**3. Role of regulatory neutrophils in GVHD protection**

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+

cell proliferation and IFN-γ production in response to polyclonal activators, mediated by the

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

**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,

PD-L1/PD-1 pathway [90].

156 Role of Neutrophils in Disease Pathogenesis

and of IFN-.

neutrophil subtype in GVHD control (**Figure 2**).

T

GVHD is characterized by a robust adaptive immune response caused by donor T cells (present in the incoming graft) after hematopoietic stem cells transplantation (HSCT), a frequent treatment for hematopoietic disorders, and leukemia. In order to be transplanted, the receptor patient undergoes a conditioning regimen that consists of radio/chemotherapy that eliminates the disease and creates the niche for the new incoming bone marrow cells. However, the conditioning regimen also damages the epithelial cells of the patient, mainly the gastrointestinal mucosa, with barrier breakdown and microbiota extravasation. In this case, when donor cells arrive, they find an inflammatory milieu and the T cells are activated by host antigen-presenting cells (APCs) in an inflammatory context and migrate to organs such as the gut, skin, liver, and lungs. The result is the development of GVHD, which has high morbidity and mortality rates, and is the most important limitation of HSCT [91, 92]. Although it is well known that GVHD is mediated and dependent on T cells, elimination of T cells from the graft does eliminate the GVHD. However, it brings other undesired consequences as the lack of anti-leukemia response and deficient hematopoiesis with bone marrow failure [92].

As mentioned before, LDGs have been found in PBMC of stem cell donors treated with G-CSF. These cells were described to be able to inhibit IFN-γ and IL-4 production by T cells in a H<sup>2</sup> O2 dependent way [36]. In the mouse model for GVHD, the same suppressor phenotype was found, and these LDGs were able to inhibit experimental GVHD [31].

Recently, we extended these results showing, among other things, that Ly6G+ RN mediates disease inhibition. In a mouse model of hematopoietic stem cell transplantation (HSCT), after G-CSF treatment, donor spleen cells are enriched in neutrophils (from ˜2 to ˜20%) and, as related in the literature by others, Treg cells were also increased [93, 94]. However, depleting Treg cells from the graft does not alter the GVHD outcome while depleting neutrophils Ly6G+ causes a huge detriment to clinical scores and survival rates. In terms of GVHD, it is important to point that protection was long-lasting and specific, keeping the immunocompetence to reject the skin grafts from third-party mice and rejecting the leukemic cells, keeping the GVL effect. These results show that neutrophils instruct T cells toward a specific tolerant state [37].

So, looking closer to RN, it is important to note that treatment with G-CSF increased the Ly6G+ Ly6C<sup>−</sup> population but not the Ly6G<sup>−</sup> Ly6C+ or Ly6G+ Ly6C+ population consistent with the enrichment of neutrophils but not macrophages or MDSC [95, 96]. The RN has a reduction in the expression of some surface molecules, such as MHC-II, CD62L, and co-stimulatory (CD80, CD86, CD40), increased phagocytic capacity and produce large amounts of H2 O2 molecules. Under stimuli in culture, they produce low IFN-γ, TNF-α, IL-17F, IL-2, IL-12 while increasing IL-22 and the suppressor cytokine IL-10. Also, they have low arginase-1 expression and high NOS, associated with low levels of MPO, which justifies the high amount of H<sup>2</sup> O2 . Altogether, these features encompass a different subtype of suppressor neutrophils.

Confirming that IL-10 production is particularly important to GVHD suppression, transference of a G-CSF-induced neutrophil from IL-10-deficient mice in a HSCT context, abolished GVHD protection showing high mortality rates and poor clinical scores. This evidences that IL-10+ neutrophils are the agents of protection [37].

Nevertheless, the long-lasting and specific protection cannot be explained only by neutrophils suppression as it lasts for several months and the half-life of neutrophils does not exceed 6 days. When analyzing the amount of Treg cells, in spleen and mesenteric lymph nodes (mLN) of the hosts, it was found that 4 days after HSCT, Tregs were increased on mLN but not on spleens. Moreover, 25 days after HSCT, both spleen and MLN show Treg increase, suggesting a systemic increase in Treg. In this way, when Treg cells are depleted early after the transplantation, the GVHD protection is abolished, showing Treg induction within the host right after transplantation. These specific suppressor effects are compatible with the antigen-specific suppression previously observed with Treg cells [97, 98]. The ability of neutrophils to suppress T cell activation is reinforced by the fact that Tregs are less sensitive than conventional T cells to H2 O2 suppression [99].

Indeed, it's known that G-CSF treatment increases the number of Treg cells in the donor, and that these cells produce IL-10 [93]. Treg cells stimulated with LPS are able to induce IL-10 and TGFβ-producing neutrophils. It means that Treg cells have anti-inflammatory properties that influence other cells, including neutrophils, which upon contact with LPS-stimulated Tregs, may prevent the induction of the Th17 profile [100] (**Figure 3**).

Given these various functions of the Ly6G+ IL-10+ neutrophils described herein, at least three possibilities, which may act together, can be suggested to explain the specific suppression by RN in GVHD model.

At first, peroxide production can act by inhibiting T cell activation because its production is carried out with L-arginine consumption and because it acts by inhibiting TCRζ chain phosphorylation. Second, the high phagocytic capacity may contribute to the clearance of translocated gut bacteria after the conditioning regimen. The diminished bacterial load and translocation will contribute to diminished activation of allogeneic T cells leading to a mild GVHD. Third, Tregs generated in the donor after G-CSF treatment influence the generation and modulation of spleen neutrophils to an IL-10+ -producing suppressor subtype, which expresses low levels of MHC II and co-stimulation (as can be seen in **Figure 4**). After HSCT, when in contact with T cells, neutrophils with low levels of co-stimulatory molecules associated with high secretion of IL-10 favor the generation of Treg cells within the host that suppresses GVHD and maintains other functions of the immune system while maintaining immunocompetence and the GVL effect (**Figure 4**).

In addition to this, we believe that the high phagocytic capacity found in these neutrophils, coupled with the high production of H2 O2 , is related to the limitation of bacterial extravasation that occurs as a result of the barrier breaking [101]. It was recently described that neutrophils can colonize intestinal tissues 3 days after transplantation, so the Ly6G+ cell, besides polarizing T cells, could maintain the intestinal integrity [102]. In fact, it has already been described that G-CSF decreases the effects of intestinal barrier breakage showing low levels of endotoxin in the blood and lower numbers of translocated bacteria observed in the spleen, liver, and mesenteric lymph nodes compared to those not treated with G-CSF [103]. Bacterial elimination is of utmost importance for the control of GVHD, so much that it has been described that germ-free animals develop an attenuated and very late form of GVHD [104]. Besides this, in our view, in this early phase after HSCT neutrophils polarize T cells in

Nevertheless, the long-lasting and specific protection cannot be explained only by neutrophils suppression as it lasts for several months and the half-life of neutrophils does not exceed 6 days. When analyzing the amount of Treg cells, in spleen and mesenteric lymph nodes (mLN) of the hosts, it was found that 4 days after HSCT, Tregs were increased on mLN but not on spleens. Moreover, 25 days after HSCT, both spleen and MLN show Treg increase, suggesting a systemic increase in Treg. In this way, when Treg cells are depleted early after the transplantation, the GVHD protection is abolished, showing Treg induction within the host right after transplantation. These specific suppressor effects are compatible with the antigen-specific suppression previously observed with Treg cells [97, 98]. The ability of neutrophils to suppress T cell activation is reinforced by the fact that Tregs are less sensitive

than conventional T cells to H2

158 Role of Neutrophils in Disease Pathogenesis

RN in GVHD model.

Given these various functions of the Ly6G+

and modulation of spleen neutrophils to an IL-10+

immunocompetence and the GVL effect (**Figure 4**).

coupled with the high production of H2

O2

may prevent the induction of the Th17 profile [100] (**Figure 3**).

suppression [99].

Indeed, it's known that G-CSF treatment increases the number of Treg cells in the donor, and that these cells produce IL-10 [93]. Treg cells stimulated with LPS are able to induce IL-10 and TGFβ-producing neutrophils. It means that Treg cells have anti-inflammatory properties that influence other cells, including neutrophils, which upon contact with LPS-stimulated Tregs,

IL-10+

possibilities, which may act together, can be suggested to explain the specific suppression by

At first, peroxide production can act by inhibiting T cell activation because its production is carried out with L-arginine consumption and because it acts by inhibiting TCRζ chain phosphorylation. Second, the high phagocytic capacity may contribute to the clearance of translocated gut bacteria after the conditioning regimen. The diminished bacterial load and translocation will contribute to diminished activation of allogeneic T cells leading to a mild GVHD. Third, Tregs generated in the donor after G-CSF treatment influence the generation

expresses low levels of MHC II and co-stimulation (as can be seen in **Figure 4**). After HSCT, when in contact with T cells, neutrophils with low levels of co-stimulatory molecules associated with high secretion of IL-10 favor the generation of Treg cells within the host that suppresses GVHD and maintains other functions of the immune system while maintaining

In addition to this, we believe that the high phagocytic capacity found in these neutrophils,

asation that occurs as a result of the barrier breaking [101]. It was recently described that neutrophils can colonize intestinal tissues 3 days after transplantation, so the Ly6G+

besides polarizing T cells, could maintain the intestinal integrity [102]. In fact, it has already been described that G-CSF decreases the effects of intestinal barrier breakage showing low levels of endotoxin in the blood and lower numbers of translocated bacteria observed in the spleen, liver, and mesenteric lymph nodes compared to those not treated with G-CSF [103]. Bacterial elimination is of utmost importance for the control of GVHD, so much that it has been described that germ-free animals develop an attenuated and very late form of GVHD [104]. Besides this, in our view, in this early phase after HSCT neutrophils polarize T cells in

O2

neutrophils described herein, at least three


cell,

, is related to the limitation of bacterial extrav-

**Figure 3.** Regulatory neutrophils IL10+ neutrophils generation. (1) G-CSF treatment of HSCT donors mobilizes neutrophils from the bone marrow to the periphery. (2) and (3) Neutrophils migrate to spleen where they found Treg cells also G-CSF stimulated. (4) In the spleen, neutrophils and T cells undergo mutual influence and polarize each other to a regulatory phenotype.

the mLN towards a regulatory phenotype, and these regulatory cells spread to GVHD target organs, as we suggest in **Figure 4**.

As we can conclude, the initial idea that neutrophils act only by responding rapidly to inflammatory stimuli and subsequently enter apoptosis causing tissue damage has been modified by the finding that activated neutrophils can perform many other functions. Studies in this regard challenge the classical view of neutrophils as fully differentiated cells and raise the question that these cells may exhibit extraordinary plasticity. In GVHD, prevention strategies that spare the recipient or that decrease tissue damage are of extreme importance [100]. In this case, the generation of a new subtype of neutrophils, capable of inhibiting GVHD maintaining the immunological competence, is paramount for the control of the disease and represents a simple approach to be applied in humans.

**Figure 4.** Potential role of regulatory neutrophils in GVHD onset. (1) After HSCT, regulatory neutrophils limit the bacterial translocation from the intestinal barrier breakdown, once they have high phagocytic capacity. (2) Production of the cytokine IL-22 contributes to epithelial healing. (3) High levels of hydrogen peroxide inhibit T effect or functions protecting the host from clonal expansion of alloreactive T cells. (4) The IL10 derived from neutrophils, polarize host Tregs and this last produces IL-10 and TGF-β comprising a mutual cycle. (5) Alternative macrophage (M2) differentiation can be influenced by the regulatory milieu.
