**1. Introduction: Regulatory neutrophils and their profile**

#### **1.1. New technologies changing the concept of a short half‐life**

Besides the known features of neutrophils as fast migrating pro-inflammatory cells, the literature has shown that this is not a homogeneous population. In fact, several different subtypes of neutrophils have been well described regarding its characteristics and mechanisms of action. However, there are still some subtypes not so well understood.

The classical, and popular, concept of neutrophils says that they are the first cells to arrive and accumulate at the site of infections where they rapidly release several toxic molecules and undergo apoptosis [1]. In the meantime, they clear the infected area through phagocytosis or "neutrophils extracellular traps" (NETs) that occur when activated neutrophils release their uncondensed chromatin and granule contents. These molecules bind to pathogens causing death, contributing to fight against infections, and causing important tissue damage. Macrophages come over after this, clean up cell debris, and amplify the response [2, 3].

However, since different and modern techniques started to be accessible to research, some functions and concepts assigned to neutrophils have changed. The paradigm of the short half-life fell with the improvement of cellular techniques that enable better stains for different phenotypic markers or gene expressions. A variety of different neutrophils subtypes that are different in their phenotype, function, package of cytokines produced, degree of maturation, and site of action have been identified. So, a number of different types of neutrophils are being described including the ones with regulatory properties (hereafter referred as regulatory neutrophils—RN) that we will look closer in this chapter [4, 5].

How can these new findings interfere in the classic view of these cells?

Previously, a half-life of ∼24 hours for human neutrophils and ∼8 hours for murine neutrophils was believed. However, recently, using *in vivo* labeling techniques with 2 H2 O, a stable isotope, it was shown that human neutrophils in homeostatic conditions present an average 5.4 days of half-life [6]. The discrepancy with previous studies is believed to be due to *ex‐vivo* manipulation and i.v. injection of neutrophils, which affects cells viability and *in vivo* distribution. Neutrophils longer half-life allows the conditions to develop phenotypic and functional alterations, including synthesis of a great number of cytokines, ability to recirculate and alter or influence other immune cells [7].

Neutrophils half-life can be dictated by several factors as inflammatory conditions, cytokines, cell interactions, PAMPS (pathogen-associated molecular pattern), and DAMPS (dangerassociated molecular pattern), which might inhibit apoptosis and prolong the cell life span [8]. Then, this longer half-life associated with all sort of stimuli paves the way for new regulatory subtypes to emerge.

### **1.2. Neutrophil subtypes generated in specific conditions**

The classical murine neutrophils can be identified by Ly6G<sup>+</sup> expression, while human neutrophils have to accomplish the expression of CD14, CD15, and CD16, always associated with a visual inspection that must identify a band or hypersegmented nucleus with a light pink cytoplasm, full of granules [4].

**1. Introduction: Regulatory neutrophils and their profile**

of action. However, there are still some subtypes not so well understood.

Besides the known features of neutrophils as fast migrating pro-inflammatory cells, the literature has shown that this is not a homogeneous population. In fact, several different subtypes of neutrophils have been well described regarding its characteristics and mechanisms

The classical, and popular, concept of neutrophils says that they are the first cells to arrive and accumulate at the site of infections where they rapidly release several toxic molecules and undergo apoptosis [1]. In the meantime, they clear the infected area through phagocytosis or "neutrophils extracellular traps" (NETs) that occur when activated neutrophils release their uncondensed chromatin and granule contents. These molecules bind to pathogens causing death, contributing to fight against infections, and causing important tissue damage. Macrophages come over after this, clean up cell debris, and amplify the response [2, 3].

However, since different and modern techniques started to be accessible to research, some functions and concepts assigned to neutrophils have changed. The paradigm of the short half-life fell with the improvement of cellular techniques that enable better stains for different phenotypic markers or gene expressions. A variety of different neutrophils subtypes that are different in their phenotype, function, package of cytokines produced, degree of maturation, and site of action have been identified. So, a number of different types of neutrophils are being described including the ones with regulatory properties (hereafter referred as regulatory

Previously, a half-life of ∼24 hours for human neutrophils and ∼8 hours for murine neutrophils was believed. However, recently, using *in vivo* labeling techniques with 2

stable isotope, it was shown that human neutrophils in homeostatic conditions present an average 5.4 days of half-life [6]. The discrepancy with previous studies is believed to be due to *ex‐vivo* manipulation and i.v. injection of neutrophils, which affects cells viability and *in vivo* distribution. Neutrophils longer half-life allows the conditions to develop phenotypic and functional alterations, including synthesis of a great number of cytokines, ability to

Neutrophils half-life can be dictated by several factors as inflammatory conditions, cytokines, cell interactions, PAMPS (pathogen-associated molecular pattern), and DAMPS (dangerassociated molecular pattern), which might inhibit apoptosis and prolong the cell life span [8]. Then, this longer half-life associated with all sort of stimuli paves the way for new regula-

phils have to accomplish the expression of CD14, CD15, and CD16, always associated with

H2 O, a

expression, while human neutro-

**1.1. New technologies changing the concept of a short half‐life**

146 Role of Neutrophils in Disease Pathogenesis

neutrophils—RN) that we will look closer in this chapter [4, 5].

recirculate and alter or influence other immune cells [7].

**1.2. Neutrophil subtypes generated in specific conditions**

The classical murine neutrophils can be identified by Ly6G<sup>+</sup>

tory subtypes to emerge.

How can these new findings interfere in the classic view of these cells?

Although the existence of different neutrophils subtypes is currently accepted, the basis for their classification remains obscure. Distinct surface markers or new cytokines are common characteristics used to define neutrophils, but heterogeneity can also be explained by a stage of differential activation of neutrophil subpopulations [3, 7].

In some autoimmune diseases, such as systemic vasculitis associated (ASV) with antineutrophil cytoplasmic autoantibody or systemic lupus erythematous (SLE), circulating neutrophils display an increased expression of the specific surface marker, CD177. This is a molecule compartmentalized in secondary (specific) granule, that is co-expressed with its membrane ligand proteinase 3 (mPR3) in neutrophils from ASV patients. mPR3 is one of the main targets of ANCA autoantibodies, and, in this case, CD177 is important to mPR3 expression influencing the potential of neutrophils to be activated by ANCAs that usually target mPR3. However, levels of CD177 expression in patients and their influence on ANCA have not been defined as disease biomarkers yet, the CD177<sup>+</sup> neutrophils (NB1 in humans) represent indeed a new subset [9, 10].

Importantly in SLE, where the response is driven against nuclear antigens in various target organs such as the skin, kidney, and joints, neutrophils have been described as the source of DNA antigens due to its extravasation of nuclear content when forming NETs. Besides the CD177+ NB1 neutrophils, low-density granulocytes (LDGs) are another subpopulation, which has been described in SLE. Specifically in this case, LDGs can assume a highly inflammatory profile, including the release of NETs, which amplifies disease physiopathology [11, 12]. The same LDG subtype was described in rheumatoid arthritis (RA). In RA, they show a low expression of TNFR, potentially affecting TNFi (inhibitor) treatment [13]. Beyond that, LDGs were also reported in mycobacterial infections being associated with disease severity [14]. This probably happens because it suppresses the immune response allowing mycobacterium growth. In severe asthma [15] and in interstitial lung disease in dermatomyositis [16], similar to what was described in autoimmune diseases like SLE and RA, the LDG acts worsening the pathologic condition.

The above features evidence the complex behavior of neutrophils requesting a lot more to be described about the plasticity of neutrophils in disease pathogenesis.

Apart from the uncommon profile of neutrophils in autoimmune diseases, we can highlight the phenotype of aged or senescent neutrophils. This particular subset expresses CXCR4 in high densities (CXCR4hi). CXCR4 mediates cell retention in the bone marrow along with low expression of CD62L (CD62Llow). They express high CD11b and have hypersegmented nuclei [17]. Recently, it was described that ageing neutrophils are regulated by the microbiota in a toll-like receptor (TLR)-dependent way. The microbiota is the community of microorganisms that lives within the body and in harmony with it. The most studied group in this regard is bacteria from the human gastrointestinal (GI) tract that harbors an estimated ∼1014 individuals from about 1000 species in a single individual. Close to ∼15,000 species of bacteria have already been identified from human GI samples [18]. These commensal bacteria can influence the immune system inducing a pro-inflammatory or a suppressor response, which depends on the bacteria quality and the milieu of activation. When the microbiota is depleted with the use of antibiotics, the number of aged neutrophils (that are highly activated cells) decreases, and the pathogenesis of sickle-cell disease or endotoxin-induced septic shock improves, showing that aged neutrophils have an important role in inflammatory diseases [19].

Another neutrophil subtype extremely relevant for immunology are the tumor-associated neutrophils (TAN), that are subdivided into type 1 (N1), with anti-tumor activity, and type 2 (N2), that fulfills a pro-tumor activity and will be discussed in the next subsection [20]. They have high relevance in the prognostic of some types of tumors, as colorectal [21], non-small cell lung [22], and breast where ratio of neutrophils/lymphocyte (NLR) is used as a toll to correlate with a better or poor prognostic [23].

Plasticity of TANs depends on many factors such as cytokines, chemokines, and adhesion molecules. These factors can be secreted by other immune cells or by the tumor itself [24]. The anti-inflammatory cytokine TGF-β has an important role in this scenario: in its presence, TANs can be directed to a pro-tumor N2 phenotype, and in its absence (using blocking antibodies) TANs are driven to an anti-tumor N1 phenotype [20]. On the other hand, these neutrophils can also interfere with the tumor microenvironment through the release of cytokines (TNF-α, IL-1β, IL-12), chemokines (CCL2, CCL3 and CCL5), reactive oxygen species (ROS), and growth factors, creating a diverse niche amplifying or down-regulating the inflammatory response [5, 25, 26]. Phenotypically, the N1 type presents a hypersegmentated nuclei with high expression of FAS, ICAM, and TNF-α production, making them able to activate TCD8+ lymphocytes, helping to eliminate the tumor cells [20].

These ambiguous characteristics evidence how the plasticity of neutrophils can impact in health and in pathological conditions. Until now, we covered how neutrophils can assume different subtypes and contribute to a pro-inflammatory milieu, amplifying the immune responses in autoimmune diseases as well in highly activated conditions, as the aged neutrophils in inflammatory infections. In tumor setting, the ratio neutrophil/lymphocyte can even predict the patient prognostic highlighting the importance of neutrophils in disease outcome.

In Section 1.3, we describe the profile of RN, the conditions in which they were described their mechanism of action, and the possibility to manipulate them for therapeutic usage.

#### **1.3. Regulatory neutrophil phenotypes**

As stated above, nowadays, the literature accepts that neutrophils can assume different subtypes. It is important to point here that the term "regulatory" or "suppressor" indicates the capacity of these cells to induce an anti-inflammatory response, either by interacting directly with other cells or by secreting molecules that induce polarization of other cell types.

The classification of RN subtypes is still unclear. The most reliable characterization of the suppressor neutrophil subtypes remains being their functional characteristics. Although there are some markers, such as CD62Llow/CD11blow highly associated with suppressor phenotypes and others such as CD244, CD115, CD11c, CD32, CD35, CD45, and CD66b, which can be up- regulated, there is no consensus for a specific combination of markers for suppressor neutrophils. Their phenotype heterogeneity is probably because they are modulated according to individual conditions [3, 4]. So, in this section, we discuss the literature on the different subtypes (or phenotypes) of RN.

which depends on the bacteria quality and the milieu of activation. When the microbiota is depleted with the use of antibiotics, the number of aged neutrophils (that are highly activated cells) decreases, and the pathogenesis of sickle-cell disease or endotoxin-induced septic shock improves, showing that aged neutrophils have an important role in inflammatory

Another neutrophil subtype extremely relevant for immunology are the tumor-associated neutrophils (TAN), that are subdivided into type 1 (N1), with anti-tumor activity, and type 2 (N2), that fulfills a pro-tumor activity and will be discussed in the next subsection [20]. They have high relevance in the prognostic of some types of tumors, as colorectal [21], non-small cell lung [22], and breast where ratio of neutrophils/lymphocyte (NLR) is used as a toll to

Plasticity of TANs depends on many factors such as cytokines, chemokines, and adhesion molecules. These factors can be secreted by other immune cells or by the tumor itself [24]. The anti-inflammatory cytokine TGF-β has an important role in this scenario: in its presence, TANs can be directed to a pro-tumor N2 phenotype, and in its absence (using blocking antibodies) TANs are driven to an anti-tumor N1 phenotype [20]. On the other hand, these neutrophils can also interfere with the tumor microenvironment through the release of cytokines (TNF-α, IL-1β, IL-12), chemokines (CCL2, CCL3 and CCL5), reactive oxygen species (ROS), and growth factors, creating a diverse niche amplifying or down-regulating the inflammatory response [5, 25, 26]. Phenotypically, the N1 type presents a hypersegmentated nuclei with high expression of FAS, ICAM, and TNF-α production, making them able to activate TCD8+

These ambiguous characteristics evidence how the plasticity of neutrophils can impact in health and in pathological conditions. Until now, we covered how neutrophils can assume different subtypes and contribute to a pro-inflammatory milieu, amplifying the immune responses in autoimmune diseases as well in highly activated conditions, as the aged neutrophils in inflammatory infections. In tumor setting, the ratio neutrophil/lymphocyte can even predict

In Section 1.3, we describe the profile of RN, the conditions in which they were described their

As stated above, nowadays, the literature accepts that neutrophils can assume different subtypes. It is important to point here that the term "regulatory" or "suppressor" indicates the capacity of these cells to induce an anti-inflammatory response, either by interacting directly with other cells or by secreting molecules that induce polarization of other cell types. The classification of RN subtypes is still unclear. The most reliable characterization of the suppressor neutrophil subtypes remains being their functional characteristics. Although there are some markers, such as CD62Llow/CD11blow highly associated with suppressor phenotypes and others such as CD244, CD115, CD11c, CD32, CD35, CD45, and CD66b, which can be up- regulated, there is no consensus for a specific combination of markers for suppressor neutrophils. Their phenotype heterogeneity is probably because they are modulated

the patient prognostic highlighting the importance of neutrophils in disease outcome.

mechanism of action, and the possibility to manipulate them for therapeutic usage.

diseases [19].

148 Role of Neutrophils in Disease Pathogenesis

correlate with a better or poor prognostic [23].

lymphocytes, helping to eliminate the tumor cells [20].

**1.3. Regulatory neutrophil phenotypes**

Among the subtypes described, we can highlight the granulocytic myeloid-derived suppressor cells (G-MDSCs), which are an important sub-population of circulating neutrophils [27]. MDSCs are a heterogeneous population of immature and mature cells, of myeloid origin, first described in tumor-bearing mice and comprise two groups of cells identified regarding their morphology and phenotype. Monocyte-MDSCs (M-MDSCs) are CD11b+ Ly6ChighLy6G<sup>−</sup> and have a typical monocyte morphology, and cells CD11b+ Ly6ClowLy6G+ with typical granulocytic morphology are G-MDSC [28, 29]. The G-MDSC phenotype is characterized mainly by large amounts of ROS expression and low amounts of nitric oxide synthase (NOS). The opposite is true for the M-MDSC phenotype that acts mainly expressing arginase-1 (Arg1) and NOS. MDSCs can inhibit T cell responses in many ways. After being generated as a consequence of intense inflammatory environment in the presence of factors like GM-CSF (granulocytemacrophage colony stimulating factor), G-CSF (granulocyte colony stimulating factor), VEGF, IL-6, MDSCs are recruited to the site of the primary tumor and secondary lymphoid organs (lymph nodes, spleen) by chemokines such as CCL2, CXCL12, and CXCL5 [30]. Upon arrival in the specific site, they can modify the microenvironment by secreting NOS, ARG, and/or ROS.

Although described as harmful in autoimmune diseases, it is also known that mouse and human LDGs are a heterogeneous population composed of mature and immature neutrophils with suppressive capacity. Neutrophils are classified as LDG or low-density neutrophils (LDNs) and high-density granulocytes (HDNs) depending on their density. In general, LDG or LDN cells co-purify with PBMC at the low-density layer in a ficoll gradient, rather than with the high-density layer, which is the usual for the classic neutrophils [31, 32].

It was shown in a tumor model that LDN comprises at least two different populations: one with a segmented nucleus (mature) and another with banded or ring-shaped nucleus (immature) that resembles the G-MDSC phenotype. Both can be generated from HDN in a TGF-β-dependent way. In this case, a new nomenclature was suggested to circulating mature neutrophils. The HDN that are pro-inflammatory with anti-tumor profile would be called Nc1 and its counterpart mature LDN, which shows a pro-tumor activity would be Nc2. The Nc2 has reduced expression of inflammatory molecules and inhibit TCD8<sup>+</sup> proliferation *in vitro* evidencing more than one type of RN [32].

During pregnancy, where immunosuppressive state is required to allow implantation and growth of the fetus, an important population of LDG producing arginase-1 was identified in PBMC and placentae of pregnant women and in the cord blood. Besides, these neutrophils were described as cells that released specific granules (once they increase expression of CD66b), and the azurophilic granules, where arginase-1 is stored (once CD63 is expressed). These phenotypical markers associated with others mean that these cells have been activated and are degranulated. Presence of arginase-1 collaborates to impair T cell responses once the L-arginine deprivation induced by release of arginase contributes to T cell hyporesponsiveness and immune privilege at the materno-fetal interface [33, 34].

In HIV infection, LDGs act to inhibit the immune system, worsening the condition. PBMCs from HIV-infected patients are rich in high arginase LDGs, suggesting that they are activated neutrophils that had degranulated [35].

Some years ago, our group observed that LDG was increased in the peripheral blood of G-CSF-treated donors of peripheral blood stem cells. These cells were capable to inhibit T cells IL-4 and IFN-γ production in a hydrogen peroxide (H<sup>2</sup> O2 )-dependent way [36]. In a murine model of graft versus host disease (GVHD), the main limitation of stem cell transplantation, LDGs prevented 100% mortality [31]. These cells were better characterized recently [37] and will be described at the end of this chapter.

On the other hand, in infection with methicillin-resistant *Staphylococcus aureus* strain, three different subtypes of mouse neutrophils with different susceptibilities to infection have been described. Besides the normal PMN-N (polymorphonuclear neutrophils), there are at least two distinct PMN subtypes (PMN-I and PMN-II). The suppressor subtype (PMN-II) can express TLR2/TLR4/TLR7/TLR9 and has low levels of MPO (myeloperoxidases). PMN-II is involved with the generation of alternatively activated macrophages (M2), through IL-10 and CCL2- dependent mechanisms. These M2 macrophages have anti-inflammatory properties and induce a Th2 response [38], modulating the adaptive immune response at the expense of neutrophils.

Regarding cytokines production, RN IL-10+ producing cells have been described. The IL-10 is an important cytokine, which can be produced by many different cell types, as B cells, mast cells, eosinophils, macrophages, DCs, and a large number of T cell subtypes that act regulating the synthesis of pro-inflammatory chemokines and cytokines, such as IL-1, IL-6, TNF-α, as well as nitric oxide (NO), collagenase, and gelatinase [3, 39, 40].

In murine models, several studies have shown that neutrophils produce IL-10 in response to a variety of infections, such as *S. aureus* [38], *Candida albicans* [40], *Trypanssoma cruzi* [41], and in inflammatory conditions, such as post-burn [42] and after G-CSF treatment [37]. However, these data are still a matter of conflict in the literature since just a few studies show the same phenotype in humans [43–45] and others were incapable to reproduce it [46, 47].

These differences in IL-10 production between mouse and human neutrophils may result from different factors, such as culture conditions, contaminating cells, or post-transcriptional regulation of IL-10 gene expression [47, 48].

Moreover, the cytokine IL-22 has also been described as being produced by neutrophils, besides being produced by many different cells, including Th17, Th22, NK cells, Tγδ, and ILC (innate-like lymphocytes). RN IL-22+ is mainly important for intestinal barrier maintenance exerting a local modulation and keeping the integrity of the intestinal mucosa, generating a protective response against certain extracellular pathogenic bacteria [49, 50]. IL-22 has the ability to synergize with other cytokines to induce gene expression of antimicrobial peptides, chemokines, matrix metalloproteinase, cytokines, and epithelial acute phase proteins in the skin, liver, lung, and intestine [51, 52]. Of note, it was described that neutrophil-producing IL-22 has an important role in intestinal protection in a model of colitis. The adoptive transfer of IL-22-producing neutrophils to IL-22-deficient animals was protective for dextran-induced colitis inducing the release of antimicrobial peptides RegIIIβ and S100A8 by colonic cells, protecting the intestinal barrier from microbes and helping the resolution of disease [53].

At last, as stated before, in tumor settings, neutrophils can assume ambiguous features being supportive or inhibiting the tumor growth. We described above the TAN-N1 proinflammatory neutrophils, protecting from tumor, but it is also important to highlight the TAN-N2 neutrophils that are suppressive and, in this case, harmful for the patient. N2 TANs are tumor resident neutrophils that influence the establishment, development, and spread of cancers. They can be generated in a TGF-β milieu and also by G-CSF produced by tumor cells, among others [20]. Under G-CSF stimuli, neutrophils are generated and expand, creating a pro-tumorigenic niche, being able to favor metastatic microenviroment [54]. TAN-N2 cells express arginase, contributing to inhibition of T cell responses. They release chemokines such as CCL2 and CCL5 that favor the recruitment of other cell types, including regulatory T cells (Tregs). Also, they can produce oncostatin-M that works promoting angiogenesis and neovascularization favoring tumor growth [20, 55, 56]. Depletion of neutrophils from tumor-bearing mice shows an increase in TCD8+ cells, supporting the concept that N2 acts in a suppressive way being an RN [20]. As stated above, the ratio between lymphocytes and neutrophils (NLR) is used to predict the patient prognosis in cancer patients. In breast tumors, a high NLR is associated with a poor prognostic and a shorter overall survival [57].

Some years ago, our group observed that LDG was increased in the peripheral blood of G-CSF-treated donors of peripheral blood stem cells. These cells were capable to inhibit T cells

model of graft versus host disease (GVHD), the main limitation of stem cell transplantation, LDGs prevented 100% mortality [31]. These cells were better characterized recently [37] and

On the other hand, in infection with methicillin-resistant *Staphylococcus aureus* strain, three different subtypes of mouse neutrophils with different susceptibilities to infection have been described. Besides the normal PMN-N (polymorphonuclear neutrophils), there are at least two distinct PMN subtypes (PMN-I and PMN-II). The suppressor subtype (PMN-II) can express TLR2/TLR4/TLR7/TLR9 and has low levels of MPO (myeloperoxidases). PMN-II is involved with the generation of alternatively activated macrophages (M2), through IL-10 and CCL2- dependent mechanisms. These M2 macrophages have anti-inflammatory properties and induce a Th2 response [38], modulating the adaptive immune response at the expense

is an important cytokine, which can be produced by many different cell types, as B cells, mast cells, eosinophils, macrophages, DCs, and a large number of T cell subtypes that act regulating the synthesis of pro-inflammatory chemokines and cytokines, such as IL-1, IL-6,

In murine models, several studies have shown that neutrophils produce IL-10 in response to a variety of infections, such as *S. aureus* [38], *Candida albicans* [40], *Trypanssoma cruzi* [41], and in inflammatory conditions, such as post-burn [42] and after G-CSF treatment [37]. However, these data are still a matter of conflict in the literature since just a few studies show the same phenotype in humans [43–45] and others were incapable to reproduce it

These differences in IL-10 production between mouse and human neutrophils may result from different factors, such as culture conditions, contaminating cells, or post-transcriptional

Moreover, the cytokine IL-22 has also been described as being produced by neutrophils, besides being produced by many different cells, including Th17, Th22, NK cells, Tγδ, and

nance exerting a local modulation and keeping the integrity of the intestinal mucosa, generating a protective response against certain extracellular pathogenic bacteria [49, 50]. IL-22 has the ability to synergize with other cytokines to induce gene expression of antimicrobial peptides, chemokines, matrix metalloproteinase, cytokines, and epithelial acute phase proteins in the skin, liver, lung, and intestine [51, 52]. Of note, it was described that neutrophil-producing IL-22 has an important role in intestinal protection in a model of colitis. The adoptive transfer of IL-22-producing neutrophils to IL-22-deficient animals was protective for dextran-induced colitis inducing the release of antimicrobial peptides RegIIIβ and S100A8 by colonic cells, protecting the intestinal barrier from microbes and helping the

TNF-α, as well as nitric oxide (NO), collagenase, and gelatinase [3, 39, 40].

O2

)-dependent way [36]. In a murine

producing cells have been described. The IL-10

is mainly important for intestinal barrier mainte-

IL-4 and IFN-γ production in a hydrogen peroxide (H<sup>2</sup>

will be described at the end of this chapter.

150 Role of Neutrophils in Disease Pathogenesis

Regarding cytokines production, RN IL-10+

regulation of IL-10 gene expression [47, 48].

ILC (innate-like lymphocytes). RN IL-22+

resolution of disease [53].

of neutrophils.

[46, 47].

**Figure 1.** Illustration of the most well-described regulatory neutrophils (RN) subtypes and their main mechanisms of action. TAN-N2 (Tumor associated neutrophil type 2); G-MDSC (Granulocytic myeloid derived suppressor cell); PMN-II (Polymorphonuclear type II); LDG (low-density granulocyte); NC2 (circulating neutrophils type 2); MT (mature); iMT (immature).

Despite those RN that share some similarities regarding their mechanism of action based on arginase production, H2 O2 , Treg induction by IL-10 secretion, and M2 generation, there are still some differences among the cell types that prevent them from being placed in the same general group of RN. Many authors have been trying to establish a nomenclature to these RN; however, new subsets keep being described as well as new features which make this a hard task and fill the literature with different names, many times for the same described cell. Some of the subsets are well defined as MDSC, like high-density mature, low-density mature or immature, as can be seen in **Figure 1**. In this regard, these cells can express different markers, be sensitive to diverse stimuli, and influence different cell types showing important consequences in amplification of suppressive immune response. The crosstalk of neutrophils with other cell types and the maintenance of the suppressor "tonus" will be explored in more details in Section 2 (**Figure 1**).
