Seminars in Immunology 57 (2021) 101506

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Seminars in Immunology
journal homepage: www.elsevier.com/locate/ysmim

Dimensions of neutrophil life and fate

Andrés Hidalgo a, María Casanova-Acebes b, *
a
Area of Cell and Developmental Biology, Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain
b
Cancer Immunity Group, Molecular Oncology Program, Centro Nacional de Investigaciones Oncologicas Carlos III, Madrid, Spain

A R T I C L E I N F O A B S T R A C T

Keywords: The earliest reported observations on neutrophils date from 1879 to 1880, when Paul Ehrlich utilized a set of coal
Neutrophil tar dyes to interrogate differential staining properties of the granules from white blood cells. While acidic and
Immune fate basic dyes identified eosinophils and basophils respectively, neutrophils were revealed by neutral dyes. Un­
Inflammation
knowingly, his work staining blood films set the stage for one of the most exciting features of immune cells
Granulopoiesis
Immune reprogramming
discovered in the last decade, myeloid heterogeneity. Since then, advances in live imaging and high-resolution
sequencing technologies have revolutionized how we analyze and envision those cells that Ehrich fixed in blood
smears. Neutrophil plasticity and heterotypic interactions with immune and non-immune compartments are
increasingly appreciated as an important part of their biology. In this review, we highlight early and recent work
that will help the reader to appreciate our current view of the neutrophil life cycle -from maturation to elimi­
nation-, and how neutrophils behave and dynamically modulate tissue immunity, both in steady-state and in
disease.

1. Principles of neutrophil biology granulopoiesis [11].

Recent work by Evrard et al. [12] identified the developmental tra­
1.1. Granulopoiesis and neutrophil trafficking from bone marrow to the jectory from GMP to mature neutrophils to show the presence of early
blood intermediary, committed progenitors. By using mass cytometry in
combination with the Fucci reporter mouse to allow the identification of
On a daily basis, around 1011 neutrophils are produced and released proliferating cells, the authors captured cycling neutrophil precursors
from the bone marrow (BM) in a human being, while 107 neutrophils are (preNeus) which were identified by the expression of cKit and CXCR4,
produced in a mouse [1]. Because neutrophils are short-lived -they have but lacking Ly6G and CXCR2. preNeus are found in human BM, and they
an estimated half-life of 18 h in humans [2,3] and 6–12 h in mice [4]-, expand in this organ and the spleen during sepsis and in orthotopic
constant production and turnover is required, and it is remarkable that models of pancreatic carcinoma. Two more recent studies have further
their production commits most of the anabolic capacity of the BM. shown that not only GMPs can generate neutrophils. Pietras et al. [13]
Myeloid cells arise from CMPs (common myeloid progenitors), described multipotent progenitors (MPPs) as an additional source of
which differentiate into more committed precursors or GMPs (gran­ neutrophils with specific kinetics depending on hematopoietic demands,
ulocyte-monocyte progenitor) [5]. Granulopoiesis relies on the cytokine and Zhu et al. [14], discovered a unipotent neutrophil progenitor (NeP)
granulocyte-colony stimulating factor (G-CSF/CSF3) as the main cyto­ in mouse and human BM, which expands in the blood of melanoma
kine required for neutrophil maturation and mobilization from the BM, patients and in tumor-bearing mice, suggesting that indeed this pro­
at least in conditions of stress [6–8]. Upon G-CSF signaling, highly genitor could contribute to the pool of neutrophil-like cells expanded in
proliferative neutrophil progenitors mature into Ly6G+ CXCR2+ neu­ tumors. Interestingly, recent findings have established a new reservoir of
trophils, and this process underlies the tight regulation of a transcrip­ neutrophils which arise from the skull and vertebral bone marrow and
tional program ruled by specialized transcription factors, including that might contribute to different functions and responses in the central
PU.1, C/EBPs, GATA-1, RUNX1 and GFI-1 [9,10]. For example, C/EBPα nervous system [15]. These findings open new venues to appreciate and
induces expression of the receptor for G-CSF (G-CSFR/CSF3R) on GMPs, investigate neutrophil plasticity as it relates to the location in which the
which allows signals delivered by G-CSF/CSF3 to promote progenitors are generated.

* Corresponding author.
E-mail address: [email protected] (M. Casanova-Acebes).

https://doi.org/10.1016/j.smim.2021.101506
Received 12 August 2021; Received in revised form 9 October 2021; Accepted 14 October 2021
Available online 26 October 2021
1044-5323/© 2021 Elsevier Ltd. All rights reserved.
A. Hidalgo and M. Casanova-Acebes Seminars in Immunology 57 (2021) 101506

In response to peripheral stress during acute infections, sustained- antagonistic with CXCR4 [35,36], thus playing an important role in the
low level stimulation derived from cytokines secreted by parenchymal dynamics of neutrophil mobilization or retention.
tissue stimulate the BM for neutrophil production. Granulopoiesis is Interestingly, while CXCR2 ligands (CXCL1 and CXCL2) are consti­
switched into an “emergency mode”, thereby giving rise to heteroge­ tutively expressed by the BM stroma and show circadian expression
neous populations of mature and immature neutrophils. While the BM is (unpublished observations Hidalgo laboratory), their expression is
the primary site for hematopoiesis in the steady-state, the spleen and the enhanced upon G-CSF or thrombopoietin administration [37], which
liver can also support extramedullary hematopoiesis and neutrophil could further contribute to neutrophil mobilization.
production during infection, sepsis or stress [6]. Under emergency After a single injection of G-CSF, blood neutrophil numbers increase
granulopoiesis both G-CSF independent and dependent granulopoiesis significantly [34,38]. G-CSF regulates steady-state hematopoiesis
can occur, which could generate additional neutrophil heterogeneity at including the regulation of neutrophil progenitor proliferation and
the progenitor level, although formal experimental evidences for this are functional differentiation of neutrophils [39]. Interestingly, CXCR2
missing. ligand–induced neutrophil mobilization is much faster than that elicited
A new concept in the field adds a twist in myeloid progenitor plas­ by G-CSF, with 10-fold neutrophilia occurring 30 minutes after injection
ticity; trained immunity has emerged as a novel modulator of epigenetic, [40], and combination with a CXCR4 antagonist potently and rapidly
metabolic and transcriptional reprogramming whereby myeloid pro­ mobilizes highly engraftable hematopoietic stem cells [41]. Impor­
genitors respond to foreign stimuli once they have experienced a pri­ tantly, it has been recently shown that G-CSF not only acts as a
mary challenge, typically a PAMP (pathogen-associated molecular neutrophil mobilizer, but also negatively regulates neutrophil mobili­
pattern). These epigenetic alterations can be transmitted to the progeny zation by CXCR2 ligands in the context of bacterial infections [42]. The
in the form of myeloid memory that reprograms neutrophil activity, as fast kinetics of CXCR2-induced mobilization (minutes to hours)
shown in preclinical models of cancer and during infection [16,17]. compared with G-CSF (hours to days) suggest that these are funda­
Whether secondary-educated myeloid progenitors can in turn modulate mentally distinct mechanisms of mobilization, with the CXCR2 axis
neutrophil fate and function remains an open question, in part due to the representing a “ready-to-go” path for rapid neutrophil egress even in the
lack of progeny-tracers for trained myeloid progenitors. absence of previous expansion.
Other key determinants of neutrophil biology are the commensal Studies in mice have shown that the chemokine CXCL12 acting
microbiota. Microbes regulate steady-state granulopoiesis by controlling through its receptor CXCR4 anchors neutrophils (as well as other leu­
the production of granulocyte colony stimulating factor (G-CSF), as kocytes) within the BM [31,43]. Stromal cells in the BM receive diurnal
evidenced by the observation that germ-free mice and antibiotic regimes adrenergic signals from sympathetic nerves, which through β3-mediated
exacerbate neutropenia [18,19]. Mechanistically, bacterial-derived signaling inhibit CXCL12 production, implying the existence of a
soluble factors mediate TLR4/Myd88 driven IL-17 production by stroma-neural axis that governs oscillatory day-night expression of the
innate lymphoid cells in the intestine, which potentiates G-CSF pro­ chemokine, stem cell retention [44] and other leukocytes subsets as
duction [20]. Importantly, the production of neutrophils can further be well. Conversely, cholinergic signals from the parasympathetic nervous
potentiated by soluble microbe-derived factors acting on the stromal system inhibit adrenergic input into the BM at night [45], hence
compartment of the BM niche to produce myeloid-differentiating cyto­ establishing a fine-tuned network of temporal rhythms in the BM (see
kines and mobilization of the BM neutrophil reservoir [21–23]. Neu­ more on circadian regulation below). In mice, downregulation of
trophils sense microbiota-derived signals via TLRs, NODs and the CXCL12 at daytime drives the natural, circadian egress of HSCs [44,46],
inflammasome [24]. Beyond their production, the microbiota can also and the release of neutrophils at this same time also coincides with
promote the education of inflammatory responses driven by neutrophils. decreased CXCL12 [43].
For example, germ-free neutrophils show reduced production of MPO
(myeloperoxidase), impaired migration and diminished phagocytic 1.2. Circadian regulation
killing capacity [25–27]. Thus, we propose that commensal
microbiota-neutrophil interactions have evolved to enable fully func­ Every cell in our organism harbors an “internal timer” that syn­
tional innate immune cells in order to overcome and modulate patho­ chronizes its activity with diurnal time following predictable environ­
gens invasion. mental cues (such as light or feeding), which in turn synchronize the
Another aspect that remains unexplored are the tissue-specific re­ clocks across different cells and organs. Hematopoietic stem cells, as
sponses of neutrophils toward the microbiota. Microbiota is extremely well as the majority of differentiated leukocytes, including neutrophils,
diverse in the different barrier surfaces, including the gastrointestinal follow these circadian patterns both in mice and humans [47,48]. In
and urinary tract, sexual mucosa, skin, oral and airways cavities, particular, neutrophil counts fluctuate in blood with a peak at noon and
including the lung [28]. It is conceivable that neutrophils populating their lowest levels in the afternoon/night. These circadian oscillations in
these surfaces are influenced by this local diversity, reacting in a circulating numbers are speculated to be synchronized with oscillations
microbiota-specific manner. Importantly, while the field has abundantly in PAMPs that peak at certain times of the day, and consistently
explored the neutrophil-modulating capacity of bacteria, we are still at germ-free mice are strongly neutropenic [20], however a causal and
the infancy when it comes to other components of the microbiota (i.e, mechanistic relationship between both has not been formally estab­
viruses, fungi and protozoa) in terms of evaluating their impact on lished [49]. What studies focusing on regulation of neutrophil numbers
neutrophil production, recruitment or effector functions. in blood have suggested is that their oscillatory levels are a result of a
Once mature, neutrophils traffic into the blood, although notably systemic rheostat, which integrates within the same circuit: 1) neutro­
only a small fraction of the neutrophil pool does so and an estimate 90% phil production in the BM, 2) neutrophil levels in circulation, 3)
pool remains in the BM as a mobilizable reservoir [29]. Tight circadian neutrophil clearance from blood and extravasation into tissues, and 4)
control controls the efflux of the newly-matured neutrophils into blood, elimination of neutrophils by professional phagocytes [50].
and this is believed to occur through 2 main mechanisms: I) Beyond quantity, circadian fluctuations also affect the quality of the
CXCR2-mediated mobilization and II) disruption of the CXCR4-CXCL12 cells. Indeed, neutrophils undergo phenotypic changes in the circulation
retention axis both in the marginated pool of neutrophils in the lung in what has been referred to as the aging program. This is characterized
[30], and in the BM [31]. Whereas pharmacological inhibition of CXCR4 by reduced L-selectin (CD62L) and increased CXCR4 surface levels
potently mobilizes neutrophils from the BM and lung [30,32–34], and (among other markers such as CD11b or CD49d), which facilitates the
genetic deletion of myeloid-specific CXCR4 triggers blood neutrophilia return of these cells back into the BM at the end of their lifecycle. Other
[31,35], absence of CXCR2 leads to neutrophil retention in the BM [33, prominent alterations include changes in transcriptional programs, loss
36] and signaling through this receptor appears to be mutually of granule content and alterations in cellular and nuclear morphology

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A. Hidalgo and M. Casanova-Acebes Seminars in Immunology 57 (2021) 101506

(reduced side scatter properties and nuclear hypersegmentation), all of macrophages, but has remained unclear for short-lived granulocytes.
which compromise their capacity of neutrophils to stick to inflamed Confocal and intravital imaging shed some provocative findings into this
vessels, to migrate during inflammation, and their overall cytotoxicity possibility. Using intravital imaging, Deniset et al. [59] showed that the
activity [35,51]. Hence, neutrophils display better anti-bacterial migration patterns of two subsets of neutrophils residing in the red pulp
response at night in line with increased NET-forming capacity and of the spleen differed both in homeostasis and in response to
granule content at this time. However, other factors are to be considered S. pneumoniae challenge, suggestive of at least two coexisting pop­
since homeostatic migration of neutrophils to tissues is favored by ulations. These subsets were further discriminated based on Ly6G
neutrophil aging and the circadian clock, and their preventive accu­ expression. Using flow cytometry, the authors further characterized
mulation in tissues at night-time protects from infections even if their Ly6GHI neutrophils, which express CD49dINT, CD62LHI, CD54HI and
anti-microbial function is blunted, as demonstrated in mice with a CD18HI levels but lack CD117 expression. These neutrophils are more
constitutive neutrophil aging which are more resistant to C. albicans efficient in S. pneumoniae clearance, while Ly6GINT neutrophils showed
infection [35]. As a trade-off, however, aged neutrophils within the reduced maturation features by the expression of CD117HI, CD49dHI,
vasculature create life-threatening risks as they can cause irreversible CD62LLO levels and reduced expression of CD18, and these can termi­
damage as shown in a preclinical model of ischemia and reperfusion, in nally expand and be mobilized, thus contributing to the mature
which constitutive aging leads to decreased survival in mice. Interest­ neutrophil splenic pool.
ingly, pulmonary vascular permeability and mice survival was also In the spleen, Puga et al. [60] identified a subset of neutrophils
compromised in the evening, when neutrophil numbers peak in tissues, specialized in supporting B cell maturation and antibody production,
formally showing that the functional properties of aged neutrophils are and Bogoslowski et al. [61] and Lok et al. [10] recently identified sub­
controlled by their intrinsic cell timer. populations of neutrophils with different location (lymph node inter­
In contrast, properties such as ROS production or cytokine release do stitium, high-endothelial venules and lymphatic vessels) and patrolling
not appear, surprisingly, to be affected by aging [35]. The abundance of phenotypes in murine and human lymph nodes using intravital and
such aged neutrophils peaks in the circulation coinciding with the ani­ confocal imaging of this organ. Finally, additional intravital microscopy
mal’s resting phase [46], however there is a misalignment with activity studies have reported five distinct neutrophil migratory behaviors
in humans, a diurnal species, in which aged neutrophils also peak at described upon influenza vaccination in the popliteal lymph node on
daytime [35], suggesting that other biological features underlie the high endothelial venules [62]. These included 1) flowing neutrophils that
dynamics of neutrophil aging. move inside blood vessels, with long and straight tracks; 2) arrested
Infiltration of neutrophils in most naïve tissues follows circadian neutrophils with confined tracks and short displacement and patrolling
patterns with a peak at night. Exceptions to this are the intestine and neutrophils featuring long, non-straight tracks associated with tissue
white adipose tissue (WAT) in which no clear circadian oscillations were scanning; 3) directed neutrophils, which exhibit straight tracks with lower
detected [52]. Controversy exists, however, with regards to infiltration migration speeds compared with flowing neutrophils; and finally, a
of the liver, with studies showing either no evident circadian pattern or subset of 4) swarming neutrophils that form aggregates of high density
preferential infiltration in the early morning [52,53]. Studies in the liver and large volume. All these studies suggest that persistent or transient
were of particular interest as they showed that infiltrating neutrophils neutrophil niches can be established in tissues, and that they could
entrain diurnal liver metabolism. Mechanistically, elastase-secreting imprint distinct neutrophil fates and downstream signaling based on the
neutrophils controlled BMAL1 expression in hepatocytes, which in cell types and tissue components that neutrophils encounter.
turn modulates their lipogenic metabolism. Consistently, impaired A critical assumption in the hypothesis that bona fide neutrophil di­
neutrophil migration into the liver of neutropenic mice or deficiency in versity exists in tissues is that neutrophils must persist long enough
elastase protected against fat diet-induced steatosis, further suggesting a outside the bloodstream to adopt specialized phenotypes and functions.
crosstalk between neutrophils and circadian regulation of liver homeo­ We have recently undertaken the challenge to assess with accuracy the
stasis [53]. Whether neutrophil infiltration throughout the day dictates half-life of neutrophils across naïve tissues [54]. By utilizing a new
further homeostatic organ-specific programs remains unexplored but neutrophil fate-mapper mouse, which expresses the tdTomato fluores­
raises an appealing hypothesis given the specific dwell times and tran­ cent protein under the control of the Ly6G locus in a
scriptional program of neutrophils found in different tissues [54]. tamoxifen-inducible manner (Ly6GCreERT2; Rosa26LSL− tdTom), in combi­
nation with mathematical modelling we estimated varying half-lives,
1.3. Local imprinting of neutrophils from 8 to 18 h, for neutrophils entering varying tissues. Using
high-dimensional transcriptomics, multiparametric mass cytometry and
An important finding in recent years has been the realization that epigenetic profiling we further found remarkable specialization of
neutrophils are found in virtually all tissues of our body [52,55], how­ neutrophils across tissues [54], implying that while the cells patrol a
ever their density and distribution are organ-specific. While the BM is given tissue for microbial threats, they engage into interactions with
the main reservoir of neutrophils, the spleen, lung and liver contain a resident cells (or their metabolites) and integrate an array of signals
pool of non-circulating cells inside blood vessels which are typically from the tissue. Remarkably, these phenotypic changes are matched by
referred to as marginated [56,57]. All these organs share a large capil­ new functional properties, such as support of revascularization by
lary network and the size of the marginated pool depends on both organ neutrophils infiltrating the lungs and intestine during organismal
blood flow and neutrophil transit times through those specific vascular growth or after an insult, while others properties are predicted based on
compartments. Whether unique neutrophil phenotypes are specifically the transcriptional profile but remain to be validated (such as epithelial
prone to margination or whether margination imprints a unique pro­ growth in the skin). Critically, these studies challenged the
gram to neutrophils in these organs remains to be elucidated. Either long-sustained dogma that neutrophils lack the capacity to adapt to
way, it is intriguing that short-term endotoxin treatment in humans tissues. These tissue-associated programs appear to be instructed in
rapidly mobilizes a pool of neutrophils with immune-suppressive func­ specific microenvironmental sites, as shown in the lungs, possibly by
tions [58], suggesting that these may represent already intravascular, direct physical and/or paracrine interactions, however this remains
marginated neutrophils and that this particular compartment may be untested [23,52,63,64].
endowed with distinct properties.
An outstanding question in the field has been whether neutrophils 1.4. Neutrophil aging and clearance
persist in the tissues that they infiltrate or are instead rapidly eliminated
by resident phagocytes [56]. The idea of environmental-reprogramming Neutrophil aging precedes their elimination from the circulation, or
is well-established for long-lived cells, such as lymphocytes and clearance. While previous studies reported the BM, spleen and liver as

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A. Hidalgo and M. Casanova-Acebes Seminars in Immunology 57 (2021) 101506

the unique “neutrophil cemeteries” [1], neutrophils can also be found in Interestingly, phagocytosis of aged neutrophils by BM macrophages
the intestine, lung, WAT, skin, skeletal muscle, lymph nodes, kidneys and activation of Liver X receptors (LXRα and β), a family of cholesterol-
and heart under steady-state conditions, and at least in some of these sensing transcription factors that become activated upon uptake of
organs they can be taken up by resident macrophages [52]. Whether apoptotic cells [69], were shown to be required for suppression of
neutrophils in these tissues die via apoptosis before being phagocytosed CXCL12. In contrast, in the intestine neutrophils displayed non-random
or move to a marginated pool (as shown in the lung) has not been distribution around isolated lymphoid follicles, which are surrounded
formally demonstrated. In fact, it is conceivable that neutrophils in some by CD169+ macrophages [52]. In this case, proximity to
or all of these tissues migrate back to the bloodstream or enter the IL-23-producing cells correlated with regulation of the levels of this
lymphatics for further recirculation. cytokine and downstream production of G-CSF. Indeed, we found that
We propose that neutrophil aging, the circadian program seen in the gut-infiltrating neutrophils function as regulators of systemic G-CSF
circulation, provides a solution for the tight control needed to preserve levels and subsequent mobilization of HSPCs from the BM [52]. Con­
tissue homeostasis while at the same time keeping a pool of aggressive trary to the gut, but in sharp similarity with the BM, neutrophil infil­
neutrophils to counterattack infections [35]. This is possible because tration in the murine lung follows circadian fluctuations with a peak in
both properties (tissue infiltration and antimicrobial responses) are the afternoon. Neutrophils in this organ were shown to entrain global
temporally compartmentalized during the day. For example, initial circadian transcription of lungs that predisposed for organ invasion by
studies showed that aged neutrophils upregulate CXCR4 in the morning metastatic cells [52], thereby suggesting that diurnal neutrophil clear­
before returning to the BM in the afternoon following a gradient of ance may influence the temporal dynamics of pathophysiological pro­
CXCL12 for their clearance [1,36,46]. These marrow-tropic neutrophils cesses in tissues, including cancer, susceptibility to infection, or
are phagocytosed by CD169+ tissue-resident macrophages, which in respiratory diseases such as asthma.
turn activate LXR signaling to blunt CXCL12 levels in mesenchymal cells
in the BM [46]. Neutrophil aging has since been proposed to be influ­ 2. Can we predict neutrophil fates?
enced by microbiota-derived products sensed through TLR4-signaling
[65]. Alternatively, we proposed that expression of the key circadian While the conspicuous low transcriptional rate of neutrophils has
clock transcription factor Artnl1 (BMAL1) in neutrophils is essential for limited examination of neutrophil heterogeneity using conventional
neutrophils to age, and that antagonizing expression of CXCR2 and tools [70–72], data are rapidly emerging with respect to neutrophils in
CXCR4 regulates this feedback loop through a CXCL2-autocrine tissues (see above), neutrophil maturation as well as neutrophils asso­
signaling mechanism. This model proposes that CXCR4 negatively ciated with tumors [9,12,14,73,74]. Single-cell-based technologies offer
controls cell-extrinsic aging upon binding its ligand CXCL12, whereas the opportunity to distinguish between discrete neutrophil states, and in
aging is cell-intrinsically driven by circadian-clock controlled genes. combination with lineage-tracing tools have the potential to elucidate
Aging, as it turns out, was also shown to be a pre-requisite for neutro­ differential stages of neutrophil maturation, aging and location-related
phils to enter tissues in the steady-state [35], thereby providing a uni­ phenotypes [54,64,75]. The Immgen consortium [64] has recently
fying circadian-to-tissue mechanism underlying multiple aspects of applied single-cell transcriptomics in combination with RNA velocity
neutrophil physiology. (an algorithm that compares unspliced vs spliced mRNA to decipher
Interestingly, neutrophil states in blood have been recently mapped maturation phenotypes as a function of time [76]) to provide evidence
at single cell resolution. Using scRNA sequencing a recent study [23] of a temporal sequence in mouse neutrophil trajectories [23]. Their data
described at least three neutrophil subsets in the blood of naïve and suggests a continuum of maturational neutrophil states and recapitulate
E. coli infected mice (named PMNa-c). While these neutrophils all harbor previous findings [12] whereby proliferating PreNeus serve as pre­
a polymorphonuclear fully mature nucleus and their frequencies remain cursors of a non-proliferating, immature stage to mature neutrophils.
steady even upon infection, the authors draw trajectories in which both Interestingly, the authors described within this trajectory a novel set of
neutrophil type a (S100a8− 9 and Mmp8 enriched) and type b (neutro­ transcription factors that remained active in the most mature neutrophil
phils expressing interferon-related genes, as also observed in [66]), differentiation stage (including ATF3, KLF2, C/EBPb, JUNB and JUND).
serve as precursors of the third c type/state. The latter contains an These studies further revealed that neutrophils display unique tran­
increased percentage of cells mapping to an aged-like phenotype, in scriptional profiles depending on the experimental model and recruit­
addition to enriched ribosome biogenesis, increased transcriptional and ment site, suggesting that differentiation, kinetics and niche education
translational programs and increased LPS signaling response, suggesting in different tissues may all contribute to the neutrophil phenotype, as
a fully activated state ready to be cleared from the circulation. It will be recently demonstrated in the steady-state lung [63].
important to determine whether such transcriptional maps truly corre­ Relevant questions remain as to what drives the fate of neutrophils
late with the circadian program of aged neutrophils. that will enter tissues, participate in acute inflammatory reactions, or in
As indicated above, aged neutrophils are preferentially wiped out chronic diseased states such as autoimmune diseases or cancer. Are
from the circulation into healthy tissues under steady-state conditions, these fates imprinted as they mature in the bone marrow, or acquired
whereas non-aged cells (“fresh” neutrophils) just released into the later in the periphery? We propose that some of the transcriptional
bloodstream are preferentially recruited to inflammatory sites [35]. A programs activated during normal maturation in the BM could in fact be
mechanistic explanation for this behavior was the progressive loss of relevant transcriptional hubs for later tissue- or inflammation-driven
microvilli needed for rolling as neutrophils aged, and the reduced programs. Similarly, we suggest that this late-stage transcription fac­
expression of CXCR2 receptor, which recognizes cues released from in­ tors core program may underlie the plastic phenotypes in tumors, but
flammatory sites. In addition to impaired migration capabilities, aged this needs further exploration. Exploration of this hypothetical model
neutrophils also displayed reduced granule content and consequently will be important to determine whether actionable pathways exist that
reduced potential for neutrophil extracellular trap (NET) production target polarization and curb neutrophil states, for example those medi­
[67], suggesting fundamentally different functional properties of day vs. ating resistance mechanisms in neutrophil-dominated tumors [74,77].
night neutrophils. Other studies have suggested an alternative model by which neutrophils
Phagocytosis of aged neutrophils by macrophages and dendritic cells can bypass certain maturation stages upon challenge [23,78,79].
(DCs) has been shown to inhibit transcription of Il23, a cytokine that in T Experimental examination of both models (discontinuous vs. continuous
cells regulates expression of IL-17, a cytokine that is a direct modulator transitions), and importantly developing the appropriate tools for line­
of G-CSF production [68]. This mechanism functions as a peripheral age tracing and elucidating global neutrophil trajectories and lifespans
“rheostat” that controls neutrophil production in the BM in response to will be critical to move the field forward. Herein we propose a set of new
their clearance in the periphery. genetic tools that can help tackle these critical questions (Table 1).

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A. Hidalgo and M. Casanova-Acebes Seminars in Immunology 57 (2021) 101506

Table 1
Genetic tools available to trace neutrophil fate.
Mouse Tool Traced population Inducible Refs.
CreERT2
Pdzk1ip1 R26 Cre HSC-derived Tam [96]
LSL− tdTom
recombinase progeny
CreERT2
Ms4a3 R26 Cre GMP-derived Tam [97]
LSL− tdTom
recombinase progeny
Ly6GCreERT2 Cre Immature-mature Tam [54]
R26LSL− tdTom recombinase neutrophils

3. Neutrophil rewiring in organismal aging and disease

Disease-specific inflammation generates different alarm signals that

impact host myelopoiesis and myeloid cell mobilization in different
manners and at various levels. It is conceivable that neutrophils released
in the steady-state functionally differ from progenitors that were
exposed to mobilizing and differentiation signals in the presence of
DAMPs (danger-associated molecular patterns) or PAMPs, endotoxins
and metabolites, cytokines, chemokines and stress-hormones all of Fig. 1. Dimensions of neutrophil plasticity. Neutrophil diversity may arise
which have been shown to impact myelopoiesis [[80]]. These modifier from progenitors that differentiate across different granulopoietic organs (1).
signals may dramatically alter the output number, phenotype and These progenitors can give rise to neutrophils under steady state or in inflam­
effector capabilities of neutrophils but this remains, paradoxically, matory settings, where either monoclonal or polyclonal granulopoiesis could
potentially generate diverse neutrophil functional phenotypes (2). Once
virtually unexplored. Thus, the pathophysiological context needs to be
differentiated neutrophils infiltrate the majority of tissues, where they are
defined before evaluation of neutrophil heterogeneity. Below we focus
further educated by environmental cues (3). Neutrophil heterogeneity urges the
on recent evidences that support this possibility and outline a framework identification of dysfunctional phenotypes at the organismal level during aging
where this context-specificity can be addressed in the future. and across diseases. This scheme was created with BioRender.com.
Increased numbers of neutrophils are found in healthy older adults,
potentially contributing to age-associated inflammation [81]. Impor­
3.1. Therapeutic-targeting of neutrophils
tantly, functional readouts of anti-microbial function and activation
differ in the aged. For example, formation of NETs is significantly
Neutrophil contribution to immune and non-immune driven diseases
impaired in old mice and humans [82] and impaired neutrophil
in experimental models has been formally-established by the use of
migration due to defective PI3K signaling has been reported in older
neutrophil-depleting or -blockade agents [85,90–93]. Nevertheless,
individuals [83]. More recently, work from the Nourshargh lab has
their highly conserved functions between mice and human, across the
shown that aged-driven increased levels of CXCL1 cause CXCR2 desen­
tissues examined [64,66], and alterations associated with neutropenia
sitization, in turn promoting neutrophil reverse transendothelial
even in the absence of infections are all suggestive of pivotal functions
migration that contributes to remote organ damage [84].
beyond immune defense, i.e. during baseline tissue homeostasis. Hence,
In the context of disease, recent studies have elegantly shown that
while neutrophil depletion is unadvisable in the clinic, therapeutic
obesity-associated inflammation and neutrophil over-activation is a
intervention should take into account that broad interference with
driver of breast cancer metastasis [85]. Experimental obese mice and
neutrophil functions could significantly impact organismal physiology.
humans with increased body mass indexes displayed compromised lung
In this context, identification of disease-specific programs provides an
vascular function secondary to neutrophils producing
ideal hypothetical scenario to target pathogenic activity while preser­
supra-physiological levels of reactive oxygen species (ROS) activity and
ving homeostatic functions. Critically, however, prior to committing to
exacerbated NET release. Concomitantly, these alterations in neutrophil
targeting approaches, the field is in dire need of new tools that excel at
activity favor metastatic breast cancer cell seeding and tumor progres­
identifying disease-associated trajectories and activation states of neu­
sion. Interestingly, remote signaling from distant sites can preferentially
trophils, such as those leading to immunosuppression in cancer.
impact granulocytes in the lung to favor the establishment of
Development of new single cell-resolution tools and lineage-tracing
pre-metastatic niches [86]. Metastatic priming in the lung seems to be
models can greatly accelerate this endeavor. An illustrating example
governed by a signature enriched in remodeling and immunosuppres­
of this comes from a recent study combining DNA-tagging of clonal
sive neutrophils expressing Mmp8, Mmp9, Trem1, Il1b and Arg1.
hematopoietic stem cells, thus allowing simultaneous tracing of
Whether these genetic determinants are established earlier during
HSC-progeny by state and fate [94]. Further, examination of blood
neutrophil ontogeny has been proposed recently [87] but remains to be
phenotypes as typically performed in the clinic may not be sufficient to
explored. Finally, in the context of experimental atherosclerosis,
predict patient responses, as neutrophils infiltrating tissues may be more
Silvestre-Roig et al., [88] showed that histone 4 derived from NETs
informative of disease outcome [95]. While we are still missing a model
produced by local neutrophils causes lytic death of smooth muscle cells,
that can satisfactorily track and anticipate neutrophil pathogenicity in
ultimately promoting plaque instability and further propagating
most tissues, combinatorial approaches aimed at deciphering their
inflammation.
spatial, epigenetic and signaling profiles can enlighten relevant fates and
While we are aware of the emerging interest of neutrophil pheno­
phenotypes, and will be fundamental if we ambition to manipulate
types in disease, we will not address here neutrophil plasticity in other
neutrophils in routine clinical care.
disease conditions such as neuroinflammation, autoimmunity or cancer.
For a complete coverage on neutrophil plasticity in different diseased
Acknowledgements
scenarios we redirect the reader to other reviews [89], including the
ones in this review series. In sum, this set of evidences support a model
We thank members of our labs for continued discussion and Carlos
whereby neutrophil identity and activity needs to be integrated with
Silvestre-Roig for critically reading the manuscript. AH is supported by
local signals within a tissue, but also within signals at the organismal
RTI2018-095497-B-I00 from Ministerio de Ciencia e Innovación
level that appear with age or with disease (Fig. 1).
(MICINN), HR17_00527 from Fundación La Caixa, Transatlantic

5
A. Hidalgo and M. Casanova-Acebes Seminars in Immunology 57 (2021) 101506

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