A novel BRET-Based GAP assay reveals phosphorylation-dependent regulation of the RAC-specific GTPase activating protein ARHGAP25.

ARHGAP25, a RAC-specific GTPase activating protein (GAP), is an essential regulator of phagocyte effector functions such as phagocytosis, superoxide production, and transendothelial migration. Furthermore, its complex role in tumor behavior has recently been recognized. We previously demonstrated that phosphorylation of serine 363 in ARHGAP25 regulates hematopoietic stem cells and progenitor cells in mouse bone marrow. However, the significance of other potential phosphorylation sites of ARHGAP25 remained unknown. Now, we developed a novel, real-time bioluminescence resonance energy transfer (BRET) assay to monitor the GAP activity of ARHGAP25 in vitro. Using this approach, we revealed that phosphorylation of S363 and S488, but not that of S379-380, controls ARHGAP25's RACGAP activity. On the other hand, we found in granulocyte-differentiated human PLB-985 cells that superoxide production and actin depolymerization are regulated by residues S363 and S379-380. The present data demonstrate the value of our BRET-GAP assay and show that different phosphorylation patterns regulate ARHGAP25's GAP activity and its effect on superoxide production and phagocytosis.

Inhibition and termination of physiological responses by GTPase activating proteins.

Physiological processes are strictly organized in space and time. However, in cell physiology research, more attention is given to the question of space rather than to time. To function as a signal, environmental changes must be restricted in time; they need not only be initiated but also terminated. In this review, we concentrate on the role of one specific protein family involved in biological signal termination. GTPase activating proteins (GAPs) accelerate the endogenously low GTP hydrolysis rate of monomeric guanine nucleotide-binding proteins (GNBPs), limiting thereby their prevalence in the active, GTP-bound form. We discuss cases where defective or excessive GAP activity of specific proteins causes significant alteration in the function of the nervous, endocrine, and hemopoietic systems, or contributes to development of infections and tumors. Biochemical and genetic data as well as observations from human pathology support the notion that GAPs represent vital elements in the spatiotemporal fine tuning of physiological processes.

Phosphoproteomic profiling of mouse primary HSPCs reveals new regulators of HSPC mobilization.

Protein phosphorylation is a central mechanism of signal transduction that both positively and negatively regulates protein function. Large-scale studies of the dynamic phosphorylation states of cell signaling systems have been applied extensively in cell lines and whole tissues to reveal critical regulatory networks, and candidate-based evaluations of phosphorylation in rare cell populations have also been informative. However, application of comprehensive profiling technologies to adult stem cell and progenitor populations has been challenging, due in large part to the scarcity of such cells in adult tissues. Here, we combine multicolor flow cytometry with highly efficient 3-dimensional high performance liquid chromatography/mass spectrometry to enable quantitative phosphoproteomic analysis from 200 000 highly purified primary mouse hematopoietic stem and progenitor cells (HSPCs). Using this platform, we identify ARHGAP25 as a novel regulator of HSPC mobilization and demonstrate that ARHGAP25 phosphorylation at serine 363 is an important modulator of its function. Our approach provides a robust platform for large-scale phosphoproteomic analyses performed with limited numbers of rare progenitor cells. Data from our study comprises a new resource for understanding the molecular signaling networks that underlie hematopoietic stem cell mobilization.

The neglected terminators: Rho family GAPs in neutrophils.

BACKGROUND: GTPase-activating proteins (GAPs) accelerate the rate of hydrolysis of GTP bound to small GTPases, thereby limiting the prevalence and concentration of the active, GTP-bound form of these proteins. The large number of potential GAPs acting on members of the Rho family of small GTPases raises the question of specificity or redundancy. RESULTS: In this review, we summarize experimental data obtained on the role of Rho family GAPs in neutrophils, highlight cases where more than one GAP is involved in a physiological function and show examples that GAPs can be involved not only in termination but also in initiation of cellular processes. We demonstrate that the expression-level regulation of GAPs may also occur in short-living cells such as neutrophils. Finally, we provide insight into the existence and structure of molecular complexes in which Rho family GAPs are involved. CONCLUSION: GAPs play more complex and varied roles than being simple terminators of cellular processes.

Rac GTPase Activating Protein ARHGAP25 Regulates Leukocyte Transendothelial Migration in Mice.

ARHGAP25 is a Rac-specific GTPase-activating protein that is expressed primarily in hematopoietic cells. The involvement of ARHGAP25 in regulating the recruitment of leukocytes to inflammatory sites was investigated in genetically modified mice. Using intravital microscopy, we show that Arhgap25 deficiency affects all steps of leukocyte recruitment with a predominant enhancement of transendothelial migration of neutrophilic granulocytes. Increased transmigration of Arhgap25-deficient leukocytes is demonstrated in inflamed cremaster muscle venules, in a peritonitis model, and in an in vitro chemotaxis assay. Using bone marrow chimeric mice lacking ARHGAP25 in the hematopoietic compartment, we show that enhanced migration in the absence of ARHGAP25 is due to defective leukocyte function. In search for potential mechanisms of ARHGAP25-regulated migration of neutrophils, we detected an increase in the amount of active, GTP-bound Rac and Rac-dependent cytoskeletal changes in the absence of ARHGAP25, suggesting a critical role of ARHGAP25 in counterbalancing the Rac-activating effect of nucleotide exchange factors. Taken together, using Arhgap25-deficient mice, we identified ARHGAP25 as a relevant negative regulator of leukocyte transendothelial migration.

The mitochondrial phosphate carrier: Role in oxidative metabolism, calcium handling and mitochondrial disease.

The mitochondrial phosphate carrier (PiC) is a mitochondrial solute carrier protein, which is encoded by SLC25A3 in humans. PiC delivers phosphate, a key substrate of oxidative phosphorylation, across the inner mitochondrial membrane. This transport activity is also relevant for allowing effective mitochondrial calcium handling. Furthermore, PiC has also been described to affect cell survival mechanisms via interactions with cyclophilin D and the viral mitochondrial-localized inhibitor of apoptosis (vMIA). The significance of PiC has been supported by the recent discovery of a fatal human condition associated with PiC mutations. Here, we present first the early studies that lead to the discovery and molecular characterization of the PiC, then discuss the very recently developed mouse models for PiC and pathological mutations in the human SLC25A3 gene.

Antibacterial effect of microvesicles released from human neutrophilic granulocytes.

Cell-derived vesicles represent a recently discovered mechanism for intercellular communication. We investigated their potential role in interaction of microbes with host organisms. We provide evidence that different stimuli induced isolated neutrophilic granulocytes to release microvesicles with different biologic properties. Only opsonized particles initiated the formation of microvesicles that were able to impair bacterial growth. The antibacterial effect of neutrophil-derived microvesicles was independent of production of toxic oxygen metabolites and opsonization or engulfment of the microbes, but depended on ß(2) integrin function, continuous actin remodeling, and on the glucose supply. Neutrophil-derived microvesicles were detected in the serum of healthy donors, and their number was significantly increased in the serum of bacteremic patients. We propose a new extracellular mechanism to restrict bacterial growth and dissemination.

ARHGAP25, a novel Rac GTPase-activating protein, regulates phagocytosis in human neutrophilic granulocytes.

Members of the Rac/Rho family of small GTPases play an essential role in phagocytic cells in organization of the actin cytoskeleton and production of toxic oxygen compounds. GTPase-activating proteins (GAPs) decrease the amount of the GTP-bound active form of small GTPases, and contribute to the control of biologic signals. The number of potential Rac/RhoGAPs largely exceeds the number of Rac/Rho GTPases and the expression profile, and their specific role in different cell types is largely unknown. In this study, we report for the first time the properties of full-length ARHGAP25 protein, and show that it is specifically expressed in hematopoietic cells, and acts as a RacGAP both in vitro and in vivo. By silencing and overexpressing the protein in neutrophil model cell lines (PLB-985 and CosPhoxFcγR, respectively) and in primary macrophages, we demonstrate that ARHGAP25 is a negative regulator of phagocytosis acting probably via modulation of the actin cytoskeleton.

Changing world of neutrophils.

Neutrophilic granulocytes are no longer regarded as cells involved only in the last phase of the immune response with one single-although vitally important-task: engulfing and killing of microorganisms marked by immunoglobulin or complement fragments. In recent years, it was shown that neutrophils are actively involved in initiation and organization of the adaptive immune response by releasing various cytokines, interacting with all major types of immune cells, regulating their own lifespan, and participating in the anaphylactic reaction and in several classically nonimmune functions such as hemostasis, atherogenesis, and even insulin resistance. The antibacterial effect is no longer restricted to killing and destruction of microorganisms sequestered in the phagosomal space. Bacteriostasis also occurs at certain locations of the extracellular space, by formation of neutrophil extracellular traps (NETs) that were shown in the last 2 years to have a significant role in the prevention of dissemination of microorganisms. Extracellular vesicles represent a recently discovered form of intercellular communication carried out both by lipids, proteins, and nucleic acids. In this review, we also summarize the role of neutrophil-derived extracellular vesicles in modifying the function of other cell types as well as their direct antibacterial effect that differs significantly from mechanisms applied either by neutrophils or by the NETs.

Mac-1 Receptor Clustering Initiates Production of Pro-Inflammatory, Antibacterial Extracellular Vesicles From Neutrophils.

Depending on the prevailing environmental conditions, neutrophilic granulocytes release extracellular vesicles (EV) which have either anti-inflammatory effects on other neutrophils or pro-inflammatory and antibacterial effects. In the present study we investigated the molecular mechanisms underlying the biogenesis of functionally heterogenic EVs. We show that selective stimulation of Mac-1 integrin (complement receptor 3) by specific ligands initiates the generation of EVs which are able to impair bacterial growth and to induce the secretion of the pro-inflammatory cytokine IL-8 (aEV). However, direct Mac-1 stimulation results in aEV release only if neutrophils were activated on ligand coated surfaces whereas soluble ligands are ineffective. Using total internal reflection fluorescence (TIRF) microcopy, an increased clustering of Mac-1 molecules could be visualized in neutrophils added to C3bi coated surfaces; moreover antibody induced cluster formation triggers aEV release as well. Mac-1 induced production of aEV apparently necessitates a strong calcium signal as it fully depends on the presence of extracellular calcium. However, initiation of a strong calcium signal by an ionophore only results the generation of EV devoid of any antibacterial or pro-inflammatory effect. Our results thus demonstrate that stimulation and clustering of Mac-1 is necessary and sufficient for initiation of aEV biogenesis. In contrast, an intracellular calcium signal is necessary but by itself not sufficient for the production of antibacterial and pro-inflammatory EVs.

MICy: a Novel Flow Cytometric Method for Rapid Determination of Minimal Inhibitory Concentration.

Early initiated adequate antibiotic treatment is essential in intensive care. Shortening the length of antibiotic susceptibility testing (AST) can accelerate clinical decision-making. Our objective was to develop a simple flow cytometry (FC)-based AST that produces reliable results within a few hours. We developed a FC-based AST protocol (MICy) and tested it on six different bacteria strains (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis) in Mueller-Hinton and Luria-Bertani broth. We monitored the bacterial growth by FC to define the optimal time of AST. All bacteria were tested against 12 antibiotics and the MIC values were compared to microdilution used as reference method. McNemar and Fleiss' kappa inter-observer tests were performed to analyze the bias between the two methods. Susceptibility profiles of the two methods were also compared. We found that FC is able to detect the bacterial growth after 4-h incubation. The point-by-point comparison of MICy and microdilution resulted in exact match above 87% (2642/3024) of all measurements. The MIC values obtained by MICy and microdilution agreed over 80% (173/216) within ±1 dilution range that gives a substantial inter-observer agreement with weighted Fleiss' kappa. By using the EUCAST clinical breakpoints, we defined susceptibility profiles of MICy that were identical to microdilution in more than 92% (197/213) of the decisions. MICy resulted 8.7% major and 3.2% very major discrepancies. MICy is a new, simple FC-based AST method that produces susceptibility profile with low failure rate a workday earlier than the microdilution method. IMPORTANCE MICy is a new, simple and rapid flow cytometry based antibiotic susceptibility testing (AST) method that produces susceptibility profile a workday earlier than the microdilution method or other classical phenotypic AST methods. Shortening the length of AST can accelerate clinical decision-making as targeted antibiotic treatment improves clinical outcomes and reduces mortality, duration of artificial ventilation, and length of stay in intensive care unit. It can also reduce nursing time and costs and the spreading of antibiotic resistance. In this study, we present the workflow and methodology of MICy and compare the results produced by MICy to microdilution step by step.

Neutrophils produce proinflammatory or anti-inflammatory extracellular vesicles depending on the environmental conditions.

Extracellular vesicles (EVs) are important elements of intercellular communication. A plethora of different, occasionally even opposite, physiologic and pathologic effects have been attributed to these vesicles in the last decade. A direct comparison of individual observations is however hampered by the significant differences in the way of elicitation, collection, handling, and storage of the investigated vesicles. In the current work, we carried out a careful comparative study on 3, previously characterized types of EVs produced by neutrophilic granulocytes. We investigated in parallel the modulation of multiple blood-related cells and functions by medium-sized vesicles. We show that EVs released from resting neutrophils exert anti-inflammatory action by reducing production of reactive oxygen species (ROS) and cytokine release from neutrophils. In contrast, vesicles generated upon encounter of neutrophils with opsonized particles rather promote proinflammatory processes as they increase production of ROS and cytokine secretion from neutrophils and activate endothelial cells. EVs released from apoptosing cells were mainly active in promoting coagulation. We thus propose that EVs are "custom made," acquiring selective capacities depending on environmental factors prevailing at the time of their biogenesis.

The oxidation state of phospholipids controls the oxidative burst in neutrophil granulocytes.

The activation of neutrophil granulocytes has to be carefully controlled to balance desired activity against invading pathogens while avoiding overwhelming activation leading to host tissue damage. We now show that phospholipids are potential key players in this process by either enhancing or dampening the production of reactive oxygen species (ROS) during the oxidative burst. Unoxidized phospholipids induce the production of ROS, and they also work synergistically with FMLP in potentiating the oxidative burst in neutrophil granulocytes. Oxidation of these phospholipids, however, turns them into potent inhibitors of the oxidative burst. OxPls specifically inhibit ROS production by inhibiting the assembly of the phagocyte oxidase complex but do not alter neutrophil viability, nor do they interfere with MAPK activation. Furthermore, up-regulation of the activation marker Mac-1 and phagocytosis of bacteria is not affected. Therefore, phospholipids may act as sensors of oxidative stress in tissues and either positively or negatively regulate neutrophil ROS production according to their oxidation state.

The Functional Heterogeneity of Neutrophil-Derived Extracellular Vesicles Reflects the Status of the Parent Cell.

Similar to other cell types, neutrophilic granulocytes also release extracellular vesicles (EVs), mainly medium-sized microvesicles/microparticles. According to published data, authors have reached a consensus on the physical parameters (size, density) and chemical composition (surface proteins, proteomics) of neutrophil-derived EVs. In contrast, there is large diversity and even controversy in the reported functional properties. Part of the discrepancy may be ascribed to differences in the viability of the starting cells, in eliciting factors, in separation techniques and in storage conditions. However, the most recent data from our laboratory prove that the same population of neutrophils is able to generate EVs with different functional properties, transmitting pro-inflammatory or anti-inflammatory effects on neighboring cells. Previously we have shown that Mac-1 integrin is a key factor that switches anti-inflammatory EV generation into pro-inflammatory and antibacterial EV production. This paper reviews current knowledge on the functional alterations initiated by neutrophil-derived EVs, listing their effects according to the triggering agents and target cells. We summarize the presence of neutrophil-derived EVs in pathological processes and their perspectives in diagnostics and therapy. Finally, the functional heterogeneity of differently triggered EVs indicates that neutrophils are capable of producing a broad spectrum of EVs, depending on the environmental conditions prevailing at the time of EV genesis.

Role of Mac-1 integrin in generation of extracellular vesicles with antibacterial capacity from neutrophilic granulocytes.

Production of extracellular vesicles (EVs) involved in intercellular communication is a common capacity of most cell types. Upon encountering opsonized microorganisms, neutrophilic granulocytes release EVs that compromise bacterial growth. We carried out a systematic investigation of the involvement of potential opsonin receptors in EV-generation from human and murine neutrophils. Applying flow cytometric, proteomic and functional analysis as well as using genetically modified mice, we demonstrate that formation of antibacterial EVs depends upon stimulation of the multifunctional Mac-1 integrin complex, also called as complement receptor 3 (CR3), whereas activation of immunoglobulin binding Fc receptors or pattern recognition receptors alone or in combination is ineffective. Mac-1/CR3 stimulation and downstream tyrosine kinase signalling affect both the numbers, the cargo content and the antibacterial capacity of the produced vesicles. In contrast, Mac-1/CR3 signalling is not required for spontaneous EV formation, clearly indicating the existence of separate molecular pathways in EV biogenesis. We propose that EVs are "tailor-made" with different composition and functional properties depending on the environmental circumstances.

Human Neutrophils Produce Antifungal Extracellular Vesicles against Aspergillus fumigatus.

Polymorphonuclear granulocytes (PMNs) are indispensable for controlling life-threatening fungal infections. In addition to various effector mechanisms, PMNs also produce extracellular vesicles (EVs). Their contribution to antifungal defense has remained unexplored. We reveal that the clinically important human-pathogenic fungus Aspergillus fumigatus triggers PMNs to release a distinct set of antifungal EVs (afEVs). Proteome analyses indicated that afEVs are enriched in antimicrobial proteins. The cargo and the release kinetics of EVs are modulated by the fungal strain confronted. Tracking of afEVs indicated that they associated with fungal cells and even entered fungal hyphae, resulting in alterations in the morphology of the fungal cell wall and dose-dependent antifungal effects. To assess as a proof of concept whether the antimicrobial proteins found in afEVs might contribute to growth inhibition of hyphae when present in the fungal cytoplasm, two human proteins enriched in afEVs, cathepsin G and azurocidin, were heterologously expressed in fungal hyphae. This led to reduced fungal growth relative to that of a control strain producing the human retinol binding protein 7. In conclusion, extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. This finding offers an intriguing, previously overlooked mechanism of antifungal defense against A. fumigatusIMPORTANCE Invasive fungal infections caused by the mold Aspergillus fumigatus are a growing concern in the clinic due to the increasing use of immunosuppressive therapies and increasing antifungal drug resistance. These infections result in high rates of mortality, as treatment and diagnostic options remain limited. In healthy individuals, neutrophilic granulocytes are critical for elimination of A. fumigatus from the host; however, the exact extracellular mechanism of neutrophil-mediated antifungal activity remains unresolved. Here, we present a mode of antifungal defense employed by human neutrophils against A. fumigatus not previously described. We found that extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. In the end, antifungal extracellular vesicle biology provides a significant step forward in our understanding of A. fumigatus host pathogenesis and opens up novel diagnostic and therapeutic possibilities.

Regulation of the substrate preference of p190RhoGAP by protein kinase C-mediated phosphorylation of a phospholipid binding site.

The Rho family GTPases are stringently regulated through the action of a large family of GTPase activating proteins (GAPs) that stimulate their relatively weak intrinsic GTP hydrolyzing activity. The p190RhoGAPs, which include the p190A and p190B proteins, are potent and widely expressed GAPs acting on both Rho and Rac GTPases. We have observed that several acidic phospholipids inhibit the RhoGAP activity and promote the RacGAP activity of p190 proteins. In liposome binding assays we have demonstrated that binding of p190A to phospholipids is controlled by electrostatic interactions. Using mapping techniques, we determined that a small polybasic peptide stretch within p190A is a common site for both the phospholipid binding and PKC phosphorylation. Moreover, PKC-mediated phosphorylation of two amino acids (serine-1221 and threonine-1226) within this region of p190A prevents the binding and substrate specificity regulation by phospholipids. Transfection of COS-7 cells with mutant forms of p190A either unable to bind to phospholipids or unable to become phosphorylated induced distinct morphological changes. Together, these findings reveal a novel GAP regulatory mechanism in which phosphorylation indirectly alters GTPase substrate preference by affecting the interaction with acidic phospholipids. Our observations provide a potential mechanism of Rac/Rho antagonism described in several cellular functions.

Different Calcium and Src Family Kinase Signaling in Mac-1 Dependent Phagocytosis and Extracellular Vesicle Generation.

Encountering opsonized particles by neutrophils results in phagocytosis of the particle and generation of extracellular vesicles with antibacterial property (aEV). The aim of the present study is to compare the involvement of different receptors and receptor-proximal signaling pathways in these two parallel processes. Investigating human neutrophils from peripheral blood, we show that complement receptors are decisive for both processes whereas immunoglobulin binding Fc receptors (FcR) only participate moderately in phagocytosis and pattern recognition receptors induce mild EV production but only minimal phagocytosis. Studying bone marrow derived neutrophils of genetically modified animals we verify that the involved complement receptor is CR3, also known as the ß2 integrin Mac-1. We show that genetic deletion of the adaptor molecules FcRγ chain or DAP12 does not influence either process, suggesting potential redundant function. Combined absence of the Src family kinases Hck, Fgr, and Lyn drastically impairs phagocytosis but does not influence aEV production. In contrast, deletion of PLCγ2 has no influence on phagocytosis, but reduces aEV formation. In accord with the essential role of PLCγ2, aEV biogenesis both from murine and from human neutrophils is dependent on presence of extracellular calcium. Absence of external calcium prevented the generation of antibacterial EVs, whereas the spontaneous EV formation was not influenced. We thus show that phagocytosis and biogenesis of antibacterial EVs are independent processes and proceed on different signaling pathways although the same receptor plays the critical role in both. Our data reveal the possibility in neutrophilic granulocytes to modulate aEV production without disturbing the phagocytic process.