Exploring Neutrophil Heterogeneity and Plasticity in Health and Disease

Abstract

Neutrophils, the most abundant polymorphonuclear leukocytes, are critical first responders to infection, and have historically been underappreciated in terms of their functional complexity within the immune response. Once viewed primarily as short-lived, innate immune cells with limited functional plasticity, recent research has illuminated their considerable heterogeneity and diverse functional roles, which extend beyond their involvement in steady-state immunity. This review seeks to provide an updated analysis of neutrophil development, maturation, heterogeneity, and plasticity, with a focus on how these characteristics influence immune modulation in both healthy and diseased tissues. Beginning with the origin of neutrophils, we explore their maturation into effector cells and their evolving roles in immune defense under homeostatic and disease-associated conditions. We then delve into their heterogeneity, discussing recent breakthroughs in neutrophil research that challenge the traditional view of neutrophils as a uniform population. We address the significant advances that have been made in identifying distinct neutrophil subsets, the emerging complexities of their plasticity, and the challenges that remain in fully understanding their functional diversity. Finally, we highlight future directions and opportunities for continued exploration in this rapidly advancing field, shedding light on how these insights could open new avenues for therapeutic interventions.

Keywords: heterogeneity; neutrophils; plasticity; therapeutics; tissue microenvironment.

Figure 1

The stages of neutrophil development, from hematopoietic stem cells in the bone marrow to mature neutrophils in the circulation. Neutrophil differentiation occurs in two phases: mitotic (hematopoietic stem cells (HSCs) → common myeloid progenitors (CMPs) → granulocyte-monocyte progenitors (GMPs) → pro-neutrophils (proNeus)), and postmitotic (pre-neutrophils (preNeus) → myelocytes → metamyelocytes → immature neutrophils (immNeus) → mature neutrophils (maNeus)). Key lineage markers are indicated. Neutrophils express the C-X-C chemokine receptor CXCR2 when they are freshly released from the bone marrow (fresh maNeus), which helps them to respond to chemokines like IL-8 and migrate to sites of infection or inflammation. As neutrophils age in circulation, they upregulate CXCR4 and downregulate CXCR2 (aged maNeus), promoting their return to the bone marrow for clearance. Neutrophil trafficking follows circadian rhythms, with CXCR2⁺ fresh maNeus released from the bone marrow during the active phase for immune defense, while CXCR4⁺ aged maNeus are cleared back to the bone marrow via the CXCR4-CXCL12 axis during the resting phase, regulated by core clock genes. Neutrophils also contain primary (azurophilic) granules with enzymes like myeloperoxidase (MPO), neutrophil elastase (NE) for microbial killing, secondary (specific) granules with lactoferrin and collagenase for reactive oxygen species (ROS) production, and tertiary (gelatinase) granules with metalloproteinases for tissue migration. Secretory vesicles play a key role in replenishing membrane enzymes, such as alkaline phosphatase and ATPase, by delivering them to the cell surface following cellular activation.

Figure 2

Neutrophil effector functions. Neutrophils play a crucial role in immune defense by engulfing pathogens or cellular debris, forming a phagolysosome to degrade the ingested material. They also release anti-microbial granules into the phagosome or the extracellular space to eliminate infections. Neutrophil extracellular traps (NETs) are triggered by inflammatory signals, immune complexes, bacteria, fungi, and protozoa. These web-like structures are composed of decondensed chromatin and granule proteins. NETs can serve two functions: vital, where neutrophils retain their functionality, while releasing NETs to trap and kill pathogens; or suicidal, where neutrophils die and release both their intracellular contents and the NETs. Neutrophils can behave as antigen-presenting cells, directly interacting with T cells through MHC II-TRC binding, CD80-CD28 interaction, and the secretion of GM-CSF, IFN-γ, IL-3, and TNF-ɑ. Additionally, neutrophils can influence T cell and macrophage polarization by secreting cytokines and granule content. IL-12 promotes Th1 differentiation, IL-4 favors Th2 differentiation, and azurocidin promotes M1 macrophage proliferation.

Figure 3

Neutrophil plasticity and transcriptional heterogeneity during maturation and tissue adaptation. Neutrophils go through multiple differentiation stages in the bone marrow, with each stage displaying unique transcriptional profiles. The lifespan of neutrophils is influenced by circadian clocks, with their activity and survival varying throughout the day, typically peaking during the night. Neutrophils exhibit distinct phenotypic and functional states, shaped by both intrinsic and extrinsic factors, in steady-state and disease contexts. Their heterogeneity extends to peripheral blood and tissues, where they specialize in response to environmental cues. Epigenetic and transcriptomic analyses have revealed that this variability reflects functional plasticity, suggesting that neutrophil subsets may not be entirely distinct, but may dynamically adjust based on stimuli.

Figure 4

Therapies targeting neutrophils and their functions. This figure summarizes therapeutic strategies aimed at modulating key neutrophil effector functions, including lifespan regulation, NETosis, degranulation, ROS production, phagocytosis, mobilization, recruitment, reprogramming, and cytokine production. The latter can be attenuated by anti-TNF and anti-IL-6 therapies. Targeting neutrophil lifespan can involve treatment with caspases-lysosomal membrane permeabilization-oxidant-necroptosis inhibition plus granulocyte colony-stimulating factor (CLON-G) or PI3-kinase inhibitors. NETosis can be inhibited using Cl-amidine and protein-arginine deiminase (PAD)4 inhibitors. Degranulation and excessive ROS production can be suppressed using selective inhibitors for myeloperoxidase (MPO), neutrophil elastase (NE), and ROS production. Phagocytic capacity can be enhanced via resolvin E1 (RvE1). Neutrophil mobilization and recruitment can be regulated by G-CSF and CXCR2/4 antagonists, providing potential avenues for therapeutic intervention in inflammatory and immune-mediated diseases. Reprogramming strategies, including inhibition of anaerobic glycolysis, of ferroptosis, or of the purinergic receptor P2RX1, aim to modulate neutrophil metabolism, non-apoptotic cell death, and activation.