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.

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.

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 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.

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.

New flow cytometry-based method for the assessment of the antibacterial effect of immune cells and subcellular particles.

Techniques currently used for assessment of bacterial count or growth are time-consuming, offer low throughput, or they are complicated or expensive. The aim of the present work was to elaborate a new method that is able to detect the antibacterial effect of cells, subcellular particles, and soluble compounds in a fast, cost, and labor effective way. Our proposed technique is based on flow cytometry (FC) optimized for detection of small particles and on fluorescently labeled bacteria. It allows direct determination of the bacterial count in 3 hours. The effect of various human phagocytes and extracellular vesicles on gram-positive and gram-negative bacteria is investigated in parallel with the new, FC-based method, with colony counting and with our previous, OD-based method. Comparing the killing effect of wild type and NADPH oxidase-deficient murine neutrophils presents an example of detection of a clinically important deficiency. Strong correlation was obtained between the results of the different techniques, but the reproducibility of the FC-based test was superior to the OD-based test. The major advantages of the new technique are: rapidity, low cost, high throughput, and simplicity.

Functionally and morphologically distinct populations of extracellular vesicles produced by human neutrophilic granulocytes.

EVs in the microvesicle size range released during spontaneous death of human neutrophils were characterized and their properties compared with previously described EVs with antibacterial effect (aEVs, generated on specific activation) or produced spontaneously (sEVs). The 3 vesicle populations overlapped in size and in part of the constituent proteins were stained with annexin V and were impermeable to PI. However, none of them produced superoxide. In contrast, remarkable differences were observed in the morphology, abundance of proteins, and antibacterial function. EVs formed spontaneously in 30 min (sEVs) were more similar to EVs released during spontaneous death in 1-3 d than to EVs formed in 30 min on stimulation of opsonin receptors (aEVs). Spontaneously generated EVs had no antibacterial effect despite their large number and protein content. We hypothesized 2 parallel mechanisms: one that proceeds spontaneously and produces EVs without antibacterial effect and another process that is triggered by opsonin receptors and results in differential sorting of proteins into EVs with antibacterial capacity. Our results call attention to the functional and morphologic heterogeneity within the microvesicle/ectosome fraction of EVs.

Effect of storage on physical and functional properties of extracellular vesicles derived from neutrophilic granulocytes.

AIM: To carry out a systematic study on the effect of different storage conditions on the number as well as the physical and functional properties of antibacterial extracellular vesicles (EVs) derived from human neutrophilic granulocytes. METHODS: Production of EVs with antibacterial properties was initiated by opsonized Zymosan A particles. The number of released fluorescent EVs was determined by flow cytometry following careful calibration. Physical properties and size of EVs were investigated by flow cytometry, dynamic light scattering and electron microscopy. Functional properties of EVs were tested by bacterial survival assay. RESULTS: Storage at +20°C or +4°C resulted in a significant decrease of EV number and antibacterial effect after 1 day. Storage at -20°C did not influence the EV number up to 28 days, but induced a shift in EV size and almost complete loss of antibacterial function by 28 days. Storage at -80°C had no significant effect either on EV number or size and allowed partial preservation of the antibacterial function up to 28 days. Snap-freezing did not improve the results, whereas the widely used cryoprotectants induced EV lysis. CONCLUSION: Storage significantly alters both the physical and functional properties of EVs even if the number of EVs stays constant. If storage is needed, EVs should be kept at -80°C, preferably not longer than 7 days. For functional tests, freshly prepared EVs are recommended.

Role of Rac GTPase activating proteins in regulation of NADPH oxidase in human neutrophils.

Precise spatiotemporal regulation of O2(-)-generating NADPH oxidases (Nox) is a vital requirement. In the case of Nox1-3, which depend on the small GTPase Rac, acceleration of GTP hydrolysis by GTPase activating protein (GAP) could represent a feasible temporal control mechanism. Our goal was to investigate the molecular interactions between RacGAPs and phagocytic Nox2 in neutrophilic granulocytes. In structural studies we revealed that simultaneous interaction of Rac with its effector protein p67(phox) and regulatory protein RacGAP was sterically possible. The effect of RacGAPs was experimentally investigated in a cell-free O2(-)-generating system consisting of isolated membranes and recombinant p47(phox) and p67(phox) proteins. Addition of soluble RacGAPs decreased O2(-) production and there was no difference in the effect of four RacGAPs previously identified in neutrophils. Depletion of membrane-associated RacGAPs had a selective effect: a decrease in ARHGAP1 or ARHGAP25 level increased O2(-) production but a depletion of ARHGAP35 had no effect. Only membrane-localized RacGAPs seem to be able to interact with Rac when it is assembled in the Nox2 complex. Thus, in neutrophils multiple RacGAPs are involved in the control of O2(-) production by Nox2, allowing selective regulation via different signaling pathways.

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.

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.