TABLE 1.

Neutrophil subsets and their relevance in homeostatic and pathological settings

Neutrophil subset	Frequently co‐expressed markers and/or morphological features	Known functions of the markers defining the subset	Prevalence and role in health and disease
CD177+	Proteinase 3 (PR3).	CD177 is a glycoprotein expressed on neutrophil plasma membrane and specific granules. It promotes the interaction of neutrophils with endothelial cells and their extravasation. However, genetic ablation in mouse or human neutrophils did not affect cell migratory capacity. PR3 is a serine protease mainly stored in neutrophil azurophilic granules, but it also presents on the cell surface of a subset of neutrophils in the peripheral blood of healthy individuals.	Humans: 45%–65% of circulating neutrophils during homeostasis. Frequency of circulating CD177+ neutrophils is augmented in inflammatory conditions, including asthma, IBD, sepsis, severe bacterial infections, cancer and anti‐neutrophil cytoplasmic autoantibody (ANCA)‐dependent vasculitis. In the latter, PR3 is the main target of the autoantibodies.
OLFM4+	_	Olfm4 was initially identified as a gene highly induced in myeloid stem cells by G‐CSF treatment. OLFM4 is located in specific granules. It acts as a negative regulator of neutrophil bactericidal activity, and its genetic ablation in mice boosted response against S. aureus or E. coli and improved survival in a model of sepsis.	Humans: 20%–25% of circulating neutrophils during homeostasis. In vitro, OLFM4+ neutrophils display a higher ability to produce NETs compared with OLFM4− neutrophils. OLFM4+ neutrophils are increased in sepsis and have been suggested to contribute to autoimmune responses in ANCA‐dependent vasculitis. OLFM4 levels are increased in the serum of asthmatic patients and inversely correlate with asthma control state.
CXCR4hi CD62Llow	CD11bhi, CD49dhi; hypersegmented nucleus and reduced size (in senescent neutrophils).	CXCR4 mediates neutrophil retention in the haematopoietic compartment by binding to CXC12, which is constitutively expressed by bone marrow stromal cells. Its expression is low on neutrophils freshly released from the bone marrow, but increases in senescent neutrophils, which are targeted for clearance. CD62L (L‐selectin) is involved in neutrophil attachment to the endothelium at sites of inflammation. It is downregulated with ageing and rapidly shed after neutrophil activation. The integrins CD11b and CD49d are also involved in neutrophil adhesion to the endothelium and migration to sites of inflammation.	Mouse: 55 ± 14% of circulating neutrophils in ZT5 (i.e. 5 h post‐initiation of the light period). Humans: ~60% of circulating neutrophils at night‐time. CXCR4hi CD62Llow frequency is increased in several pathological settings, including asthma, COPD and CF. CXCR4hi neutrophils display higher proclivity to form NETs (both in humans and in mice).
CD63+ (otherwise known as A2 or ‘GRIM’ neutrophils in CF airways)	CD11bhi, CD66hi, MHC II+, CD16low, CD14low ↑ Arginase 1 [ARG1] (in CF patients)	CD63 is a tetraspanin involved in the retention of neutrophil elastase (NE) into primary granules; CD63 mobilization to the cell surface correlates with extensive NE release to the extracellular space. CD66 is involved in the modulation of neutrophil adhesion and effector function. MHC II is an antigen‐presenting molecule essential for development and activation of CD4+ T cells. CD16 is a low‐affinity Fc receptor. CD14 acts as a co‐receptor for TLR4. ARG1 is an enzyme that metabolizes arginine and is stored in gelatinase granules.	In CF patients, neutrophils isolated from sputum display robust mobilization of CD63 to the surface compared with circulating neutrophils; this is accompanied by an upregulation of CD11b, CD66 and MHC II. Conversely, CD14 and CD16 are downregulated, and this is believed to contribute to the blunted phagocytic ability observed in airway PMNs isolated from CF patients as opposed to healthy individuals. Neutrophils isolated from the BAL of CF patients also feature increased ARG1 activity, which can block T‐cell proliferation. Frequency of CD63+ neutrophils is increased in sputum from asthmatic children (compared with healthy children). CD63 surface expression is also increased in senescent/apoptotic neutrophils.
CD64+	PD‐L1 (in COVID‐19 patients)	CD64 is the high‐affinity Fcγ‐receptor I; it binds to opsonized pathogens and immune complexes, thus triggering phagocytosis. PD‐L1 binds to PD‐1 on T cells and inhibits their activation, proliferation, cytokine secretion and survival.	CD64 surface expression is increased upon bacterial infection in both mice and humans. It is used as a marker of sepsis. Upregulated on blood neutrophils upon IAV and RSV infection, as well as during the delayed asthmatic response. A subpopulation of immature CD64+ PD‐L1+ neutrophils has been associated with more severe disease in COVID‐19 patients.
CD49d+	Cysteinyl leukotriene receptor 1 [CysLTR1] (in patients with viral respiratory tract infections, including IAV, IBV, RSV HRV, HMPV and HPIV). Can be co‐expressed with CXCR4 (possible link to intrinsic ageing?).	CD49d is the α4 subunit of the VLA−4 integrin, which mediates the interaction with VCAM−1 on endothelial cells. CysLTR1 is a receptor for cysteinyl leukotrienes.	Mouse: Upon infection with Sendai virus, CD49d+ neutrophils induce FcεRI expression on lung dendritic cells. Humans: CD49d and CysLTR1 co‐expressing neutrophils are elevated in nasal lavage from patients with acute respiratory viral infections. CD49d+ neutrophil frequency is significantly increased in the peripheral blood and nasal lavage of atopic patients (compared with non‐atopic) and increases further upon allergen challenge.
CD49dhiCXCR4hi VEGFR1hi	↑MMP‐9	VEGFR1 binds to VEGF‐A and VEGF‐B. MMP‐9 is a metalloproteinase involved in the degradation of extracellular matrix.	CD49dhiCXCR4hiVEGFR1hi neutrophils are believed to support angiogenesis during development, normal tissue repair and pathogenic states characterized by tissue hypoxia. For instance, they are involved in the revascularization of transplanted tissues, as well as in tumour neovascularization, where MMP‐9 secretion facilitates vessel penetration.
ICAM‐1hiCXCR1low	↑ROS production	ICAM‐1 is an adhesion molecule involved in leucocyte trafficking. CXCR1 is a receptor for ELR+ chemokines.	ICAM‐1hiCXCR1low neutrophils are able to reverse transmigrate from the extravascular space back into the circulation. Their frequency is increased in rheumatoid arthritis and ischaemia/reperfusion damage. They have been suggested to contribute to systemic propagation of inflammation. They have been observed also in patients with COPD.
Low‐density neutrophils (LDNs), multiple subsets	CD15hi, CD14low, CD10variable, CD16variable; CD11bhi, CD66bhi (in SLE, HIV, asthma and cancer); CD33hi (in cancer).	CD15, also known as Lewis x, is expressed in mature human neutrophils. It is considered to be involved in cell–cell interactions, phagocytosis and stimulation of degranulation. CD16 is the low‐affinity FCγRIII; its expression is acquired at the band cell stage during neutrophil differentiation, but it is downregulated during apoptosis and activation. CD10, also known as neutral endopeptidase, starts being expressed at the segmented stage. CD10 is a marker of neutrophil maturation. CD66b, also known as CEACAM8, is GPI‐anchored, highly glycosylated protein belonging to the carcinoembryonic Ag supergene family. It is located in lipid rafts, where it physically associates with CD11b, and it is considered an activation marker. CD33 (Siglec‐3) is a transmembrane receptor whose binding by sialic acids leads to activation of ITIM on the cytosolic portion of the protein and inhibition of apoptosis.	Opposite to standard neutrophils (normal density neutrophils), LDNs appear in the low‐density Ficoll gradient fraction together with mononuclear cells. They present as a mixed population of variable maturity. LDNs are present in a variety of pathological settings characterized by chronic inflammation and/or immunosuppression, such as systemic lupus erythematosus (SLE), rheumatoid arthritis, cancer, psoriasis, asthma, malaria, tuberculosis, HIV infection, obesity and pregnancy. LDNs are also observed in septic patients, as well as in those with fungal (e.g. Aspergillus fumigatus and C. albicans) and viral infections (particularly from HBV and HCV). LDN properties appear to be context‐dependent. In SLE and psoriasis, LDNs display heightened inflammatory activity, with enhanced cytokine secretion and NETosis, but reduced phagocytic ability. In cancer and HIV, as well as in pregnant women, they show an immunosuppressive phenotype, characterized by low arginase activity and impaired capacity to drive CD8 proliferation and IFN‐γ production.
PMN‐I and PNM‐II (in the context of infection by methicillin‐resistant Staphylococcus aureus [MRSA])	PMN‐I: CD49d+, CD11b−, TRL5+, TRL8+, secrete IL‐12 and CCL3, drive classical activation of macrophages. PMN‐II: CD49d−, CD11b+, TRL7+, TRL9+, secrete IL‐10 and CCL2, drive alternative activation of macrophages.	_	Mouse: PMN‐I were isolated from MRSA‐resistant hosts, whereas PMN‐II were isolated from MRSA‐sensitive hosts.

For a more extensive discussion of different neutrophil subsets, refer to references [1, 3, 4, 8].

Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; HMPV, human metapneumovirus; HPIV, human parainfluenza virus; HRV, human rhinovirus; IAV, influenza A virus; IBD, irritable bowel disease; IBV, infectious bronchitis virus; ICAM‐1, intercellular adhesion molecule‐1; OLFM4, olfactomedin 4; PMN, polymorphonuclear cells; RSV, respiratory syncytial virus; VCAM‐1, vascular cell adhesion molecule‐1; VEGF, vascular endothelial growth factor; VEGFR1, vascular endothelial growth factor receptor 1; VLA‐4, very late antigen‐4.