrophils rely heavily on lipophagy-mediated FAO. However, upon maturation, they shift their metabolic program toward glycolysis^103,112. Short-chain fatty acids and oxidative stress resulting from FAO can reduce neutrophil viability¹¹³. In contrast, fatty acid synthase–mediated lipogenesis supports neutrophil survival by promoting ether lipid synthesis, which helps maintain membrane integrity¹¹⁴.

In summary, there is a need for further investigation into how neutrophils selectively utilize metabolic precursors in distinct pathways in response to external stimulus or internal “aging” signaling, ultimately influencing their function, survival, or death.

Summary

Neutrophil death is a highly heterogeneous process, encompassing multiple forms that are differentially regulated by various triggers³¹ (Table 1) These processes are governed by complex regulatory mechanisms that control the balance between distinct cellular fates, especially within the context of programmed cell death. This variability expands the functional plasticity of neutrophils, allowing them to adapt to diverse signals that vary in duration and intensity. It remains unclear whether neutrophils are intrinsically preprogrammed to undergo a specific type of cell death in response to a given cue or if they rely on rapid stimuli-triggered de novo transcription and translation to determine their fate. With various neutrophil death mechanisms being revealed, several strategies have been developed to extend their lifespan. CLON-G treatment, a combination of caspase inhibition, lysosomal membrane permeabilization prevention, oxidant suppression, necroptosis inhibition, and G-CSF, can prolong neutrophil survival for up to five days. Notably, neutrophils treated with CLON-G retain their infection-clearing capacity upon transfer into neutropenic mice, comparable to freshly isolated neutrophils²⁵.

Table 1.

Summary of modes of neutrophil death in response to stimulation

Simulation/mode of death	Apoptosis	Pyroptosis	Necroptosis	Ferroptosis	NETosis
Adhesion	Focal adhesion kinase engagement 39
Type 1-fimbriated Escherichia coli	Cathepsin mediated Bid cleavage41
Phagocytosis of pathogens	ROS dependent upregulation of proapoptotic proteins42,48
Protease proteinase 3	Caspase-9 activation43	Caspase-3 activation cleaves GSDME62
Cytochrome c/ Apaf-1	Apoptotic Caspase activation46,47
Fas ligand	Fas162,163
TNF-related apoptosis-inducing ligand	TRAIL-R2 and R3 receptors163,164
TNF-a (high doses)	mitoROS production165,166		RIPK3 (when apoptosis was blocked)74
Lipopolysaccharide (LPS)+ Nigericin		GSDMD localization to primary granlues60			Cytosolic LPS-GSDMD167
Pseudomonas aeruginosa		NLRC4/NLRP3-GSDMD168,169
Burkholderia thailandensis		NLRC4-GSDMD170
Leishmania mexicana		NLRP1-GSDMD171
Aspergillus fumigatus		NLRP3- GSDM-D172
Cell adhesion molecule ligation			ROS-RIPK373
Birinapant+z-VAD-fmk			RIPK3 (when apoptosis was blocked)79
SLE IgG/ IFN-α				Gpx4 down-regulation85
RSL3				Gpx4 inhibition 86
PMA					PKC activation88,101
Ionomycin					Calcium influx-PAD4 activation101,173
Various Bacteria					ROS-PKC (PAD4?) activation90,101,174
Candida albicans					β-glucan, PKC acivation89,96,101
Platelets					Neutrophil-platelet aggregation98
IgG Immune complex					Fc receptor signlaing175,176

Neutrophil function after death

Neutrophil aging and clearance via efferocytosis

As neutrophils age, they undergo a series of transformations, including changes in cell marker expression. These changes are characterized by increased levels of CXCR4 and CD18, alongside decreased levels of CD62L^115–117. Functionally, they show a heightened tendency to migrate to inflamed tissue, an accumulation of intracellular ROS but decreased ROS production in response to extracellular stimuli, and improved phagocytic capabilities^116,118. While aged neutrophils are thought to provide better protection against infections, they might cause more severe tissue damage in sterile injuries¹¹⁹. The primary factors driving neutrophil aging remain largely unknown, but some reports suggest links to spontaneous neutrophil activation, circadian rhythms, or the microbiome ^119,120. Mechanistically, the CXCL2-CXCR2 axis has been implicated in driving aging, whereas CXCR4 antagonizes it¹¹⁹. While the elevated CXCR4 would guide the aged neutrophil back to the bone marrow where their altered apoptotic regulatory factors accelerate their progression toward terminal apoptosis¹¹⁵. Subsequently, the aging neutrophil exhibits a downregulation of “don’t eat me” signals, while “find-me” and “eat-me” signals are enhanced¹²¹. This promotes engulfment by resident or circulating macrophages, ensuring clearance and completing the cycle to maintain homeostasis ^12,34,122. The efferocytosis of dying neutrophils or apoptotic bodies induces an immunosuppressive signaling via TGF-β and IL-10 secretion^123,124. Given the transformations observed during neutrophil aging, it is plausible that aged neutrophils may acquire novel roles or functions.

NETs

The short lifespan and rapid clearance of neutrophils have prompted speculation about their extended functions beyond their expected duration of life. The concept of neutrophil functions during aging or even after death was first highlighted with the discovery of NETs ⁸⁸. In this process, activated neutrophils discharge a combination of chromatin, granule proteins, and histones, forming a web-like structure to trap and kill bacteria, thus prolonging the functionality of neutrophils even after their death. Since this revelation, the field of NETs has seen expansive growth, with a multitude of NET variants being characterized ^{92,95,101,125}. Beyond their primary role in anti-microbial defense, NETs have been implicated in a range of pathophysiological scenarios, including autoimmune diseases such as SLE and RA ^126–128, gallstone formation ¹²⁹, vasculitis, vaso-occlusion, diabetes, atherosclerosis, gout ^92,130,131, and thombosis¹³². Furthermore, NET DNA or protein components can lead to regional inflammation and by increasing its immunogenicity leading to autoantibody production^133,134. Depending on the context, NETs can have either pro-tumor or anti-tumor function. NETs can exert cytotoxic effects, directly targeting tumor cells or indirectly via other cells. However, they can form a physical barrier to shield tumor mass or serve as a direct chemoattractant for tumor metastasis Furthermore, their inflammatory, matrix-degrading, and angiogenic properties often support tumor progression^135–139.

DAMP release

Following neutrophil lytic cell death, cellular contents, known as damage-associated molecular patterns (DAMPs), are released into the extracellular space to augment host immune response¹⁴⁰. These DAMPs contribute to harmful pro-inflammatory responses and localized tissue damage, potentially leading to end-organ failure, the onset of chronic autoimmune diseases like SLE and rheumatoid arthritis, or even fatal outcomes^141–143.

Many intracellular molecules, including alarmins, ATP, uric acid, yest-to be secreted cytokines, and even F-actin, can be released into the extracellular space, acting as DAMPs and further driving inflammation^143,144. Protein DAMPs, such as HMGB1, have been shown to stimulate TNF-α secretion by monocytes^144,145. Extracellular RNA can also promote inflammation via TLR3 signaling and is speculated to be released alongside other DAMPs during neutrophil lytic death. Histones and DNA from NETs are also released as DAMPs during neutrophil cell death. Upon neutrophil activation, peptidylarginine deiminase 4 (PAD4) catalyzes histone citrullination, weakening DNA-histone interactions. This process drives chromatin depolymerization, leading to the release of histones and other cellular components⁸⁸. Once in circulation, free histones exert cytotoxic effects by inducing membrane bending, leading to rupture, and contribute to inflammation through the activation of PRRs ^146,147. Additionally, granule proteins such as MPO, neutrophil elastase, and MMP9 contribute to tissue damage by degrading and remodeling the extracellular matrix (ECM)^148–150. Presumably, intact granules may also be released during neutrophil cell death, from which highly reactive granule proteins disperse in a more prolonged fashion. Finally, many components released during the death of other cell types may also act as DAMPs in neutrophils. For instance, during cell activation or death, mitochondria can release DAMPs (mitoDAMPs), including proteins, mtDNA, and lipids, into the extracellular space. These mitoDAMPs initiate inflammatory responses by activating pathways such as TLR and MAP and contribute to inflammasome activation¹⁵¹, playing a role in diseases such as sepsis, ischemic stroke, and neurological pathologies^152–155. During cell death, ER components such as calreticulin have been shown to be expressed on the cell surface, facilitating immunogenicity and promoting the phagocytosis of tumor cells¹⁵⁶. Interestingly, calreticulin has also been shown to be expressed on the neutrophil surface, and when neutrophils are activated, they shed calreticulin into the extracellular space, where it binds to C1q and activates the complement pathway¹⁵⁷.

Of note, some studies suggest that aging or dying neutrophils also release lipoxins and resolvins, which promote tissue repair and homeostasis. These molecules may enhance neutrophil efferocytosis by macrophages and facilitate NET clearance via AMPK signaling^158–160.

LAND-Vs

We have recently characterized a novel class of vesicles, designated Large Aging Neutrophil-Derived Vesicles (LAND-Vs), which are formed during neutrophil spontaneous death. These vesicles diverged from conventional vesicle classifications due to their unique size, structure, and biogenesis pathway. LAND-Vs are equipped with surface “don’t eat-me” signals, which effectively prevent efferocytotic clearance by phagocytes, facilitating their accumulation during the resolution phase of neutrophilic inflammation. Notably, CD55 on the surface of LAND-Vs elicits potent anti-inflammatory effects by inhibiting the complement activation cascade. This modulation not only diminishes neutrophil recruitment but also alleviates tissue damage, underscoring their potential in regulating inflammation resolution after facilitating inflammation and host defense¹⁶¹. This finding exhibits an active anti-inflammatory function of neutrophils during aging which is distinct from the passive functions of neutrophil death and transcends the short lifespan of these cells. The functions of LAND-Vs could extend beyond regulating inflammation, potentially modulate other neutrophil-mediated immune responses such as tissue repair, coagulation, or angiogenesis. LAND-Vs may also interact with the surrounding cells and tissue during inflammation or tumorous conditions.

Outlook

Although neutrophil aging ultimately leads to cell death, it involves distinct transcriptional and protein regulation, resulting in biological functions that differ from those of fresh neutrophils. This suggests that neutrophils could retain functional capacity during their terminal stages, contributing to regulatory processes beyond their immediate demise. While neutrophil death is governed by distinct triggers and mechanisms, it is plausible that throughout the process of aging, after neutrophils are committed to die, they may continue to perform active functions rather than merely preparing for clearance or death. Therefore, it is essential to define the characteristics of neutrophil aging and identify the internal and external triggers of this process. Establishing a clear definition of the stages of neutrophil aging at the molecular level, beyond mere phenotypical changes, will provide deeper insights into the mechanisms and functions of neutrophil aging, rather than viewing it solely as a transition to cell death.

Figure 1. Immune modulation by aging and dying neutrophils.

This schematic illustrates the transition from fresh to aged neutrophils and their functional outcomes after cell death. Fresh neutrophils undergo changes in surface protein expression, including increased CXCR4 and CD18 and decreased CD62L, with the CXCL2-CXCR2 axis intrinsically driving their aging. As neutrophils age, they can undergo different post-mortem fates. One pathway is neutrophil extracellular trap (NET) formation (NETosis), in which aged neutrophils release antimicrobial chromatin structures that aid in pathogen clearance but also contribute to tissue damage, vascular occlusion, tumor metastasis, and autoimmunity. Alternatively, apoptotic neutrophils can be cleared via efferocytosis by macrophages, a process associated with the release of immunosuppressive cytokines such as TGF-β and IL-10, which help resolve inflammation. Additionally, neutrophil lytic death leads to the release of damage-associated molecular patterns (DAMPs), including RNA, DNA, proteins (e.g., high-mobility group box 1 protein [HMGB1]), mitochondria, cytokines, and granules, all of which modulate immune responses and inflammation. Neutrophil aging is also linked to the release of large aging neutrophil-derived vesicles (LAND-Vs), which regulate inflammation and promote homeostasis.

Highlights:

• Neutrophils are the most abundant but short-lived immune cells, with their lifespan extendable under inflammatory or tumor conditions.

• Neutrophil death occurs through diverse pathways, depending on the type of stimulation, leading to distinct inflammatory outcomes.

• Neutrophil aging is not merely a step toward death but may also impart additional functions, such as LAND-Vs.

• Even after death, neutrophils continue to influence their surrounding environment, facilitating various biological processes.