Isolation methods determine human neutrophil responses after stimulation

Studying neutrophils is challenging due to their limited lifespan, inability to proliferate, and resistance to genetic manipulation. Neutrophils can sense various cues, making them susceptible to activation by blood collection techniques, storage conditions, RBC lysis, and the isolation procedure itself. Here we assessed the impact of the five most used methods for neutrophil isolation on neutrophil yield, purity, activation status and responsiveness. We monitored surface markers, reactive oxygen species production, and DNA release as a surrogate for neutrophil extracellular trap (NET) formation. Our results show that neutrophils isolated by negative immunomagnetic selection and density gradient methods, without RBC lysis, resembled untouched neutrophils in whole blood. They were also less activated and more responsive to milder stimuli in functional assays compared to neutrophils obtained using density gradients requiring RBC lysis. Our study highlights the importance of selecting the appropriate method for studying neutrophils, and underscores the need for standardizing isolation protocols to facilitate neutrophil subset characterization and inter-study comparisons

Démêler les NETs : activation des neutrophiles en physiopathologie - des déclencheurs aux cibles

Human neutrophils are the most abundant white blood cells and are at the forefront of defending our body against infections. One of their most intriguing antimicrobial mechanisms is the ejection of net-like DNA structures decorated with antimicrobial nuclear and granular proteins, called neutrophil extracellular traps (NETs), that can trap and possibly kill pathogens. On the other hand, the inappropriate release of NETs by neutrophils bares a large potential for host damage, contributing to autoimmune and inflammatory disease. Therefore, gaining a deep understanding of NET formation may pave the way for novel therapeutic strategies and advance our knowledge of such diseases. While the association of NETs with pathologies has been repeatedly documented, the roles of many NET stimuli, signal transduction molecules, and cellular events leading to NET formation remain controversial. During my thesis, I aimed to untangle the NETs by precisely understanding how NETs are generated: What stimuli robustly induce NETs? What intracellular pathways and cellular events are involved? And, how do NET compositions differ depending on the trigger? Very early it became clear that the isolation method used to obtain human neutrophils for my studies largely impacted the outcome of my experiments. I therefore started to compare the five most commonly used neutrophil isolation methods and demonstrated that only neutrophils in a low-activation state, resembling "untouched" neutrophils from blood can respond to biological NETosis stimuli and undergo lytic cell. Thus, the use of different neutrophil isolation protocols and methods may partially explain inter-study discrepancies in response to the same NETosis stimuli. The results underscore the need for standardized neutrophil isolation protocols. I then undertook an in-depth characterization on NET generation. I first compared 31 reported NETosis stimuli side-by-side to identify a set of biologically relevant stimuli. Surprisingly, only 12 (39%) robustly induced NETosis in our settings. To investigate the underlying molecular pathways, I then used inhibitors of various intracellular signaling molecules, which allowed me to identify several distinct NETosis pathways. To investigate the cellular events leading to NET formation, I performed confocal time-lapse microscopy on neutrophils labeled with fluorescent markers of organelles and stimulated with defined stimuli. Neutrophils undergo a series of events that can include ER vesiculation, chromatin decondensation, progressive plasma membrane permeabilization, and finally plasma membrane rupture with release of extracellular DNA, that depends on the stimuli used and varies in its intensity and kinetics. Finally, I performed mass spectrometry analysis to determine the composition of isolated NETs. My preliminary results seem to indicate that different NETosis stimuli induce a specific "NETome" similar to a fingerprint. Taken together, my thesis contributes to a better understanding of the process of NET formation and highlights the necessity to comparability of research conditions. The new knowledge will help to advance diagnostic and possibly therapeutic approaches for NET-related diseases.Les neutrophiles humains sont les leucocytes les plus abondants et sont en première ligne pour défendre notre corps contre les infections. L'un de leurs mécanismes antimicrobiens les plus intrigants est l'éjection de filaments d'ADN en forme de filet, ornés de protéines nucléaires et granulaires antimicrobiennes. Ces structures, appelées pièges extracellulaires des neutrophiles (NETs), servent à piéger puis à tuer les agents pathogènes. Le revers de la médaille est que ces propriétés antimicrobiennes sont également potentiellement toxiques pour l'hôte lorsque les neutrophiles libèrent des NETs de manière inappropriée, contribuant ainsi aux maladies auto-immunes et inflammatoires. Par conséquent, une compréhension approfondie de la formation des NETs pourrait ouvrir la voie à de nouvelles stratégies thérapeutiques et enrichir nos connaissances sur ces maladies. Bien que l'association des NETs avec un grand nombre de pathologies ait été documentée à maintes reprises, la nature des agents déclencheurs des NETs, des signaux de transduction et des événements cellulaires impliqués reste sujet à débat. Durant ma thèse, j'ai cherché à « démêler les NETs » en investiguant précisément comment ils sont générés: -quels stimuli induisent les NETs de manière robuste -quelles voies intracellulaires et quels événements cellulaires sont impliqués -comment la composition des NETs varie en fonction du déclencheur Assez rapidement dans ma thèse, il est devenu évident que la méthode d'isolement utilisée pour obtenir les neutrophiles humains influençait grandement les résultats de mes expériences. J'ai donc commencé par comparer les cinq méthodes d'isolement des neutrophiles les plus couramment utilisées. J¿ai pu démontrer que seuls les neutrophiles faiblement activés, ressemblant aux neutrophiles "intacts" du sang, peuvent répondre et subir une mort cellulaire lytique (en tant qu'indicateur de NETose) en réponse aux stimuli biologiques de la NETose. Ainsi, l'utilisation de différents protocoles et méthodes d'isolement des neutrophiles pourrait en partie expliquer les divergences entre études en réponse aux mêmes stimuli de NETose. Les résultats soulignent la nécessité de protocoles d'isolement des neutrophiles standardisés. J'ai ensuite entrepris une caractérisation approfondie de la génération de NET. J'ai d'abord comparé 31 stimuli de NETose publiés pour identifier un ensemble de stimuli biologiquement pertinents. Étonnamment, seulement 12 (39%) ont induit la NETose de manière robuste dans nos conditions. Pour étudier les voies moléculaires sous-jacentes, j'ai ensuite utilisé des inhibiteurs de diverses molécules de signalisation intracellulaire, ce qui m'a permis d'identifier quatre voies différentes de NETose. Pour étudier les événements cellulaires menant à la formation de NET, j'ai utilisé une approche par microscopie à balayage confocal en temps réel sur des neutrophiles marqués avec des marqueurs fluorescents des organites et stimulés avec des stimuli définis. Selon la stimulation et en fonction du déclencheur, les neutrophiles subissent une série d'événements qui peuvent inclure une vésiculation du réticulum endoplasmique, une décondensation de la chromatine, une perméabilisation progressive de la membrane plasmique, et finalement une rupture de la membrane plasmique avec libération d'ADN extracellulaire. Cette séquence d'événements cellulaires varie en intensité et en cinétique en fonction des stimuli. Enfin, pour déterminer si différents stimuli de NET conduisent à des NET avec une composition spécifique au stimulus, j'ai effectué une analyse par spectrométrie de masse « label-free ». Mes résultats préliminaires semblent indiquer que différents stimuli de NETose induisent un "NETome" spécifique, semblable à une empreinte digitale. Dans l'ensemble, ma thèse contribue à une meilleure compréhension du processus de formation des NETs et de la biologie des neutrophiles in vitro, ainsi qu'à la comparabilité des condition

The role of neutrophils in antibody-driven autoimmune cytopenias

International audienceAutoimmune cytopenias are a consequence of autoantibodies that target blood cell lineages and mark them for their accelerated destruction, mostly through phagocytosis by monocytes and macrophages and complement activation. Neutrophils, although equipped with Fc and complement receptors and effector mechanisms that are critical in other autoimmune conditions, remained long time overlooked. Recent reports however propose a new and possibly critical role for neutrophils. In this review we gathered available evidences on the contribution of neutrophils to the development, onset and consequences of autoantibody-dependent cytopenias

DataSheet_1_Isolation methods determine human neutrophil responses after stimulation.docx

Studying neutrophils is challenging due to their limited lifespan, inability to proliferate, and resistance to genetic manipulation. Neutrophils can sense various cues, making them susceptible to activation by blood collection techniques, storage conditions, RBC lysis, and the isolation procedure itself. Here we assessed the impact of the five most used methods for neutrophil isolation on neutrophil yield, purity, activation status and responsiveness. We monitored surface markers, reactive oxygen species production, and DNA release as a surrogate for neutrophil extracellular trap (NET) formation. Our results show that neutrophils isolated by negative immunomagnetic selection and density gradient methods, without RBC lysis, resembled untouched neutrophils in whole blood. They were also less activated and more responsive to milder stimuli in functional assays compared to neutrophils obtained using density gradients requiring RBC lysis. Our study highlights the importance of selecting the appropriate method for studying neutrophils, and underscores the need for standardizing isolation protocols to facilitate neutrophil subset characterization and inter-study comparisons.</p

Specificity of mouse and human Fcgamma receptors and their polymorphic variants for IgG subclasses of different species

International audienceImmunoglobulin G (IgG) is the predominant antibody class generated during infections and used for the generation of therapeutic antibodies. Antibodies are mainly characterized in or generated from animal models that support particular infections, respond to particular antigens or allow the generation of hybridomas. Due to the availability of numerous transgenic mouse models and the ease of performing bioassays with human blood cells in vitro, most antibodies from species other than mice and humans are tested in vitro using human cells and/or in vivo using mice. In this process, it is expected, but not yet systematically documented, that IgG from these species interact with human or mouse IgG receptors (FcRs). In this study, we undertook a systematic assessment of binding specificities of IgG from various species to the families of mouse and human FcRs, including their polymorphic variants. Our results document the specific binding patterns for each of these IgG (sub)classes, reveal possible caveats of antibody-based immunoassays, and will be a useful reference for the transition from one animal model to preclinical mouse models or human cell-based bioassays