2.1 Multidimensional landscape of neutrophil heterogeneity

Neutrophil heterogeneity manifests across multiple dimensions: maturation state, density, surface marker expression, and functional polarization. One well-established axis of heterogeneity relates to the neutrophil maturation continuum. Contrary to the traditional view of neutrophils as terminally differentiated cells, they exhibit a spectrum of maturity ranging from immature progenitors to aged, senescent cells.¹⁵ Immature neutrophils, characterized by their banded nuclear morphology or low-density properties, are mobilized from the bone marrow during emergency granulopoiesis and display enhanced inflammatory and oxidative burst capacities.^{16, 17} In contrast, aged neutrophils, marked by increased CXCR4 and decreased CD62L surface expression, exhibit impaired antimicrobial functions but a heightened propensity for neutrophil extracellular trap (NET) formation and immunomodulatory roles.^{18, 19}

Neutrophils can be polarized into distinct functional states reminiscent of the M1/M2 paradigm in macrophages. Evidence supports the existence of proinflammatory “N1” neutrophils and anti-inflammatory “N2” neutrophils, which differentially contribute to the initiation and resolution of inflammatory responses.²⁰ N1 and N2 neutrophils can be distinguished immunophenotypically based on their differential expression of surface markers. N1 neutrophils, primed by inflammatory stimuli like LPS or IFN-γ, exhibit increased expression of activation markers such as CD11b, CD66b, and CD64.^{21, 22} In contrast, N2 neutrophils, induced by anti-inflammatory factors like transforming growth factor-β (TGF-β), interleukin-4 (IL-4), or glucocorticoids, display higher levels of CD16, CD163, and CD206, which are typically associated with an immunoregulatory phenotype.^{23, 24} Additionally, N1 neutrophils have been shown to express higher levels of the chemokine receptor CXCR2, which mediates their recruitment to sites of inflammation. On the other hand, N2 neutrophils exhibit increased expression of the chemokine receptor CXCR4, which is involved in their homing to the bone marrow and lymphoid organs, where they can exert immunomodulatory functions. This functional polarization enables neutrophils to orchestrate both the initiation and resolution phases of inflammatory responses.

Neutrophil heterogeneity also extends to their buoyancy, with low-density neutrophils (LDNs) and normal-density neutrophils (NDNs) exhibiting distinct phenotypic and functional properties. LDNs, enriched in inflammatory conditions, display an activated phenotype with enhanced production of reactive oxygen species (ROS), cytokines, and NETs, and increased expression of adhesion molecules and chemokine receptors.^{25, 26} In contrast, NDNs predominate in healthy individuals and exhibit more robust antimicrobial functions, including phagocytosis and degranulation.^{24, 27}

Recent single-cell transcriptomic analyses have further expanded the landscape of neutrophil heterogeneity, identifying distinct subsets based on their gene expression profiles.^{4, 28} These subsets encompass proinflammatory, anti-inflammatory, and immunoregulatory populations with unique functional capabilities, such as differential cytokine production, phagocytic capacity, and interactions with other immune cells.^{29, 30} Moreover, neutrophils acquire tissue-specific phenotypes and functions depending on the microenvironment they encounter.³¹ For instance, tumor-associated neutrophils (TANs) exhibit distinct transcriptional signatures and functional properties that promote tumor progression, including producing proangiogenic factors, matrix-remodeling enzymes, and immunosuppressive mediators.^{32, 33} Similarly, neutrophils in the lung microenvironment during acute respiratory distress syndrome (ARDS) display a proinflammatory phenotype characterized by increased production of cytokines, chemokines, and NETs, contributing to lung injury.^{34, 35}

2.2 Microenvironmental drivers of neutrophil plasticity

The remarkable plasticity of neutrophils, enabling their transition between distinct phenotypic and functional states, is governed by a complex interplay of microenvironmental signals.^{21, 36} Key factors shaping neutrophil plasticity include inflammatory mediators, metabolic cues, and tissue-specific signals.

Inflammatory mediators, such as cytokines, chemokines, and pathogen-associated molecular patterns, DAMPs, are pivotal drivers of neutrophil polarization and functional reprogramming.³⁷ Proinflammatory stimuli like LPS, tumor necrosis factor-alpha (TNF-α), and IFN-γ promote the acquisition of an N1 phenotype characterized by enhanced antimicrobial functions and proinflammatory mediator production.^{20, 21} Conversely, anti-inflammatory signals like TGF-β, IL-4, and glucocorticoids induce an N2 phenotype, favoring immunomodulatory and proresolving functions.²⁰

Metabolic cues, including nutrient availability, oxygen tension, and metabolic intermediates, also profoundly influence neutrophil plasticity.⁴⁷ Hypoxic microenvironments in inflamed or neoplastic tissues stabilize hypoxia-inducible factor-1alpha (HIF-1α), driving a metabolic shift toward glycolysis and promoting proinflammatory neutrophil phenotypes.^{38, 39} In contrast, normoxic conditions favor oxidative phosphorylation and anti-inflammatory neutrophil polarization.⁴⁰ Metabolic intermediates like succinate, fumarate, and itaconate can modulate neutrophil functions by influencing epigenetic landscapes and signaling pathways.^41-43

Tissue-specific signals, including ECM components, stromal cell-derived factors, and organ-specific metabolites, further shape neutrophil plasticity.⁴⁴ For instance, tumor-derived factors like granulocyte-colony-stimulating factor (G-CSF), vascular endothelial growth factor (VEGF), and hypoxia influence the phenotype and function of TANs.^{20, 32} The gut microbiome has also been implicated in modulating neutrophil phenotypes and functions by producing metabolites and modulating inflammatory pathways.^{45, 46}

2.3 Molecular mechanisms underpinning neutrophil plasticity

The plasticity of neutrophils is underpinned by dynamic alterations in their transcriptional programs, mediated by a complex network of signaling pathways and epigenetic modifications. Metabolic reprogramming plays a central role in governing neutrophil plasticity, with proinflammatory stimuli inducing a shift toward aerobic glycolysis.⁴² At the same time, anti-inflammatory cues promote oxidative phosphorylation and fatty acid oxidation.⁴¹ These metabolic adaptations are orchestrated by transcription factors like HIF-1α and signaling pathways such as PI3K/Akt/mTOR³⁸.

Signaling cascades involving mitogen-activated protein kinases (MAPKs), nuclear factor-kappa B (NF-κB), and signal transducer and activator of transcription (STAT) proteins integrate environmental cues and drive transcriptional programs that shape neutrophil phenotypes and functions.²¹ For instance, the p38 MAPK pathway is crucial for inducing proinflammatory neutrophil responses,³⁶ while the STAT3 pathway promotes anti-inflammatory polarization.²⁰

Epigenetic mechanisms, including histone modifications, DNA methylation, and noncoding RNAs, also regulate neutrophil plasticity by modulating the accessibility of gene regulatory regions and establishing distinct transcriptional programs.⁴⁷ For example, the inhibition of histone deacetylases has promoted an anti-inflammatory neutrophil phenotype.³⁷ MicroRNAs (miRNAs), such as miR-223, have been implicated in modulating neutrophil phenotypes and functions by targeting proinflammatory genes.⁴⁸

Posttranslational modifications, including protein phosphorylation, acetylation, and ubiquitination, further fine-tune the activity and stability of essential signaling proteins involved in neutrophil polarization and functional reprogramming.⁴³ For instance, the ubiquitination and degradation of IκBα, an inhibitor of NF-κB, is a critical step in activating proinflammatory neutrophil responses.³²

Emerging evidence also suggests that neutrophils can undergo a form of innate immune memory, termed “trained immunity,” in response to certain stimuli.⁴⁴ This phenomenon involves epigenetic reprogramming that enhances or suppresses neutrophil responses to subsequent challenges, potentially contributing to their functional plasticity.⁴⁰

In conclusion, the past decade has witnessed a remarkable evolution in our understanding of neutrophil biology, revealing a complex landscape of heterogeneity and plasticity. Neutrophils comprise a spectrum of phenotypic and functional states dynamically shaped by diverse microenvironmental cues, metabolic adaptations, and epigenetic modifications. This multidimensional heterogeneity enables neutrophils to fulfill various roles beyond their canonical antimicrobial functions, including immunomodulation, tissue repair, and even the shaping of adaptive immune responses. The molecular mechanisms underpinning neutrophil plasticity involve a complex interplay of signaling pathways, transcriptional regulators, and epigenetic factors that fine-tune neutrophil phenotypes and functions context-dependently (Figure 1).

2.4 Developmental trajectory of neutrophil subsets

Neutrophils have traditionally been viewed as a homogeneous population of short-lived, terminally differentiated cells with limited developmental plasticity. However, recent advances in single-cell technologies and lineage tracing approaches have unveiled a complex landscape of neutrophil heterogeneity, with distinct subsets exhibiting diverse phenotypes, functions, and developmental trajectories.⁴⁹ This section explores the current understanding of neutrophil development, focusing on the emergence of neutrophil subpopulations and the molecular mechanisms governing their differentiation and functional specialization.

2.4.3 Molecular mechanisms governing neutrophil subset differentiation

The molecular mechanisms underlying the emergence of neutrophil subsets during development are complex and multifaceted, involving the interplay of transcriptional regulators, epigenetic modifiers, and microenvironmental cues. Recent studies have begun to unravel the key players and pathways that drive the differentiation and functional specialization of neutrophil subpopulations.

One of the critical transcriptional regulators of neutrophil differentiation is the CCAAT/enhancer-binding protein (C/EBP) family, particularly C/EBPα and C/EBPε.⁷¹ These transcription factors play essential roles in the commitment of myeloid progenitors to the granulocytic lineage and the subsequent maturation of neutrophils.⁷² Interestingly, the relative expression levels of C/EBPα and C/EBPε have been shown to influence the balance between immature and mature neutrophil subsets.⁷³ High C/EBPα expression promotes the generation of immature neutrophils, while increased C/EBPε expression favors the production of mature neutrophils.

Another key transcriptional regulator of neutrophil development is growth factor independence 1 (Gfi1). Gfi1 is essential for the differentiation and survival of neutrophils, as evidenced by the severe neutropenia observed in Gfi1-deficient mice.⁷⁴ Recent studies have revealed that Gfi1 also plays a role in the functional specialization of neutrophil subsets. For example, a study by Ordoñez-Rueda et al.⁷⁵ demonstrated that Gfi1 regulates the expression of granule proteins and receptors in a subset-specific manner. The authors found that Gfi1 deficiency led to the selective depletion of a mature neutrophil subset characterized by high expression of the Fc receptor FcγRIII and the granule protein Ngp.

Epigenetic mechanisms, such as DNA methylation and histone modifications, also contribute to the establishment and maintenance of neutrophil subset identities. For instance, a study by Ronnerblad et al.⁷⁶ identified distinct DNA methylation patterns associated with different stages of neutrophil differentiation. The authors found that the promoter regions of genes involved in neutrophil effector functions, such as granule proteins and respiratory burst enzymes, underwent progressive demethylation during the course of differentiation. Moreover, they observed subset-specific methylation signatures, with LDNs exhibiting a hypomethylated profile compared with NDNs, suggesting that epigenetic remodeling may underlie the functional specialization of these subsets.

The microenvironment in which neutrophils develop and reside also plays a crucial role in shaping their phenotypic and functional properties.⁷⁷ Neutrophils are exposed to a wide array of soluble factors, cell–cell interactions, and physicochemical cues that can influence their differentiation and specialization. For example, the bone marrow niche provides a complex milieu of cytokines, growth factors, and stromal cell interactions that support neutrophil development and modulate their functional attributes.⁷⁸ In particular, the balance between G-CSF and GM-CSF signaling has been shown to influence the generation of distinct neutrophil subsets, with G-CSF favoring the production of mature, terminally differentiated neutrophils, while GM-CSF promotes the expansion of immature, proinflammatory subsets.⁷⁹

In conclusion, the unraveling of neutrophil heterogeneity and the delineation of distinct developmental trajectories have revolutionized our understanding of neutrophil biology and opened new avenues for therapeutic intervention. The identification of specialized neutrophil subsets with unique functional properties has revealed the complex and multifaceted roles of these cells in health and disease.⁸⁰ As we continue to dissect the molecular mechanisms underlying neutrophil subset differentiation and specialization, we will be better positioned to develop targeted therapies that harness the full potential of these versatile immune cells. The journey ahead promises to yield exciting insights into the developmental origins of neutrophil heterogeneity and to transform our approach to the treatment of neutrophil-mediated diseases (Figure 2).

3.1 Orchestration of neutrophil recruitment and migration

Efficient neutrophil recruitment to sites of infection or injury is a critical determinant of host defense and inflammatory responses. This complex process involves a tightly regulated cascade of events, including neutrophil release from bone marrow niches, margination within blood vessels, adhesion to activated endothelium, transendothelial migration, and chemotactic navigation through tissues.^{36, 81} Recent studies have shed light on the intricate signaling networks that govern each step of this migratory odyssey.

The mobilization of neutrophils from bone marrow reservoirs is triggered by inflammatory mediators, such as complement component C5a, IL-8, and G-CSF.^{82, 83} These stimuli activate neutrophil GPCRs, propagating signals through PLC, PI3K, and MAPK cascades.⁸⁴ Downstream effectors, including the Rho GTPases Rac1/2 and Cdc42, orchestrate actin cytoskeletal remodeling, facilitating neutrophil detachment from bone marrow stromal cells.^{85, 86} Concurrently, G-CSF-mediated activation of the transcription factor STAT3 suppresses the expression of the retention chemokine CXCL12, further promoting neutrophil egress from the bone marrow.^{83, 87}

As neutrophils enter the circulation, they undergo priming in response to inflammatory cues, enhancing their responsiveness to subsequent stimuli.⁸⁸ Selectin-mediated rolling along activated endothelium triggers signaling through Src family kinases (SFKs), Syk, and PLCγ2, leading to the upregulation of integrin activation states.⁸⁹ Chemokine receptors, such as CXCR2, bind to immobilized IL-8 gradients on the endothelial surface, propagating Gαi signals that amplify integrin affinity through the adaptor proteins talin-1 and kindlin-3.^{90, 91} Additionally, PI3Kγ generates phosphatidylinositol (3,4,5)-trisphosphate (PIP3) lipids that stabilize integrin clustering and promote firm adhesion.^{92, 93}

Neutrophil extravasation into tissues involves a complex interplay of adhesion receptors, chemokine sensing, and cytoskeletal remodeling. Engagement of endothelial intercellular adhesion molecule-1 (ICAM-1) triggers “outside-in” signaling through the neutrophil β2 integrin Mac-1, activating SFKs, Vav guanine nucleotide exchange factors (GEFs), and Rac/Cdc42 to coordinate actin polymerization.^{85, 94} Simultaneously, GPCR sensing of chemokine gradients, such as IL-8 and leukotriene B4 (LTB4), activates PI3Kγ, generating PIP3 domains that direct cell polarity and protrusion through Rac, Cdc42, and WAVE regulatory complexes.^{95, 96} Rho/ROCK signaling mediates neutrophil tail detachment during transendothelial migration.⁹⁷

Within tissues, neutrophils integrate multiple guidance cues, including chemokines, bacterial peptides, and ECM interactions, to navigate along chemotactic gradients. GPCRs activate Gαi proteins, such as Gαi2, which inhibit cAMP production, promoting cell polarity through Rac/Cdc42 and actin branching via WAVE complexes.^{96, 98} Gβγ subunits also activate PI3Kγ, generating PIP3 domains that recruit Rac GEFs and WAVE regulators to drive leading-edge protrusion.^{95, 99} Simultaneously, receptor-activated PLC generates IP3 to induce calcium fluxes that activate PKC isoforms and the Arp2/3 actin nucleator N-WASP.¹⁰⁰ Negative feedback loops involving phosphatases, such as PTEN, restrict protrusive activity to the leading edge, ensuring directional persistence.¹⁰¹

Recent studies have further elucidated the intricate signaling networks governing neutrophil migration. The atypical Rho GTPase RhoU has emerged as a critical regulator of neutrophil polarization and directional migration, acting through the Wiskott–Aldrich syndrome protein (WASP) Homology 2 And Myosin-binding protein nucleation-promoting factor to stimulate Arp2/3-mediated actin polymerization at the leading edge.¹⁰² Additionally, the lipid chemoattractant S1P has been shown to induce neutrophil migration through S1P receptor signaling, activating Rac and Cdc42 GTPases and promoting integrin-mediated adhesion.¹⁰³

Integrating multiple guidance cues during neutrophil chemotaxis involves complex crosstalk between signaling pathways. For instance, the PI3K and MAPK pathways converge on the pseudokinase TRIB1, which acts as a molecular hub to coordinate neutrophil polarization and directional migration. Moreover, the Wnt/β-catenin pathway has been implicated in regulating neutrophil trafficking, with Wnt ligands modulating the expression of adhesion molecules and chemokine receptors.¹⁰⁴

Dysregulation of these signaling circuits can manifest in pathological contexts, such as impaired chemotaxis in chronic granulomatous disease or excessive neutrophil infiltration driving chronic inflammatory disorders.^{21, 36} Targeting key signaling nodes, such as PI3Kγ or Rho GTPases, represents a promising therapeutic strategy to modulate neutrophil migration in diverse disease settings¹⁰⁵ (Table 1).

3.2 Signaling in antimicrobial functions

Neutrophils deploy a potent antimicrobial arsenal upon reaching sites of infection or tissue damage, including phagocytosis, degranulation, oxidative burst, and NET formation.^{81, 107} Various signaling pathways tightly regulate These diverse effector functions to ensure adequate pathogen clearance while minimizing collateral tissue damage.⁸⁴

Phagocytosis, the engulfment of microbes or cellular debris, is initiated by ligating opsonic receptors, such as FcγRs or CRs.¹⁰⁸ These receptors activate SFKs, including Hck, Fgr, and Lyn, which propagate signals through Syk and PLCγ2 to induce cytoskeletal rearrangements necessary for particle internalization.^{85, 109} Activated Rho GTPases, such as Rac1 and Cdc42, coordinate actin polymerization and membrane remodeling to form the phagocytic cup.¹¹⁰ Concurrently, PI3K generates PIP3 lipids that recruit and activate GEFs and effector proteins, further promoting actin assembly and phagosome formation.¹¹¹

Following phagocytosis, neutrophils employ oxidative and nonoxidative mechanisms to eliminate internalized pathogens. The oxidative burst rapidly generates ROS by the NADPH oxidase complex, which assembles on the phagosomal membrane.¹¹² The activation of the NADPH oxidase requires the phosphorylation and translocation of its cytosolic subunits, p47phox and p67phox, mediated by PKC and MAPK signaling.¹¹³ Rac2, a member of the Rac GTPase family, plays a crucial role in regulating NADPH oxidase assembly and activity.¹¹⁴

Degranulation, which releases preformed antimicrobial proteins and proteases from neutrophil granules, is another crucial effector mechanism.¹¹⁵ The mobilization of specific granule subsets is differentially regulated by calcium signaling and Rab GTPases.^{116, 117} Elevation of cytosolic calcium, triggered by PLC-mediated IP3 generation or calcium influx through store-operated channels, activates PKC and calcium-dependent proteases like calpain, promoting granule fusion with the plasma membrane or phagosome.^{118, 119} Rab27a and Rab27b have been implicated in the differential exocytosis of neutrophil granule subsets, with Rab27a regulating azurophilic granules and Rab27b controlling specific and gelatinase granules.^{120, 121}

NET formation, a recently discovered antimicrobial strategy, involves the extrusion of decondensed chromatin decorated with granule-derived proteins to trap and kill microbes.¹²² The signaling pathways triggering NET formation, or NETosis, are complex and context-dependent.¹²³ In response to stimuli like phorbol esters or calcium ionophores, PKC activation and calcium influx induce the translocation of neutrophil elastase (NE) and myeloperoxidase (MPO) from granules to the nucleus, where they promote chromatin decondensation.^{124, 125} Rac2 and ROS generated by the NADPH oxidase are also critical for NETosis, with ROS-mediated activation of the protein arginine deiminase 4 (PAD4) leading to histone citrullination and chromatin unfolding.^{126, 127} In contrast to the PKC- and ROS-dependent pathway, NETosis induced by bacteria or immune complexes involves a distinct signaling cascade requiring integrin engagement, Src kinases, and Syk activation.^{128, 129}

Recent studies have provided further insights into the molecular mechanisms regulating neutrophil antimicrobial functions. The cyclin-dependent kinase (CDK) inhibitor roscovitine has been shown to enhance phagocytosis and bacterial killing by neutrophils, suggesting a novel role for CDKs in modulating phagocytic signaling.¹³⁰ Additionally, the cGAS–STING pathway, traditionally associated with viral sensing, has been implicated in regulating NET formation, with STING activation promoting NETosis in response to cytosolic DNA.¹³¹

Negative regulatory mechanisms maintain the intricate balance between effective antimicrobial responses and excessive inflammation. For instance, the phosphatase SHIP-1 breaks PI3K signaling, limiting phagocytosis and oxidative burst.¹³² The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptors, such as SIRP-α and PIR-B, recruit phosphatases like SHP-1 to attenuate ITAM-mediated activation signals.^{133, 134} Dysregulation of these inhibitory pathways has been linked to neutrophil hyperactivation and tissue damage in various inflammatory disorders.¹³⁵

Targeting signaling pathways involved in neutrophil antimicrobial functions offers therapeutic opportunities for infectious and inflammatory diseases. For example, pharmacological inhibition of PI3Kγ has shown promise in reducing neutrophil-mediated tissue injury in sepsis and acute lung injury (ALI) models.¹³⁶ Similarly, modulating the activity of Rho GTPases or their downstream effectors may provide a means to fine-tune neutrophil responses in various disease contexts^{137, 138} (Table 2).

3.3 Signaling in neutrophil cell fate

In addition to their antimicrobial functions, neutrophils play a crucial role in shaping the inflammatory milieu and resolving inflammation. The lifespan and clearance of neutrophils are tightly regulated by signaling pathways that control apoptosis, survival, and efferocytosis.¹³⁹ Moreover, recent studies have highlighted the active contribution of neutrophils to the resolution of inflammation through the production of specialized proresolving mediators (SPMs) and the adoption of distinct proresolving phenotypes.¹⁴⁰

Neutrophil apoptosis is a tightly regulated process that ensures the timely removal of effete cells and prevents excessive inflammation. The intrinsic apoptotic pathway, triggered by intracellular stress or damage, is regulated by the balance between proapoptotic (e.g., Bax, Bak) and antiapoptotic (e.g., Mcl-1, Bcl-2) members of the Bcl-2 family.¹⁴¹ Survival signals, such as GM-CSF and IL-8, promote neutrophil survival by activating PI3K/Akt and MAPK pathways, which upregulate the expression of antiapoptotic proteins.^{142, 143} In contrast, death receptors like Fas and TNF-related apoptosis-inducing ligand (TRAIL) receptors initiate the extrinsic apoptotic pathway, activating caspase-8 and downstream effector caspases.¹⁴⁴

The clearance of apoptotic neutrophils, or efferocytosis, is mediated by phagocytes like macrophages and is crucial for the resolution of inflammation.¹⁴⁵ Apoptotic neutrophils release “find-me” signals, such as ATP and lysophosphatidylcholine (LPC), which attract phagocytes through purinergic receptors and G2A, respectively.^{146, 147} The exposure of “eat-me” signals, including phosphatidylserine (PS) and calreticulin, on the surface of apoptotic neutrophils, engages receptors like TIM-4 and LRP1 on phagocytes, triggering Rac-dependent actin rearrangements and subsequent engulfment.^{148, 149}

Recent studies have underscored the active role of neutrophils in promoting inflammation resolution through the production of SPMs, such as lipoxins, resolvins, and protectins, which exert potent anti-inflammatory and proresolving effects.¹⁵⁰ The biosynthesis of SPMs is regulated by enzymes like 5-lipoxygenase (5-LOX) and 15-LOX, whose expression and activity are modulated by cytokines and lipid mediators.¹⁵¹ SPMs act through specific G protein-coupled receptors (GPCRs), such as ALX/FPR2 and GPR32, to inhibit neutrophil infiltration, enhance efferocytosis, and promote tissue repair.^{152, 153}

Moreover, during the resolution phase of inflammation, neutrophils can transition from a proinflammatory to a proresolving phenotype, driven by local mediators such as annexin A1 and resolvin E1, which engage specific receptors and activate signaling pathways that reprogram neutrophil gene expression and function.^{154, 155} Proresolving neutrophils exhibit enhanced apoptosis, increased production of anti-inflammatory cytokines like IL-10, and the ability to promote tissue repair by releasing growth factors and proangiogenic mediators.

The manipulation of signaling pathways regulating neutrophil cell fate and phenotype represents an attractive therapeutic strategy for inflammatory diseases. For instance, promoting neutrophil apoptosis and efferocytosis through the modulation of Bcl-2 family proteins or PS receptors could facilitate the resolution of chronic inflammation.^{156, 157} Additionally, harnessing the proresolving properties of SPMs and their receptors may offer a novel approach to treating inflammatory conditions characterized by impaired resolution, such as atherosclerosis and chronic obstructive pulmonary disease (COPD)¹⁵⁸ (Table 3).

In conclusion, the past decade has witnessed significant advances in understanding the signaling pathways governing neutrophil functions, from their recruitment and migration to their antimicrobial activities and cell fate decisions. These signaling networks exhibit remarkable complexity and plasticity, enabling neutrophils to mount tailored responses to diverse inflammatory challenges while maintaining tissue homeostasis. Dysregulation of these pathways underlies the pathogenesis of various infectious, inflammatory, and autoimmune diseases, highlighting the therapeutic potential of targeting neutrophil signaling. As our knowledge of the molecular mechanisms controlling neutrophil functions continues to expand, it opens up new avenues for developing precision therapies that harness the multifaceted roles of these essential immune cells in health and disease (Figure 3).

4.1 Neutrophil plasticity maintaining immune homeostasis

Neutrophils are the most abundant leukocytes in the human blood and play a crucial role in innate immunity. Traditionally, neutrophils have been considered short-lived effector cells that rapidly migrate to sites of inflammation, where they perform their primary functions, such as phagocytosis, degranulation, and release of NETs.³⁶ However, recent studies have revealed that neutrophils exhibit remarkable plasticity and heterogeneity, adapting their phenotype and function in response to various microenvironmental cues.^{15, 47} Single-cell transcriptome profiling has further highlighted the heterogeneity of neutrophils in homeostasis and infection, revealing distinct subpopulations with specialized functions.⁴ Moreover, tissue environments can shape neutrophil fates and functions, leading to the co-option of neutrophils for tissue-specific roles.³¹

4.2 Neutrophil plasticity in the maintenance of microbiome homeostasis

Neutrophils are the most abundant circulating leukocytes in humans and play a crucial role in innate immunity. As the first line of defense against invading pathogens, neutrophils employ a diverse arsenal of antimicrobial strategies, including phagocytosis, degranulation, and the release of NETs.¹²² While traditionally viewed as short-lived effector cells with a primary function in pathogen elimination, recent evidence suggests neutrophils also play a critical role in shaping and maintaining the host microbiome.

4.3 Neutrophil plasticity in the regulation of healthy metabolic homeostasis

Neutrophils play a crucial role in maintaining metabolic homeostasis, as demonstrated by their involvement in regulating various aspects of metabolism.^{200, 201} These cells exhibit remarkable plasticity in response to metabolic cues, adapting their phenotypes and functions to meet the specific needs of the tissue microenvironment.^{202, 203} Furthermore, neutrophil activity and metabolism are subject to circadian regulation, adding another layer of complexity to their role in metabolic homeostasis.^{204, 205}

5.1 Neutrophil plasticity in acute tissue injuries

Acute tissue injuries, such as those resulting from ischemia–reperfusion, trauma, or chemical insults, trigger a rapid and robust inflammatory response characterized by the massive infiltration of neutrophils.³⁶ While neutrophils play a crucial role in the initial stages of tissue repair by clearing cellular debris and releasing growth factors, their excessive activation and prolonged presence can exacerbate tissue damage and impair the resolution of inflammation.¹⁴⁰

In the context of ALI and ARDS, neutrophils have been shown to exhibit remarkable plasticity, adopting distinct phenotypes that contribute to the pathogenesis of these conditions.²³⁴ Upon exposure to proinflammatory stimuli, such as LPS or cytokines like IL-8 and TNF-α, neutrophils acquire a hyper-activated phenotype characterized by enhanced production of ROS, proteases, and proinflammatory mediators.²³⁵ These activated neutrophils cause direct tissue damage by releasing cytotoxic molecules and exacerbate inflammation by recruiting additional immune cells to the injury site.²³⁶

Moreover, neutrophils in ALI/ARDS exhibit an increased propensity for NET formation, known as NETosis.²³⁷ NETs, composed of decondensed chromatin decorated with antimicrobial proteins, can trap and kill pathogens and contribute to lung injury by promoting alveolar–capillary barrier dysfunction, thrombosis, and fibrosis.¹²³ The molecular mechanisms driving NET formation in ALI/ARDS involve the activation of key signaling pathways, such as the Raf–MEK–ERK cascade and the PI3K–Akt axis, which induce the translocation of NE and MPO to the nucleus, facilitating chromatin decondensation.²³⁸

In contrast to the detrimental effects of hyper-activated neutrophils, emerging evidence suggests that neutrophils can acquire an anti-inflammatory and proresolving phenotype during the resolution phase of ALI/ARDS.²³⁹ These neutrophils, often referred to as “N2” or “polymorphonuclear myeloid-derived suppressor cells,” exhibit increased expression of immunomodulatory molecules like arginase-1, TGF-β, and IL-10.²⁴⁰ N2 neutrophils suppress T cell proliferation, promote the efferocytosis of apoptotic cells, and facilitate tissue repair, thereby contributing to the resolution of inflammation and the restoration of lung homeostasis.²⁴¹

The plasticity of neutrophils has also been implicated in the pathogenesis of AKI, a common complication of sepsis, ischemia–reperfusion, and nephrotoxic insults.²⁴² In AKI, neutrophils infiltrate the kidney and acquire a proinflammatory phenotype, releasing ROS, proteases, and cytokines that damage renal tubular cells and promote microvascular dysfunction.²⁴³ Additionally, neutrophils in AKI exhibit enhanced NET formation, which can obstruct renal tubules, promote thrombosis, and exacerbate inflammation.²⁴⁴

Recent studies have highlighted the molecular mechanisms driving neutrophil plasticity in AKI. The activation of TLRs, mainly TLR4, by DAMPs released from injured renal cells has been shown to induce a proinflammatory neutrophil phenotype.²⁴⁵ Furthermore, the chemokine receptor CXCR2 and its ligands, such as CXCL1 and CXCL2, play a crucial role in neutrophil recruitment and activation in AKI.²⁴⁶ Targeting these signaling pathways may offer therapeutic opportunities to modulate neutrophil plasticity and attenuate renal injury.²⁴⁷

In the context of ischemic stroke, neutrophils have been shown to exhibit distinct phenotypes that contribute to both brain injury and repair.²⁴⁸ In the acute phase of stroke, neutrophils acquire a proinflammatory phenotype characterized by releasing ROS, proteases, and proinflammatory cytokines, which exacerbate blood–brain barrier disruption, neuronal death, and cerebral edema.²⁴⁹ Moreover, neutrophils in ischemic stroke display an increased propensity for NET formation, which can promote thrombosis, microvascular occlusion, and neuroinflammation.²⁵⁰

However, recent evidence suggests neutrophils can also acquire a neuroprotective phenotype in the subacute and chronic phases of stroke.²⁵¹ These neutrophils exhibit increased expression of anti-inflammatory mediators, such as IL-10 and TGF-β, and promote the clearance of cellular debris, thereby facilitating tissue repair and functional recovery.²⁵² The molecular mechanisms underlying this phenotypic switch involve the activation of the transcription factor Nrf2, which induces the expression of antioxidant and anti-inflammatory genes.²⁵³

5.2 Neutrophil plasticity in chronic tissue injuries

Chronic tissue injuries, such as those associated with nonhealing wounds, COPD, and inflammatory bowel disease (IBD), are characterized by persistent inflammation and impaired tissue repair. Neutrophil plasticity has emerged as a key factor in the pathogenesis of these conditions, with distinct neutrophil phenotypes contributing to the perpetuation of inflammation and the failure of resolution.^{14, 254} The role of neutrophils in chronic tissue injuries has been extensively studied, highlighting their potential as therapeutic targets.

In the context of nonhealing wounds, such as diabetic foot ulcers and pressure ulcers, neutrophils exhibit a dysfunctional phenotype characterized by impaired phagocytosis, reduced ROS production, and decreased chemotactic ability.²⁵⁵ These functional deficits are attributed to the chronic exposure of neutrophils to the inflammatory milieu of the wound, which induces a state of cellular senescence and exhaustion.²⁵⁶ Senescent neutrophils secrete proinflammatory mediators, such as IL-1β and TNF-α, and matrix-degrading enzymes, like MMPs, which impair wound healing by promoting persistent inflammation and ECM degradation.^{257, 258} The impact of senescent neutrophils on wound healing has been demonstrated in both diabetic and nondiabetic ulcers.

Moreover, neutrophils in nonhealing wounds exhibit an increased propensity for NET formation, which can further exacerbate tissue damage and delay wound closure.²⁵⁹ NETs have been shown to trap and degrade growth factors, such as platelet-derived growth factor (PDGF) and VEGF, impairing angiogenesis and granulation tissue formation.²⁶⁰ Targeting the molecular pathways that drive neutrophil dysfunction and NET formation, such as the PI3K–Akt axis and the NADPH oxidase complex, may offer therapeutic strategies to promote wound healing.^{261, 262}

In COPD, a chronic inflammatory lung disease characterized by airflow obstruction and emphysema, neutrophils play a central role in the pathogenesis of the disease.²⁶³ Neutrophils in COPD exhibit a hyper-activated phenotype, with increased production of ROS, proteases, and proinflammatory cytokines.^{264, 265} These mediators cause direct damage to the lung parenchyma, leading to alveolar destruction and airway remodeling.²⁶⁶ Additionally, neutrophils in COPD display an enhanced capacity for NET formation, which can further exacerbate lung injury and contribute to the development of autoimmunity.²⁶⁷ The role of neutrophil-derived proteases and NETs in the progression of COPD has been highlighted in recent studies.

Recent studies have identified key molecular pathways that drive neutrophil plasticity in COPD. The activation of the NLRP3 inflammasome, a multiprotein complex that regulates the production of IL-1β and IL-18, has been implicated in the induction of a proinflammatory neutrophil phenotype.²⁶⁸ Furthermore, the PI3K–Akt–mTOR signaling axis has been shown to promote neutrophil survival and activation in COPD, contributing to the persistence of inflammation.²⁶⁹ Targeting these pathways may provide novel therapeutic approaches to modulate neutrophil plasticity and attenuate the progression of COPD. The potential of targeting the NLRP3 inflammasome and PI3K–Akt–mTOR signaling in COPD has been explored in recent studies.

In IBD, a group of chronic inflammatory disorders affecting the gastrointestinal tract, neutrophils have been implicated in the pathogenesis of the disease.²⁷⁰ Neutrophils in IBD exhibit a proinflammatory phenotype, with increased production of ROS, proteases, and cytokines that damage the intestinal epithelium and disrupt the mucosal barrier.²⁷¹ Moreover, neutrophils in IBD display an enhanced capacity for NET formation, which can further exacerbate intestinal inflammation and promote the development of colorectal cancer.²⁷² The contribution of neutrophil-derived factors and NETs to the pathogenesis of IBD has been underscored by recent findings.

Recent studies have shed light on the molecular mechanisms driving neutrophil plasticity in IBD. The activation of the IL-23/IL-17 axis, a critical pathway in the pathogenesis of IBD, has been shown to promote a proinflammatory neutrophil phenotype.²⁷³ Additionally, the dysregulation of the Wnt/β-catenin signaling pathway in neutrophils has been implicated in the impairment of intestinal wound healing and the perpetuation of inflammation.²⁷⁴ Targeting these pathways may offer therapeutic opportunities to modulate neutrophil plasticity and promote mucosal healing in IBD.²⁷⁵

5.3 Neutrophil plasticity in fibrotic diseases

Fibrotic diseases, characterized by the excessive deposition of ECM and the progressive loss of organ function, represent a significant healthcare burden worldwide.²⁷⁶ Recent studies have highlighted the critical role of neutrophil plasticity in the pathogenesis of various fibrotic conditions, including idiopathic pulmonary fibrosis (IPF), liver fibrosis, and systemic sclerosis (SSc).^{2, 277, 278}

In IPF, a chronic and progressive interstitial lung disease, evidence suggests that neutrophils are implicated in the initiation and progression of fibrosis.²⁷⁹ Neutrophils in IPF exhibit a profibrotic phenotype characterized by the increased production of TGF-β, a potent inducer of fibroblast activation and ECM deposition.²⁸⁰ Moreover, neutrophils in IPF display an enhanced capacity for NET formation, promoting the differentiation of fibroblasts into myofibroblasts and stimulating collagen production.^{279, 281}

Recent studies have identified key molecular pathways that drive neutrophil plasticity in IPF. The activation of the NLRP3 inflammasome in neutrophils has been shown to promote the release of profibrotic mediators, such as IL-1β and TGF-β.²⁸² Additionally, the PI3K–Akt signaling axis has been implicated in the induction of a profibrotic neutrophil phenotype, with studies demonstrating that inhibiting this pathway attenuates fibrosis in preclinical models of IPF.²⁸³

In liver fibrosis, a common outcome of chronic liver injuries, accumulating evidence suggests that neutrophils contribute to the initiation and progression of fibrosis.²⁸⁴ Neutrophils in liver fibrosis exhibit a profibrotic phenotype, with increased production of TGF-β and PDGF, which promote the activation and proliferation of hepatic stellate cells (HSCs), the primary source of ECM in the fibrotic liver.^{285, 286} Moreover, neutrophils in liver fibrosis display an enhanced capacity for NET formation, stimulating the differentiation of HSCs into myofibroblasts and promoting collagen production.²⁸⁷

Recent studies have shed light on the molecular mechanisms driving neutrophil plasticity in liver fibrosis. The activation of the CXCR4/CXCL12 axis has been shown to promote the recruitment and activation of neutrophils in the fibrotic liver, contributing to the progression of fibrosis.²⁸⁸ Additionally, the dysregulation of the Hedgehog signaling pathway in neutrophils has been implicated in the induction of a profibrotic phenotype, with studies showing that inhibiting this pathway attenuates liver fibrosis in preclinical models.²⁸⁹

Emerging evidence implicates neutrophils in the pathogenesis of SSc, a chronic autoimmune disorder characterized by skin and organ fibrosis.²⁷⁷ Neutrophils in SSc exhibit a profibrotic phenotype, with increased production of TGF-β and connective tissue growth factor, which promote the activation and differentiation of fibroblasts into myofibroblasts.²⁹⁰ Moreover, neutrophils in SSc display an enhanced capacity for NET formation, which can stimulate the production of autoantibodies and promote the activation of fibroblasts.²⁹¹

Recent studies have identified key molecular pathways that drive neutrophil plasticity in SSc. The activation of the TLR4 signaling pathway in neutrophils has been shown to promote the release of profibrotic mediators, such as TGF-β and IL-6.²⁹² Additionally, the dysregulation of the Wnt/β-catenin signaling pathway in neutrophils has been implicated in the induction of a profibrotic phenotype, with studies demonstrating that the inhibition of this pathway attenuates skin fibrosis in preclinical models of SSc.²⁹³

In conclusion, neutrophil plasticity plays a crucial role in the pathogenesis of various tissue damage diseases, including acute injuries, chronic injuries, and fibrotic conditions. Neutrophils exhibit remarkable phenotypic and functional adaptability in response to the microenvironmental cues encountered in these pathological states, acquiring distinct phenotypes that can either exacerbate tissue damage or promote tissue repair and resolution of inflammation. The molecular mechanisms driving neutrophil plasticity involve a complex interplay of signaling pathways, transcriptional regulators, and epigenetic modifications. These provide potential therapeutic targets for modulating neutrophil functions in tissue damage diseases (Figure 4).

5.4 Neutrophil plasticity in metabolic diseases

Emerging evidence suggests that neutrophils play a crucial role in the pathogenesis of metabolic diseases, such as nonalcoholic fatty liver disease (NAFLD) and its more severe form, NASH.^{294, 295} NASH is characterized by hepatic steatosis, inflammation, and progressive fibrosis, which can lead to cirrhosis and hepatocellular carcinoma.^{296, 297}

Recent studies have highlighted the involvement of neutrophils in the progression of NAFLD to NASH. In the context of NASH, neutrophils exhibit a proinflammatory phenotype, with increased production of ROS, proteases, and proinflammatory cytokines, which contribute to hepatocyte damage and the perpetuation of inflammation.²⁹⁸ Moreover, neutrophils in NASH display an enhanced capacity for NET formation, which can further exacerbate liver injury and promote fibrosis.²⁹⁵

The plasticity of neutrophils in NASH is regulated by various factors, including lipid mediators, cytokines, and metabolic stress. Free fatty acids, particularly saturated fatty acids, have been shown to activate neutrophils through TLR4 signaling, inducing a proinflammatory phenotype.²⁹⁹ Additionally, the CXCR2 ligands CXCL1 and CXCL2, which are upregulated in the liver during NASH, promote neutrophil recruitment and activation.^{300, 301}

Recent studies have identified key molecular pathways that drive neutrophil plasticity in NASH. The NLRP3 inflammasome, a multiprotein complex that regulates the production of IL-1β and IL-18, has been implicated in the induction of a proinflammatory neutrophil phenotype in NASH.^{298, 302} Furthermore, the receptor-interacting protein kinase 3 (RIPK3), a key regulator of necroptosis, has been shown to promote neutrophil infiltration and activation in NASH.³⁰³

Targeting neutrophil plasticity in NASH represents a promising therapeutic strategy. Inhibition of CXCR2 signaling has been shown to attenuate neutrophil recruitment and ameliorate liver inflammation and fibrosis in preclinical models of NASH.³⁰⁴ Additionally, pharmacological inhibition of RIPK3 has been found to reduce neutrophil infiltration and improve liver function in experimental NASH.³⁰³

In conclusion, neutrophil plasticity plays a crucial role in the pathogenesis of metabolic diseases like NASH. Neutrophils acquire a proinflammatory and profibrotic phenotype in response to lipid mediators, cytokines, and metabolic stress, contributing to liver injury and fibrosis. Targeting the molecular pathways that drive neutrophil plasticity in NASH, such as CXCR2 and RIPK3 signaling, may offer novel therapeutic opportunities for the treatment of this prevalent and progressive metabolic disorder.

5.5 Neutrophil heterogeneity and plasticity in aging

Aging is associated with significant changes in the immune system, a phenomenon known as immunosenescence, which contributes to increased susceptibility to infections, reduced vaccine efficacy, and a higher incidence of chronic inflammatory diseases in the elderly population.^{305, 306} Neutrophils, as key players in innate immunity, undergo substantial alterations in their phenotype and function during the aging process, which may have important implications for age-related immune dysfunction.^{307, 308}

Recent studies have revealed that neutrophil heterogeneity and plasticity are significantly influenced by aging. Elderly individuals exhibit a higher proportion of immature neutrophils in the circulation, characterized by a banded nuclear morphology and reduced expression of surface markers like CD16.³⁰⁹ These immature neutrophils display impaired phagocytic capacity, reduced ROS production, and diminished chemotactic ability compared with their mature counterparts.³¹⁰ The increased presence of immature neutrophils in the elderly may reflect a compensatory mechanism to maintain neutrophil numbers in the face of declining bone marrow function.^{311, 312}

Moreover, aging is associated with the emergence of distinct neutrophil subsets with altered functional profiles. For instance, a subset of LDNs has been found to accumulate in the circulation of elderly individuals.³¹³ These LDNs exhibit a proinflammatory phenotype, with increased production of cytokines like IL-8 and TNF-α, and enhanced NET formation.³¹⁴ The expansion of proinflammatory neutrophil subsets in the elderly may contribute to the chronic low-grade inflammation (inflammaging) that characterizes the aging process.

Neutrophil plasticity is also affected by aging, with elderly neutrophils displaying reduced responsiveness to microenvironmental cues and impaired ability to switch between proinflammatory and proresolving phenotypes.³¹⁵ This loss of plasticity may hinder the resolution of inflammation and tissue repair in the elderly, leading to prolonged inflammatory responses and delayed wound healing.³¹⁶

The molecular mechanisms underlying age-related changes in neutrophil heterogeneity and plasticity are complex and multifaceted. Alterations in signaling pathways, such as decreased PI3K/Akt activation and increased p38 MAPK signaling, have been implicated in the functional deficits of elderly neutrophils.^{317, 318} Additionally, epigenetic modifications, such as changes in histone acetylation and DNA methylation patterns, may contribute to the altered transcriptional profiles and functional states of neutrophils during aging.³¹⁹

Targeting age-related changes in neutrophil heterogeneity and plasticity may offer novel therapeutic strategies for improving immune function and reducing the burden of age-related diseases. For instance, interventions aimed at restoring the balance between immature and mature neutrophil subsets, such as G-CSF administration or mTOR inhibition, have shown promise in enhancing neutrophil function in elderly individuals.^{320, 321} Moreover, modulating the polarization of neutrophils toward a proresolving phenotype, through the use of SPMs or other immunomodulatory agents, may promote the resolution of inflammation and tissue repair in the aging population.

5.6 Neutrophil heterogeneity and plasticity in tumor progression

Neutrophils play a complex and multifaceted role in the tumor microenvironment, exhibiting both protumorigenic and antitumorigenic functions depending on their phenotypic state and the specific context of the tumor.^{77, 322} The heterogeneity and plasticity of neutrophils in cancer have gained increasing attention in recent years, as they may offer novel targets for cancer immunotherapy.

TANs exhibit distinct phenotypes and functions compared with their circulating counterparts, reflecting the profound influence of the tumor microenvironment on neutrophil plasticity.³²³ TANs can be broadly classified into two subsets: N1 (antitumorigenic) and N2 (protumorigenic) neutrophils, which mirror the M1/M2 polarization paradigm of macrophages.^{324, 325} N1 neutrophils display an activated phenotype, with increased expression of proinflammatory cytokines, enhanced ROS production, and potent antitumor cytotoxicity.³²⁶ In contrast, N2 neutrophils exhibit an immunosuppressive and proangiogenic phenotype, characterized by the production of growth factors like VEGF and matrix-degrading enzymes that promote tumor growth and metastasis.³²⁷

The polarization of TANs toward an N1 or N2 phenotype is regulated by a complex interplay of signals within the tumor microenvironment. Factors like TGF-β, IL-10, and G-CSF have been shown to promote the N2 phenotype, while IFN-γ and TNF-α favor the N1 state.³²⁸ Moreover, the hypoxic and metabolically challenging conditions within the tumor can influence neutrophil plasticity, with HIF-1α driving the acquisition of protumorigenic functions in TANs.³²⁹

In addition to the N1/N2 classification, recent single-cell transcriptomic analyses have revealed further heterogeneity within the TAN population, identifying distinct subsets with unique gene expression profiles and functional properties.^{330, 331} For instance, a subset of TANs expressing high levels of programmed death-ligand 1 (PD-L1) has been identified in several cancer types, which may contribute to the immunosuppressive microenvironment and facilitate tumor immune escape.^{332, 333}

The plasticity of TANs has important implications for cancer progression and response to therapy. The ability of neutrophils to switch between protumorigenic and antitumorigenic states in response to microenvironmental cues suggests that targeting the pathways that regulate neutrophil plasticity may offer a means to reprogram TANs toward an antitumor phenotype. For example, inhibiting TGF-β signaling or enhancing IFN-γ responses may promote the polarization of TANs toward an N1 state, enhancing their antitumor functions.^{20, 334}

Moreover, the heterogeneity of TANs may influence the efficacy of cancer immunotherapies, such as immune checkpoint inhibitors. The presence of immunosuppressive neutrophil subsets, like PD-L1-expressing TANs, may limit the effectiveness of these therapies by inhibiting T cell responses.^{335, 336} Targeting these specific neutrophil subpopulations, in combination with existing immunotherapies, may enhance therapeutic outcomes in cancer patients.

In conclusion, neutrophil heterogeneity and plasticity play crucial roles in shaping the immune response in aging and cancer. Age-related changes in neutrophil phenotypes and functions contribute to immunosenescence and the development of chronic inflammatory conditions in the elderly. In the context of cancer, the polarization of neutrophils toward protumorigenic or antitumorigenic states in response to microenvironmental cues has profound implications for tumor progression and response to therapy.³³⁷ Unraveling the molecular mechanisms that govern neutrophil heterogeneity and plasticity in these contexts may pave the way for novel therapeutic strategies aimed at harnessing the power of neutrophils to promote healthy aging and combat cancer.

6.1 Neutrophil-mediated regulation of the wound healing cascade

The wound-healing process is a highly coordinated and dynamic cascade involving multiple cellular and molecular events. Neutrophils are among the first immune cells to infiltrate the wound site, where they combat potential pathogens and actively regulate various stages of the wound-healing cascade.³⁶

In the initial inflammatory phase, neutrophils release many cytokines, chemokines, and proteolytic enzymes that shape the local microenvironment. These factors orchestrate the recruitment of additional immune cells, such as macrophages and lymphocytes, and initiate the clearance of cellular debris and foreign materials.¹⁴ Neutrophil-derived antimicrobial peptides, such as the human cathelicidin LL-37 and human α-defensins, exhibit potent antimicrobial activities, modulate the inflammatory response, and promote angiogenesis, a crucial process for wound healing.^{338, 339}

As the inflammatory phase subsides, neutrophils are pivotal in transitioning to the proliferative phase. By releasing matrix metalloproteinases (MMPs), including MMP-2 and MMP-9, and NE, neutrophils facilitate the degradation of the provisional ECM, creating a permissive environment for the migration and proliferation of endothelial cells, fibroblasts, and keratinocytes.^{340, 341} Concurrently, neutrophils produce various growth factors, such as VEGF, fibroblast growth factor (FGF), and TGF-β, which stimulate angiogenesis, fibroblast activation, and ECM deposition.^342-344

During the remodeling phase, neutrophils contribute to the resolution of inflammation and the maturation of the newly formed tissue. Neutrophil-derived serine proteases, including NE and cathepsin G, participate in the degradation and remodeling of the ECM, particularly at the epidermal–dermal junction, facilitating the formation of a mature scar.^{345, 346} Additionally, neutrophils produce SPMs, such as lipoxins, resolvins, and protectins, which promote the clearance of apoptotic cells and the resolution of inflammation.^{150, 151}

Recent studies have shed light on the dynamic phenotypic changes that neutrophils undergo during wound healing. In the early inflammatory phase, neutrophils exhibit a proinflammatory phenotype characterized by enhanced production of ROS, cytokines, and proteases.¹²² Moreover, neutrophils can form extracellular traps (NETs) that further contribute to bacterial killing and modulation of the inflammatory response.³⁴⁷ However, as the healing progresses, neutrophils can adopt an anti-inflammatory and proresolving phenotype, marked by increased production of SPMs and anti-inflammatory cytokines like IL-10.³⁴⁸ This phenotypic plasticity enables neutrophils to orchestrate the transition from the inflammatory to the proliferative and remodeling phases, ensuring a coordinated and timely progression of the wound-healing cascade^{140, 179} (Figure 5).

6.2 Neutrophil-mediated regulation of angiogenesis and vascular remodeling

Angiogenesis, forming new blood vessels from pre-existing vasculature, is a critical process in tissue repair and regeneration, ensuring the delivery of oxygen, nutrients, and immune cells to the site of injury or regeneration.³⁴⁹ Neutrophils have emerged as critical regulators of angiogenesis, exerting both proangiogenic and antiangiogenic effects depending on the context and their phenotypic state.³⁵⁰

In the early stages of wound healing, neutrophils promote angiogenesis by releasing various proangiogenic factors. Neutrophil-derived VEGF, a potent inducer of endothelial cell proliferation and migration, is crucial in initiating the angiogenic response.³⁵¹ Moreover, neutrophils secrete hepatocyte growth factor (HGF) and FGF-2, which further stimulate endothelial cell proliferation and tube formation.^{352, 353} Additionally, neutrophils secrete matrix-remodeling enzymes, such as MMPs and elastase, which facilitate the degradation of the basement membrane and ECM, creating a permissive environment for endothelial cell invasion and sprouting.^{342, 354}

Neutrophils also contribute to the maturation and stabilization of newly formed blood vessels through the production of angiopoietins and the recruitment of pericytes and smooth muscle cells.^{344, 355} Neutrophil-derived PDGF-BB promotes pericyte recruitment and vessel stabilization.³⁵⁶ Furthermore, neutrophil-derived SPMs, such as lipoxin A4 and resolvin D1, have enhanced endothelial cell proliferation, migration, and tube formation, supporting the later stages of angiogenesis.^{357, 358}

Conversely, neutrophils can also exert antiangiogenic effects under certain conditions. During chronic inflammation or in the context of tumor angiogenesis, neutrophils can release factors that inhibit angiogenesis, such as angiostatin, endostatin, and thrombospondin-1.^359-361 NE has been shown to generate angiostatin from plasminogen, thus inhibiting angiogenesis.³⁵⁹ These antiangiogenic factors counteract the proangiogenic signals, limiting excessive vascular growth and promoting vascular normalization.³⁶²

The proangiogenic or antiangiogenic functions of neutrophils are influenced by their phenotypic state and the local microenvironment. Proinflammatory neutrophils, characterized by increased production of ROS and proinflammatory cytokines, tend to promote angiogenesis,^{363, 364} while anti-inflammatory or proresolving neutrophils exhibit antiangiogenic properties.^{20, 365} The polarization of neutrophils toward a proangiogenic phenotype can be induced by hypoxia, growth factors, and cytokines present in the wound microenvironment.^{21, 366} This functional plasticity allows neutrophils to fine-tune the angiogenic response during tissue repair and regeneration, ensuring an appropriate balance between vascular growth and resolution.³⁶⁷

6.3 Neutrophil-mediated regulation of stem cell behavior and tissue regeneration

Emerging evidence suggests neutrophils are pivotal in regulating stem cell behavior and tissue regeneration processes.^{368, 369} By releasing various soluble factors and direct cell–cell interactions, neutrophils can modulate the proliferation, differentiation, and migration of stem and progenitor cells, thereby influencing tissue regeneration and homeostasis.^{19, 355} Neutrophils have been shown to regulate the behavior of various stem cell populations, including HSCs, mesenchymal stem cells, and tissue-specific stem cells.

In the context of skeletal muscle regeneration, neutrophils have been shown to regulate the behavior of muscle stem cells, known as satellite cells.³⁷⁰ Neutrophil-derived factors, such as VEGF, HGF, and LIF, promote satellite cells' activation, proliferation, and migration to the injury site.^371-373 Additionally, neutrophils release proteases, including elastase and cathepsin G, which facilitate the degradation of the ECM and create a permissive environment for satellite cell migration and myofiber formation.^{374, 375} Neutrophil depletion has been shown to impair skeletal muscle regeneration, highlighting their crucial role in this process.³⁷⁶

In the liver, neutrophils play a crucial role in the regenerative response following injury or partial hepatectomy.³⁷⁷ Neutrophil-derived cytokines, such as IL-6 and TNF-α, stimulate the proliferation of hepatocytes and liver progenitor cells, initiating the regenerative process.^{378, 379} Furthermore, neutrophils release proteases and ROS that activate MMPs, facilitating the remodeling of the ECM and creating a permissive environment for hepatocyte proliferation and migration.³⁸⁰ Neutrophil infiltration has been observed during the early stages of liver regeneration, and their depletion impairs the regenerative response.³⁸¹

Neutrophils also contribute to intestinal epithelium regeneration following injury or inflammation.³⁸² Neutrophil-derived factors, such as HGF and GM-CSF, promote the proliferation and migration of ISCs and their progeny.³⁸³ Additionally, neutrophils release antimicrobial peptides, such as α-defensins, which exhibit antimicrobial properties and stimulate the proliferation and migration of intestinal epithelial cells, supporting epithelial regeneration.^{384, 385} Neutrophil infiltration is a hallmark of intestinal inflammation, and their presence has been associated with enhanced epithelial regeneration.¹⁹⁹

The regulatory effects of neutrophils on stem cell behavior and tissue regeneration are not limited to the tissues above but extend to various other organs and systems, including the skin, lung, and nervous system.³⁸⁶ In these contexts, neutrophils modulate the behavior of resident stem and progenitor cells through the release of soluble factors, proteases, and direct cell–cell interactions, thereby influencing tissue homeostasis and regenerative processes.³⁸⁷ For example, in the skin, neutrophils promote wound healing by stimulating the proliferation and migration of epidermal stem cells and keratinocytes.¹⁴

6.4 Neutrophil-mediated regulation of ECM remodeling

The ECM is a complex network of structural and functional proteins that provides structural support, regulates cell behavior, and serves as a reservoir for growth factors and cytokines.³⁸⁸ Neutrophils play a pivotal role in the dynamic remodeling of the ECM during tissue repair and regeneration, contributing to the degradation, deposition, and reorganization of ECM components.³⁸⁶

Neutrophils are a rich source of various proteolytic enzymes, including MMPs, serine proteases (such as NE and cathepsin G), and heparanase, which collectively degrade and remodel the ECM.^{389, 390} These enzymes facilitate the degradation of the provisional ECM, creating a permissive environment for cell migration, proliferation, and angiogenesis during the early stages of wound healing and tissue regeneration.^{391, 392}

In addition to ECM degradation, neutrophils contribute to the deposition and reorganization of ECM components. Neutrophils release various ECM proteins, such as fibronectin, vitronectin, and tenascin-C, which serve as scaffolds for cell migration and tissue remodeling.^{393, 394} Furthermore, neutrophils produce growth factors, such as TGF-β and PDGF, which stimulate the production of ECM components by fibroblasts and other stromal cells.³⁹⁵

Endogenous inhibitors, such as TIMPs and α1-antitrypsin, tightly regulate neutrophils' ECM-remodeling activities.³⁹⁶ These inhibitors modulate the proteolytic activities of MMPs and serine proteases, respectively.^{397, 398} This fine-tuned balance between ECM degradation and deposition is crucial for maintaining tissue homeostasis and preventing excessive tissue damage or fibrosis.^{399, 400}

Recent studies have highlighted the role of neutrophil-derived extracellular vesicles (EVs) in ECM remodeling.^{401, 402} Neutrophil EVs carry a cargo of proteolytic enzymes, growth factors, miRNAs,⁴⁰³ and signaling molecules that can modulate the behavior of target cells, such as fibroblasts and endothelial cells, and influence ECM remodeling processes.⁴⁰⁴ These EVs serve as vehicles for the intercellular transfer of bioactive molecules, enabling neutrophils to exert their regulatory effects on ECM remodeling and tissue repair in a paracrine manner.^{403, 405}

6.5 Neutrophil-mediated regulation of inflammation resolution and tissue homeostasis

While neutrophils are traditionally associated with initiating and propagating inflammatory responses, accumulating evidence suggests that they also play a crucial role in the resolution of inflammation and restoring tissue homeostasi.^{140, 406} This dual functionality of neutrophils is essential for maintaining a delicate balance between protective inflammatory responses and excessive tissue damage.⁴⁰⁷

During the resolution phase of inflammation, neutrophils undergo phenotypic and functional changes, transitioning from a proinflammatory state to an anti-inflammatory and proresolving state.¹⁶⁴ This transition is mediated by various endogenous and exogenous factors, including SPMs, such as lipoxins, resolvins, and protectins.^{150, 408} SPMs are derived from polyunsaturated fatty acids and are crucial in regulating the resolution of inflammation.⁴⁰⁹

Neutrophils are not only targets of SPMs but also active producers of these bioactive lipids.⁴⁰⁹ Through the enzymatic activity of LOXs and cyclooxygenases, neutrophils can synthesize SPMs from polyunsaturated fatty acids, such as arachidonic acid and docosahexaenoic acid.⁴¹⁰ These SPMs exert potent anti-inflammatory and proresolving effects by inhibiting neutrophil recruitment, promoting neutrophil apoptosis and efferocytosis (clearance of apoptotic cells), and stimulating the production of anti-inflammatory cytokines like IL-10.^{411, 412} SPMs also promote the polarization of macrophages toward an anti-inflammatory phenotype.

In addition to SPMs, neutrophils contribute to the resolution of inflammation by producing other anti-inflammatory mediators, such as annexin A1 and lactoferrin.¹⁵⁶ Annexin A1, released by neutrophils during apoptosis, inhibits neutrophil recruitment and promotes macrophages’ clearance of apoptotic cells.^{178, 413} Lactoferrin, a multifunctional protein in neutrophil granules, exhibits anti-inflammatory properties by inhibiting the production of proinflammatory cytokines and promoting the resolution of inflammation.^{414, 415} These mediators work in concert with SPMs to promote the resolution of inflammation.¹⁷⁸

Furthermore, neutrophils play a crucial role in clearing apoptotic cells and cellular debris, a process essential for restoring tissue homeostasis.^{164, 416} Neutrophils can directly phagocytose apoptotic cells and cellular debris or release “find-me” signals that attract professional phagocytes, such as macrophages, to the site of inflammation.^{145, 416} This clearance process is critical for preventing the release of intracellular contents and the perpetuation of inflammatory responses.⁴¹⁷ Effective clearance of apoptotic cells by neutrophils and macrophages is essential for the resolution of inflammation and the prevention of autoimmunity.⁴¹⁶

Recent studies have also highlighted the role of NETs in resolving inflammation While NETs are initially released as a defense mechanism against pathogens, they can also promote the resolution of inflammation by serving as a scaffold for the degradation of inflammatory mediators and the clearance of apoptotic cells.^{418, 419} However, excessive or dysregulated NET formation can contribute to tissue damage and the perpetuation of inflammatory responses, underscoring the importance of tightly regulating this proces.^{123, 420} The balance between the beneficial and detrimental effects of NETs is crucial for maintaining tissue homeostasis and preventing chronic inflammation.

In summary, neutrophils play multifaceted roles in tissue repair and regeneration, extending far beyond their traditional antimicrobial functions. Through the release of various soluble factors, proteolytic enzymes, and direct cell–cell interactions, neutrophils orchestrate the wound healing cascade, regulate angiogenesis and vascular remodeling, modulate stem cell behavior and tissue regeneration, and contribute to the dynamic remodeling of the ECM. Moreover, neutrophils actively participate in the resolution of inflammation and the restoration of tissue homeostasis, highlighting their versatility and plasticity in adapting to diverse microenvironmental cues. This newfound appreciation of neutrophils as critical players in tissue repair and regeneration opens up exciting avenues for developing novel therapeutic strategies targeting neutrophil functions in various pathological conditions, including chronic inflammatory disorders, tissue injury, and regenerative medicine.

7.1 Small molecule inhibitors

Small molecule inhibitors have been extensively explored for modulating neutrophil responses by targeting key signaling pathways, enzymes, and effector mechanisms. These compounds offer the advantages of high specificity, tunability, and potential for oral administration.

7.2 Biologics and targeted therapies

Biologic agents, including monoclonal antibodies, cytokines, and receptor antagonists, have emerged as powerful tools for selectively targeting neutrophil subpopulations or modulating specific neutrophil functions.⁴³⁴

7.3 Cell-based therapies

Cell-based therapies have emerged as a promising approach for treating various diseases, including cancer, inflammatory disorders, and wound healing. Neutrophils, the most abundant type of white blood cells, have gained increasing attention as potential therapeutic agents due to their unique functions and abilities to target specific sites of inflammation or injury.⁴⁵¹ Neutrophils possess a remarkable capacity to migrate to sites of inflammation, where they release a variety of bioactive molecules, such as cytokines, chemokines, and growth factors, which can modulate the immune response and promote tissue repair.⁴⁵²

Recent studies have explored the potential of using neutrophils as drug delivery vehicles for targeted therapy.⁹ By exploiting the natural homing ability of neutrophils, researchers have developed strategies to load neutrophils with therapeutic agents, such as drugs, nanoparticles, or genetic material, which can then be delivered to specific sites of interest. For example, neutrophils have been used to deliver anticancer drugs to tumor sites, resulting in enhanced therapeutic efficacy and reduced systemic side effects.⁴⁵³

In addition to their role as drug carriers, neutrophils have also been found to release EVs, which can mediate intercellular communication and modulate the immune response. Neutrophil-derived EVs have been shown to contain a variety of bioactive molecules, such as proteins, lipids, and nucleic acids, which can influence the behavior of recipient cells.⁴⁰⁵ For instance, neutrophil-derived EVs have been found to promote the proliferation and migration of breast cancer cells by releasing oncogenic miRNAs.

The ability to reprogram neutrophils has also opened up new possibilities for cell-based therapies. By modulating the phenotype and function of neutrophils, researchers aim to enhance their therapeutic potential and minimize potential side effects. For example, neutrophils have been shown to undergo temporal polarization following myocardial infarction, which can influence their role in tissue repair and regeneration.⁴⁵⁴

The use of nanoparticles has further expanded the therapeutic potential of neutrophils. Nanoparticles can be designed to target specific receptors or pathways in neutrophils, enabling the modulation of their function and behavior. For instance, nanoparticles coated with neutrophil membranes have been shown to effectively treat cancer metastasis by mimicking the natural targeting ability of neutrophils.⁴⁵⁴

Despite the promising potential of neutrophil-based therapies, several challenges remain to be addressed. One of the main concerns is the potential off-target effects of neutrophils, which can lead to tissue damage and exacerbate inflammation.²⁶⁶ Additionally, the short lifespan of neutrophils and their sensitivity to environmental factors can limit their therapeutic efficacy.⁴⁵⁵

To overcome these challenges, researchers are exploring various strategies to enhance the stability and functionality of neutrophils. For example, genetic engineering approaches have been used to modify neutrophils to express specific receptors or proteins that can enhance their targeting ability or prolong their lifespan.⁴⁵⁶ Additionally, the use of biomaterials, such as hydrogels or scaffolds, can provide a supportive microenvironment for neutrophils, enabling their sustained release and function at the target site.⁴¹⁶

7.4 Nanomedicine platforms

Nanotechnology has emerged as a powerful tool for modulating neutrophil responses and enabling targeted delivery of therapeutic payloads. Nanoparticles can be engineered with specific surface modifications, drug payloads, and imaging capabilities to target neutrophils or selectively modulate their phenotypes and functions.^{9, 457}

7.5 Disease-specific treatment protocols

The therapeutic targeting of neutrophils has been explored across various disease contexts, with diverse treatment protocols and strategies tailored to specific pathological mechanisms and clinical needs.^1-3

7.6 Preclinical and clinical studies on neutrophil-targeted therapies

The growing understanding of neutrophil biology and its role in various pathological conditions has led to an increasing number of preclinical studies and clinical trials aimed at modulating neutrophil functions for therapeutic benefit. This section provides an overview of key preclinical animal experiments and clinical trials targeting neutrophils, organized by the main pathways and mechanisms of action.

8.1 Key advances and their implications

8.2 Future directions and challenges