Regulatory T cells contribute to the immunosuppressive phenotype of neutrophils in a mouse model of chronic lymphocytic leukemia.

BACKGROUND: Impaired neutrophil activity is an important issue in chronic lymphocytic leukemia (CLL), as it contributes to a dysfunctional immune response leading to life-threatening infections in patients. Some features typical of CLL neutrophils, e.g., the B-cell-supportive secretion profile, have already been described. However, most of these studies were performed on cells isolated from peripheral blood. It is still unclear which molecular factors and cell types are involved in shaping neutrophil function and phenotype in the CLL microenvironment. Since regulatory T cells (Treg) play an important role in CLL progression and influence the activity of neutrophils, we investigated the crosstalk between Treg and neutrophils in the spleen using a murine model of CLL. METHODS: In this work, we used an Eµ-TCL1 mouse model of human CLL. For our in vivo and ex vivo experiments, we inoculated wild-type mice with TCL1 leukemic cells isolated from Eµ-TCL1 transgenic mice and then monitored disease progression by detecting leukemic cells in peripheral blood. We analyzed both the phenotype and activity of neutrophils isolated from the spleens of TCL1 leukemia-bearing mice. To investigate the interrelation between Treg and neutrophils in the leukemia microenvironment, we performed experiments using TCL1-injected DEREG mice with Treg depletion or RAG2KO mice with adoptively transferred TCL1 cells alone or together with Treg. RESULTS: The obtained results underline the plasticity of the neutrophil phenotype, observed under the influence of leukemic cells alone and depending on the presence of Treg. In particular, Treg affect the expression of CD62L and IL-4 receptor in neutrophils, both of which are crucial for the function of these cells. Additionally, we show that Treg depletion and IL-10 neutralization induce changes in the leukemia microenvironment, partially restoring the "healthy" phenotype of neutrophils. CONCLUSIONS: Altogether, the results indicate that the crosstalk between Treg and neutrophils in CLL may play an important role in CLL progression by interfering with the immune response.

Secretomes of M1 and M2 macrophages decrease the release of neutrophil extracellular traps.

The release of neutrophil extracellular traps (NETs) can be either beneficial or detrimental for the host, thus it is necessary to maintain a balance between formation and clearance of NETs. Multiple physiological factors eliciting NET release have been identified, yet the studies on natural signals limiting NET formation have been scarce. Accordingly, our aim was to analyze whether cytokines or immune cells can inhibit NET formation. To that end, human granulocytes were incubated with interleukin (IL)-4, IL-10, transforming growth factor beta-2 or adenosine and then stimulated to release NETs. Additionally, neutrophils were cultured in the presence of natural killer (NK) cells, regulatory T cells (Tregs), pro-inflammatory or anti-inflammatory macrophages (M1 or M2 macrophages), or in the presence of NK/Tregs/M1 macrophages or M2 macrophages-conditioned medium and subsequently stimulated to release NETs. Our studies showed that secretome of M1 and M2 macrophages, but not of NK cells and Tregs, diminishes NET formation. Co-culture experiments did not reveal any effect of immune cells on NET release. No effect of cytokines or adenosine on NET release was found. This study highlights the importance of paracrine signaling at the site of infection and is the first to show that macrophage secretome can regulate NET formation.

Lack of Functional P110δ Affects Expression of Activation Marker CD80 but Does Not Influence Functions of Neutrophils.

Neutrophils are specialized immune cells that are essential constituents of the innate immune response. They defend the organism against pathogens through various mechanisms. It was reported that phosphatidylinositols are key players in neutrophil functions, especially in the activity of class-I phosphoinositide 3-kinases (PI3Ks). P110δ, one of the PI3K subunits, is mostly expressed in immune cells, and its activity plays an important role in inflammatory responses. The aim of this study was to investigate the role of p110δ in neutrophil antimicrobial functions, activation status and cytokine production. To this end, we used bone marrow and splenic neutrophils isolated from a murine model expressing catalytically inactive p110δD910A/D910A. The level of phagocytosis and degranulation, the expressions of activation markers and cytokine production were determined by flow cytometry. ROS generation and NET release were assessed by fluorometry and fluorescent microscopy. We observed a significantly higher percentage of CD80-positive cells among the splenic granulocytes and found granulocytes subpopulations of differing phenotypes between WT and p110δD910A/D910A mice by multiparametric tSNE analysis. Moreover, we detected some differences in the expressions of activation markers, intracellular production of cytokines and bacterial killing. However, we did not observe any alterations in the selected neutrophil functions in p110δ mutant mice. Altogether, our data suggest that the catalytic p110 subunit(s), other than p110δ, is a key player in most neutrophil functions in mice. A follow-up study to correlate these in vitro results with in vivo observations is highly recommended.

Iron excess affects release of neutrophil extracellular traps and reactive oxygen species but does not influence other functions of neutrophils.

Neutrophils apply several antimicrobial strategies including degranulation, phagocytosis, the generation of reactive oxygen species (ROS) and the release of neutrophil extracellular traps (NETs) to fight pathogens. Iron is considered to be an invaluable constituent of host immune defense and plays a dual role in immunity. It is a well-known component of antimicrobial proteins and is a necessary microelement for pathogen survival. The aim of this study was to broaden the knowledge regarding the impact of iron on the function of neutrophils. Neutrophils from healthy blood donors and patients with mild iron-deficiency anemia and HL-60 cells differentiated toward granulocyte-like cells were incubated with Fe2+ , Fe3+ or holo-transferrin (holo-Tf). Moreover, we isolated murine neutrophils of HFE gene knockout (KO) mice and mice fed iron-deficient, iron-equivalent and high-iron diets. We analyzed the release of NETs, phagocytosis, degranulation of azurophilic granules, ROS release, bactericidal activity of granulocytes against Escherichia coli and neutrophil elastase (NE) activity. We show that holo-Tf inhibits the release of NETs stimulated by phorbol 12-myristate 13-acetate by inhibiting NE activity. Studies performed in mice models reveal that iron overload inhibits the release of NETs and ROS production in neutrophils isolated from HFE KO mice and mice fed a high-iron diet. No impact of a low-iron diet on neutrophil phagocytosis, ROS production or release of NETs was observed. Our study underscores the physiological significance of iron in neutrophil function, specifically in the release of NETs.

Zinc Supplementation Modulates NETs Release and Neutrophils' Degranulation.

Zinc plays an important physiological role in the entire body, especially in the immune system. It is one of the most abundant microelements in our organism and an essential component of enzymes and antibacterial proteins. Zinc levels were reported to be correlated with the intensity of innate immunity responses, especially those triggered by neutrophils. However, as the results are fragmentary, the phenomenon is still not fully understood and requires further research. In this study, we aimed to perform a comprehensive assessment and study the impact of zinc on several basic neutrophils' functions in various experimental setups. Human and murine neutrophils were preincubated in vitro with zinc, and then phagocytosis, oxidative burst, degranulation and release of neutrophil extracellular traps (NETs) were analyzed. Moreover, a murine model of zinc deficiency and zinc supplementation was introduced in the study and the functions of isolated cells were thoroughly studied. We showed that zinc inhibits NETs release as well as degranulation in both human and murine neutrophils. Our study revealed that zinc decreases NETs release by inhibiting citrullination of histone H3. On the other hand, studies performed in zinc-deficient mice demonstrated that low zinc levels result in increased release of NETs and enhanced neutrophils degranulation. Overall, it was shown that zinc affects neutrophils' functions in vivo and in vitro. Proper zinc level is necessary to maintain efficient functioning of the innate immune response.

Overexpression of ATG5 Gene Makes Granulocyte-Like HL-60 Susceptible to Release Reactive Oxygen Species.

Neutrophils represent the first line of defense against pathogens using various strategies, such as phagocytosis, production of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. Recently, an autophagy-independent role of autophagy related (ATG) gene 5 in immune cells, including neutrophils, was emphasized. Our aim was to investigate the role of ATG5 protein in neutrophils' antimicrobial functions, proliferation and apoptosis. To this end, we used genetically modified human promyelocytic leukemia (HL-60) cells overexpressing ATG5, differentiated toward granulocyte-like cells with all-trans retinoic acid (ATRA) and dimethylformamide. The level of differentiation, phagocytosis, proliferation and apoptosis were determined by flow cytometry. ROS production and NETs release was assessed by fluorometry and fluorescent microscopy. ATG5 gene expression was evaluated by real-time PCR, whereas the protein level of ATG5 and LC3-II was determined by Western blot. We did not observe the induction of autophagy in differentiated HL-60 cells overexpressing ATG5. The increased expression of ATG5 affects the differentiation of HL-60 cells with ATRA, ROS production and phagocytosis. However, we did not detect changes in NETs release. Moreover, ATG5 protects differentiated HL-60 cells from apoptosis but does not cause changes in proliferation rate.

Convolutional Neural Networks-Based Image Analysis for the Detection and Quantification of Neutrophil Extracellular Traps.

Over a decade ago, the formation of neutrophil extracellular traps (NETs) was described as a novel mechanism employed by neutrophils to tackle infections. Currently applied methods for NETs release quantification are often limited by the use of unspecific dyes and technical difficulties. Therefore, we aimed to develop a fully automatic image processing method for the detection and quantification of NETs based on live imaging with the use of DNA-staining dyes. For this purpose, we adopted a recently proposed Convolutional Neural Network (CNN) model called Mask R-CNN. The adopted model detected objects with quality comparable to manual counting-Over 90% of detected cells were classified in the same manner as in manual labelling. Furthermore, the inhibitory effect of GW 311616A (neutrophil elastase inhibitor) on NETs release, observed microscopically, was confirmed with the use of the CNN model but not by extracellular DNA release measurement. We have demonstrated that a modern CNN model outperforms a widely used quantification method based on the measurement of DNA release and can be a valuable tool to quantitate the formation process of NETs.

Nitric oxide and peroxynitrite trigger and enhance release of neutrophil extracellular traps.

Despite great interest, the mechanism of neutrophil extracellular traps (NETs) release is not fully understood and some aspects of this process, e.g. the role of reactive nitrogen species (RNS), still remain unclear. Therefore, our aim was to investigate the mechanisms underlying RNS-induced formation of NETs and contribution of RNS to NETs release triggered by various physiological and synthetic stimuli. The involvement of RNS in NETs formation was studied in primary human neutrophils and differentiated human promyelocytic leukemia cells (HL-60 cells). RNS (peroxynitrite and nitric oxide) efficiently induced NETs release and potentiated NETs-inducing properties of platelet activating factor and lipopolysaccharide. RNS-induced NETs formation was independent of autophagy and histone citrullination, but dependent on the activity of phosphoinositide 3-kinases (PI3K) and myeloperoxidase, as well as selective degradation of histones H2A and H2B by neutrophil elastase. Additionally, NADPH oxidase activity was required to release NETs upon stimulation with NO, as shown in NADPH-deficient neutrophils isolated from patients with chronic granulomatous disease. The role of RNS was further supported by increased RNS synthesis upon stimulation of NETs release with phorbol 12-myristate 13-acetate and calcium ionophore A23187. Scavenging or inhibition of RNS formation diminished NETs release triggered by these stimuli while scavenging of peroxynitrite inhibited NO-induced NETs formation. Our data suggest that RNS may act as mediators and inducers of NETs release. These processes are PI3K-dependent and ROS-dependent. Since inflammatory reactions are often accompanied by nitrosative stress and NETs formation, our studies shed a new light on possible mechanisms engaged in various immune-mediated conditions.