**2. Changes in the immune response under neutrophils influence**

As mentioned earlier, neutrophils are recruited to different tissues after injury or infection and, in this sites, encounter resident and/or recruited leukocytes, which promote interactions that influence each other mutually. Under neutrophil influence, other cell types acquire regulatory properties worsening or improving the host condition. On the other hand, neutrophils can suffer influences that polarize them to a suppressor phenotype.

#### **2.1. Neutrophil influence in macrophage polarization**

In *Nippostrongylus brasiliensis* infection, neutrophils acquire a N2 phenotype that secrete high amount of IL-13 that in turn is essential to provide helper functions to promote alternatively activated M2 macrophage polarization with long-lived profile. The M2 macrophages mediate parasitic larval damage during recall responses in the lung and are essential to nematode damage and clearance [58]. The presence of these M2 macrophages also impairs host antibacterial resistance against sepsis. In thermally injured mice, PMN-II displays an immunosuppressive phenotype with production of CCL2 and IL-10. These induce macrophages conversion to M2 favoring *Enterococcus faecalis* translocation [59]. The same mechanism was also described in humans with severe burn injuries [60].

#### **2.2. NK, NKT, and ILCs suppression by neutrophils**

Neutrophils are also able to inhibit NK cell activity. *In vitro* co-culture assay demonstrated that in the presence of neutrophils and G-MDSCs, there was a significant decrease in NKp30 expression that led to a reduced NK cell cytotoxicity against *Aspergillus fumigatus*. Moreover, activation markers CD69 and CD137 expression and secretion of the effector molecule IFN-γ were also decreased in NK cells incubated with neutrophils or G-MDSCs before the infection with *A. fumigatus* [61]. In vaccinia virus infection, the G-MDSC subset was responsible for the NK function and proliferation inhibition mediated by ROS [62]. A crucial role of primary tumor-mobilized neutrophils and NK crosstalk in the establishment of lung pre-metastatic niches was described. In this case, they are able to inhibit NK cell-mediated clearance of metastatic cells and simultaneously promoting intraluminal survival and extravasation at the metastatic site. *In vitro* functional assay of lung NK cells obtained from tumor- bearing mice showed that these cells were significantly less responsive to the NKG2D or NKp46, NK- activating receptors (measured by expression of CD107 and IFN-γ) than the naïve mice [63]. Reciprocally, human NK cells (resting or activated by IL-12) were able to induce neutrophil apoptosis dependent on cell-cell contact and caspases, which overcome the antiapoptotic effect of GM-CSF. Involvement of the activating NK cell receptor NKp46 and the Fas pathway was observed in this process [64]. This regulatory effect of NK cells on neutrophils was also observed in the DSS-induced colitis model. However, in this model, NK cells significantly lowered the percentage of apoptotic neutrophils in co-cultured assay. The regulatory effect is dependent on down-regulation of the inflammatory neutrophil functions (decrease in IL-6 and increase in IL-10 production) and is largely dependent on direct NK cell-neutrophil contact, via their inhibitory receptor NKG2A [65].

Mouse and human invariant NKT (iNKT) cells were also inhibited by contact with live neutrophils. iNKT cells from mice with acute inflammatory neutrophilia (as in peritonitis) display decrease in T-bx21 and GATA3 expression and diminished cytokine production compared with those from control mice. *In vitro* assay demonstrated that cell-cell contact between iNKT and neutrophils is required for the inhibitory effect and that this encounter impairs the cytotoxicity capacity of iNKT cells [66].

The relationship between neutrophils and innate lymphoid cells (ILC3) was shown in human decidua during pregnancy. The ILCs are important effectors of innate immunity present in small amounts in lymphoid tissues and enriched at barrier surfaces, such as the skin, lung, intestine, and mucosal-associated lymphoid tissues. Characterized by the absence of recombination activating gene (RAG)-dependent rearranged antigen receptors, lack of myeloid cell and dendritic cell phenotypical markers and lymphoid morphology, ILCs undergo neither clonal selection nor expansion when stimulated. These cells reflect the phenotypes and functions of T lymphocytes and NK cells. There are at least three subtypes of ILCs, which are named ILC1, ILC2, and ILC3. They represent the innate counterparts of CD4+ Th1, Th2, and Th17, respectively [67–69]. It has been observed that the numbers of Natural Cytotoxic Receptors positive ILC 3 (NCR+ ILC3) infiltrating decidual tissues positively correlate with those of infiltrating neutrophils. Neutrophils are present in human decidua during the first trimester of normal pregnancy but not in spontaneous miscarriages decidua. *In vitro* assays show that decidual NCR+ ILC3 release CXCL8 and GM-CSF and can induce neutrophil migration and survival, respectively. Moreover, NCR+ ILC3-derived GM-CSF induces expression of HB-EGF and of IL1rα in neutrophils that have anti-inflammatory activity and helps to mediate trophoblast invasion, pointing out a possible role of these cells in the early phases of pregnancy [70].

#### **2.3. Dendritic cells on the neutrophils target**

Despite those RN that share some similarities regarding their mechanism of action based on

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

**2. Changes in the immune response under neutrophils influence**

can suffer influences that polarize them to a suppressor phenotype.

**2.1. Neutrophil influence in macrophage polarization**

**2.2. NK, NKT, and ILCs suppression by neutrophils**

humans with severe burn injuries [60].

As mentioned earlier, neutrophils are recruited to different tissues after injury or infection and, in this sites, encounter resident and/or recruited leukocytes, which promote interactions that influence each other mutually. Under neutrophil influence, other cell types acquire regulatory properties worsening or improving the host condition. On the other hand, neutrophils

In *Nippostrongylus brasiliensis* infection, neutrophils acquire a N2 phenotype that secrete high amount of IL-13 that in turn is essential to provide helper functions to promote alternatively activated M2 macrophage polarization with long-lived profile. The M2 macrophages mediate parasitic larval damage during recall responses in the lung and are essential to nematode damage and clearance [58]. The presence of these M2 macrophages also impairs host antibacterial resistance against sepsis. In thermally injured mice, PMN-II displays an immunosuppressive phenotype with production of CCL2 and IL-10. These induce macrophages conversion to M2 favoring *Enterococcus faecalis* translocation [59]. The same mechanism was also described in

Neutrophils are also able to inhibit NK cell activity. *In vitro* co-culture assay demonstrated that in the presence of neutrophils and G-MDSCs, there was a significant decrease in NKp30 expression that led to a reduced NK cell cytotoxicity against *Aspergillus fumigatus*. Moreover, activation markers CD69 and CD137 expression and secretion of the effector molecule IFN-γ were also decreased in NK cells incubated with neutrophils or G-MDSCs before the infection with *A. fumigatus* [61]. In vaccinia virus infection, the G-MDSC subset was responsible for the NK function and proliferation inhibition mediated by ROS [62]. A crucial role of primary tumor-mobilized neutrophils and NK crosstalk in the establishment of lung pre-metastatic niches was described. In this case, they are able to inhibit NK cell-mediated clearance of

, Treg induction by IL-10 secretion, and M2 generation, there are

arginase production, H2

152 Role of Neutrophils in Disease Pathogenesis

details in Section 2 (**Figure 1**).

O2

The crosstalk between neutrophils and dendritic cells (DC) can be deleterious for DC functions in *Leishmania major* infection. When neutrophils from ear dermis of C57BL/6-infected mice were cultured with bone marrow-derived dendritic cells, they were engulfed by the DC. These DCs show a significant reduction in expression of MHC class II, CD40, and CD86, as well as an inhibited capacity to stimulate T CD8+ lymphocytes proliferation and IFN-γ production. These effects were mediated by the tyrosine kinase receptor "Mer" expressed on DCs [71].

The immunosuppressive effect of apoptotic and necrotic neutrophils was also observed in humans' DC. *In vitro*, DC phagocytes apoptotic/necrotic neutrophils and display upregulation of CD83 and MHC II. However, a decrease of important molecules that stimulate T lymphocytes as CD40, CD80, and CD86 was observed showing that apoptotic/necrotic neutrophils are able to induce a suppressor immune response through DC modulation [72].

DCs infected with *Mycobacterium bovis* (BCG) produced high levels of CXCL1 and CXCL2 that attract neutrophils. In this process, the close contact mediated by CD11b between DCs and neutrophils induces the production of large amounts of the immunosuppressive cytokine IL-10 via MyD88 and Syk pathways in neutrophils. These IL-10+ neutrophils specifically shut down IL-17A production by Th17 cells. This mechanism could break IL-17A production and avoid exacerbated neutrophil recruitment modulating inflammation [73].

MPO is an enzyme found in neutrophils azurophilic granules and is important for intracellular pathogen killing. It was demonstrated that MPO is deposited by neutrophils in lymph nodes, where it interacts with DCs (by catalytic activity through various ROS and DC Mac-1). In this way, MPO is involved in DC changes during the induction of adaptive immunity. DC display reduced activation, defect in uptake/processing antigens, and inhibited migration to LNs by reduced expression of CCR7, leading to reduced adaptive immune response [74].

Neutrophil elastase is a serine proteinase stored in neutrophils azurophilic granules that can damage endothelial cells and cleave endothelial cell-associated adhesion molecules. As MPO, elastase shows modulating effect on DCs. In presence of elastase, immature DC increases the expression of TGFβ-1 and decreases the IL-6, as well their ability to allostimulate T cells. Elastase-treated dendritic cells not only inhibit the proliferation of allogeneic T cells but also increase TGF-β1 expression inducing the differentiation higher number of CD4<sup>+</sup> Foxp3+ Treg cells in MLR cultures. Together, these data suggest mechanisms by which tolerogenic DCs generated by neutrophils elastase exposure contribute to immune regulation [75, 76].

#### **2.4. Neutrophils and B cells crosstalk**

During *S. aureus* infection, neutrophils infiltrate the draining lymph nodes, occupying the medulla and interfollicular areas. These cells form transient and long-lived interactions with B lymphocytes and plasma cells inducing a decrease in B cell IgM production in a TGF- β1 dependent manner [77].

Site-specific splenic neutrophils function as professional helper cells for marginal zone B cells, specialized area in T cell-independent responses to circulating antigen, leading to the generation of affinity-matured antibodies. Neutrophils colonize the marginal zone of the spleen after postnatal mucosal colonization by microbiota. In the spleen, these neutrophils interact with local macrophages that produce IL-10, splenic sinusoidal endothelial cells, that in response to the microbial TLR ligands secrete IL-10 and neutrophil-attracting chemokines, and other STAT3-activating stromal factors, which induce modification of neutrophils phenotype, acquiring a "B cell-helper phenotype." These neutrophils B-cell helper (NBH) are divided into two subpopulation according to their molecule profile expression. NBH1 cells had intermediate expression of CD15 and CD16 and NBH2 cells had low expression of CD15 and CD16. Despite their morphological and ultrastructural similarity to NBH2 cells, NBH1 cells were more activated than NBH2 cells, as they had higher expression of CD27, CD40L, CD86, CD95, and HLA-II but lower expression of CD24. Relative to genes expression (mRNA abundance) compared with "conventional neutrophils," NBH1 and NBH2 cells had more abundant mRNA immunoregulatory molecules, such as IL-10, IL-10 receptor, arginase-1, RALDH1, iNOS, IDO, SOCS1, progranulin, and SLPI, suggesting a skew toward a regulatory profile. In fact, these neutrophils could suppress CD4 proliferation in a contact-independent way. Thus, NBH cells could function as professional MZ B cell helper cells and may suppress T cells to induce immunoglobulin responses in a T-independent manner. These NBH cells were specially characterized by their higher expression of CD40L and surface BAFF and released high amounts of BAFF, APRIL and IL-21, crucial molecules for the B cell functions [78].

#### **2.5. T cell activities under neutrophils control**

an inhibited capacity to stimulate T CD8+

154 Role of Neutrophils in Disease Pathogenesis

**2.4. Neutrophils and B cells crosstalk**

dependent manner [77].

lymphocytes proliferation and IFN-γ production.

neutrophils specifically shut

Foxp3+

Treg

These effects were mediated by the tyrosine kinase receptor "Mer" expressed on DCs [71].

The immunosuppressive effect of apoptotic and necrotic neutrophils was also observed in humans' DC. *In vitro*, DC phagocytes apoptotic/necrotic neutrophils and display upregulation of CD83 and MHC II. However, a decrease of important molecules that stimulate T lymphocytes as CD40, CD80, and CD86 was observed showing that apoptotic/necrotic neutrophils are able to induce a suppressor immune response through DC modulation [72].

DCs infected with *Mycobacterium bovis* (BCG) produced high levels of CXCL1 and CXCL2 that attract neutrophils. In this process, the close contact mediated by CD11b between DCs and neutrophils induces the production of large amounts of the immunosuppressive cytokine

down IL-17A production by Th17 cells. This mechanism could break IL-17A production and

MPO is an enzyme found in neutrophils azurophilic granules and is important for intracellular pathogen killing. It was demonstrated that MPO is deposited by neutrophils in lymph nodes, where it interacts with DCs (by catalytic activity through various ROS and DC Mac-1). In this way, MPO is involved in DC changes during the induction of adaptive immunity. DC display reduced activation, defect in uptake/processing antigens, and inhibited migration to LNs by reduced expression of CCR7, leading to reduced adaptive immune response [74].

Neutrophil elastase is a serine proteinase stored in neutrophils azurophilic granules that can damage endothelial cells and cleave endothelial cell-associated adhesion molecules. As MPO, elastase shows modulating effect on DCs. In presence of elastase, immature DC increases the expression of TGFβ-1 and decreases the IL-6, as well their ability to allostimulate T cells. Elastase-treated dendritic cells not only inhibit the proliferation of allogeneic T cells but also

cells in MLR cultures. Together, these data suggest mechanisms by which tolerogenic DCs

During *S. aureus* infection, neutrophils infiltrate the draining lymph nodes, occupying the medulla and interfollicular areas. These cells form transient and long-lived interactions with B lymphocytes and plasma cells inducing a decrease in B cell IgM production in a TGF- β1

Site-specific splenic neutrophils function as professional helper cells for marginal zone B cells, specialized area in T cell-independent responses to circulating antigen, leading to the generation of affinity-matured antibodies. Neutrophils colonize the marginal zone of the spleen after postnatal mucosal colonization by microbiota. In the spleen, these neutrophils interact with local macrophages that produce IL-10, splenic sinusoidal endothelial cells, that in response to the microbial TLR ligands secrete IL-10 and neutrophil-attracting chemokines, and other STAT3-activating stromal factors, which induce modification of neutrophils phenotype, acquiring a "B cell-helper phenotype." These neutrophils B-cell helper (NBH) are

increase TGF-β1 expression inducing the differentiation higher number of CD4<sup>+</sup>

generated by neutrophils elastase exposure contribute to immune regulation [75, 76].

IL-10 via MyD88 and Syk pathways in neutrophils. These IL-10+

avoid exacerbated neutrophil recruitment modulating inflammation [73].

Finally, we will describe one of the most important crosstalk of neutrophils, which can have long-term consequences: interactions with T cells. The mechanisms involved in the T cells suppression by neutrophils can be achieved by depletion of essential amino acids from the microenvironment, such as L-arginine, generation of ROS, or through cell-cell contact. Neutrophils produce large amounts of arginase-1 that are stocked in gelatinase granules. Release of arginase-1 requires cellular activation and degranulation of gelatinase and azurophilic granules. Arginase is an enzyme that metabolizes L-arginine into L-ornithine and urea. L-arginine is crucial to T cell proliferation, in the absence of it T lymphocytes are arrested in the G0–G1 phase of the cell cycle. Also, in the absence of L-arginine, expression of TCRζ (CD3 zeta chain) is down-regulated and cofilin dephosphorylation is impaired affecting F-actin remodeling, which is essential for T cell effector function [79–82].

Another important mediator of neutrophil-T cell inter-talk is the ROS, which are membranepermeable and act on neighboring cells. Peroxide (H2 O2 ) can suppress lymphocyte proliferation by decreasing NF-κB activation, down-regulating TCRζ and oxidating cofilin. H<sup>2</sup> O2 has a short half-life and is degraded by many endogenous anti-oxidants. Thus, a close contact between neutrophils and T lymphocyte is required for the suppressor effect. The cell-to-cell contact is mediated by expression of the integrin CD11b/CD18 in neutrophil [4, 83, 84].

The cell-cell contact mediated immunosuppression on T cells can be through a PD-L1-PD1 pathway. PD1 is a negative co-stimulatory receptor expressed primarily on activated T cells. Its main role is to limit the effector functions of T cells during inflammatory response. When engaged by one of its ligands, PD1 inhibits kinases, which reduces cytokine production and suppresses T cell proliferation [85, 86]. In volunteers who participated in a human endotoxemia clinical trial, submitted to LPS inoculums, was observed an accumulation of suppressive neutrophils (CD16hi CD62Llo) that exhibit an increased expression of PD-L1 gene and membrane-bound molecule, which was attributed a exposure and stimulation of these cells with IFN-γ and to a lesser extend IFN-α or IFN-β. These IFN-γ-treated neutrophils were able to inhibit proliferation of polyclonal-activated T cells in PD-L1 and cell-cell contact-dependent mechanism [87]. The same phenomenon was observed in murine model of sepsis [88].

In HIV-infected patients, neutrophils play an unappreciated role contributing to the chronic state of immunosuppression leading to opportunistic infection. Low-density neutrophils (which display the same phenotype of G-MDSC) from the peripheral blood of HIV-1 viremic patients express high level of PD-L1. The PD-L1 expression on neutrophils was regulated by the interaction of these cells with inactivated HIV-1 virions, IFN-α, and TLR-7 and TLR-8 ligands. These neutrophils suppress T cell function via PD-L1/PD-1 interaction and production of ROS [89]. The same suppressive function was also observed in *Burkholderia pseudomallei* infected neutrophils, that up-regulated expression of PD-L1 and was able to inhibit CD4+ T cell proliferation and IFN-γ production in response to polyclonal activators, mediated by the PD-L1/PD-1 pathway [90].

Thus, as can be seen in **Figure 2**, the subtypes of RN as well as their diverse interactions with other cell types perform an amplification of the immune response that may help or hinder the host. In Section 3, we describe in details the mechanism of action of an important regulatory neutrophil subtype in GVHD control (**Figure 2**).

**Figure 2.** Regulatory neutrophils act on immune cells playing immunosuppressive role. Neutrophil MPO and elastase induce decrease in uptake/antigens processing by DC, inhibit migration to the lymph nodes, and their ability to stimulate T cells are impaired. Also, elastase induces TGF-1 production by DC. Moreover, the uptake of apoptotic neutrophils down-modulates expression of MHCII, CD40, CD80, and CD86. The secretion cytokines IL-10, IL-13, and chemokine CCL-2 are implicated in the ability of neutrophils to induce changes in macrophages phenotypes to the "M2" antiinflammatory kind. The immunossuppression of neutrophils over T cells is mediated by the production of ROS, ARG-1, and co-inhibitory molecule PD-L1. Moreover, neutrophils secret IL-10 that exert suppression of T cells functions. TGB-1 secretion by neutrophils directly influences humoral response by decreasing IgM production. Splenic neutrophils display a particular profile, which produces high amounts of soluble factors essential to the B cells maintenance and also express IL-10. ROS production by neutrophils and cell-cell contact between ILC and neutrophils decreases cytotoxicity, reduces ILC responsiveness to activator receptor, and down-modulates expression of NKp30, CD69, CD137, CD107, and of IFN-.
