Neutrophils provide rapid and efficient defense mechanisms against invading pathogens. Upon stimulation with proinflammatory mediators, including complement factors and bacterial peptides, neutrophils respond with cellular changes in their membrane potential, intracellular pH, and cellular size. This study provides an approach to quantify these changes simultaneously using multiparametric flow cytometry, thereby revealing a typical sequence of neutrophil activation consisting of depolarization, alkalization, and increase in cellular size. Additionally, the time resolution of the flow cytometric measurement is improved in order to allow changes that occur within seconds to be monitored, and thus to enhance the kinetic analysis of the neutrophil response. The method is appropriate for the reliable semiquantitative detection of small variations with respect to an increase, no change, and decrease in those parameters as demonstrated by the screening of various proinflammatory mediators. As a translational outlook, the findings are put into context in inflammatory conditions in vitro as well as in a clinically relevant whole blood model of endotoxemia. Taken together, the multiparametric analysis of neutrophil responsiveness regarding depolarization, alkalization, and changes in cellular size may contribute to a better understanding of neutrophils in health and disease, thus potentially yielding innovative mechanistic insights and possible novel diagnostic and/or prognostic approaches.

The cellular and fluid phase-innate immune responses of many diseases predominantly involve activated neutrophil granulocytes and complement factors. However, a comparative systematic analysis of the early impact of key soluble complement cleavage products, including anaphylatoxins, on neutrophil granulocyte function is lacking. Neutrophil activity was monitored by flow cytometry regarding cellular (electro-)physiology, cellular activity, and changes in the surface expression of activation markers. The study revealed no major effects induced by C3a or C4a on neutrophil functions. By contrast, exposure to C5a or C5a des-Arg stimulated neutrophil activity as reflected in changes in membrane potential, intracellular pH, glucose uptake, and cellular size. Similarly, C5a and C5a des-Arg but no other monitored complement cleavage product enhanced phagocytosis and reactive oxygen species generation. C5a and C5a des-Arg also altered the neutrophil surface expression of several complement receptors and neutrophil activation markers, including C5aR1, CD62L, CD10, and CD11b, among others. In addition, a detailed characterization of the C5a-induced effects was performed with a time resolution of seconds. The multiparametric response of neutrophils was further analyzed by a principal component analysis, revealing CD11b, CD10, and CD16 to be key surrogates of the C5a-induced effects. Overall, we provide a comprehensive insight into the very early interactions of neutrophil granulocytes with activated complement split products and the resulting neutrophil activity. The results provide a basis for a better and, importantly, time-resolved and multiparametric understanding of neutrophil-related (patho-)physiologies.

La deuxième édition de la Journée des Jeunes Chercheurs organisée par la Commission de la Recherche Translationnelle de la SRLF a eu lieu en distanciel Paris le 21 janvier 2021. Cette journée a offert aux jeunes acteurs de la Médecine Intensive Réanimation l’opportunité de présenter leurs travaux  de  recherche  en  Master  2  ou  Thèse  d’université  à  un  collège  de  chercheurs  experts.  Pour  cette  deuxième Journée, Les professeurs Pierre Asfar (Angers), Bruno Levy (Nancy), Steve Lancel (Lille) et Fabio Taccone (Bruxelle) ont accepté de venir apporter aux jeunes candidats leur soutien critique et bienveillant. Les présentations couvrant les domaines des neurosciences à l’immunologie en passant par le métabolisme, l’hémodynamique ou la pharmacologie ont illustré la richesse et la diversité de la recherche translationnelle en réanimation.