NEUTROXIA

Neutrophils are the most abundant circulating white blood cells. Due to their short lifespan in vitro (<8h), neutrophils were not well studied and remain probably the most mysterious white blood cell population. Their lifespan in vivo under basal conditions is not clearly stated. Neutrophil transfusion in neutropenic patients with multiresistant bacteria infection stands as the last therapeutic option but remains inefficient. A better comprehension of their physiology is urgently needed. Neutrophil differentiate from hematopoietic stem cells in the bone marrow. Circulating neutrophils represent only 5-10% of the reserve, which remains mainly stored in the bone marrow until its massive mobilization, stimulated by bacterial infections. In this project, the model of infection is the shigellosis (colonic mucosa destruction), induced by the pathogenic enterobacteria Shigella, since 1/ neutrophils are the most abundant immune cell recruited to infected tissue, although their antimicrobial activity is not described, 2/ the pathophysiology of the disease is not fully understood and consequently no licensed pan-Shigella vaccine is available. During their lifecycle, neutrophils evolve under low oxygen conditions (hypoxic bone marrow, quasi-anoxic blood plasma fraction – 98% oxygen is transported by red blood cells), until they transmigrate to infected organs. Oxygen exposure is hypothesized here to play an essential role in neutrophil activation and ultimately cell death induction (inflammation resolution). The oxygen-dependent modulation of neutrophil physiology is crucial, since we demonstrated that hypoxia was induced in Shigella foci of infection, not when bacteria were dispersed in the colonic mucosa, hence neutrophil face Shigella either under normoxic or hypoxic conditions in vivo. The potential oxygen-dependent modulation of neutrophil senescence is not described; the cell death induction has not been well studied or restricted to caspase-dependent apoptosis. In order to address this question, and according to these physiological considerations, a novel method of neutrophil purification and manipulation was set up under anoxic conditions. Neutrophils’ lifespan in vitro was extended; their glycolytic capacity was preserved; controlled oxygen exposure was further allowed. Preliminary results were obtained with this original breakthrough procedure. We confirmed that oxygen exposure induced neutrophil cell death and also demonstrated that mitochondria biogenesis was induced. Neutrophil were so far described as cells containing few mitochondria with a function restricted to the induction of the caspase-dependent apoptosis. Our preliminary results show that other signaling pathways (ie. caspase-indenpent) may be involved. Consequently, in this project we will study the role of mitochondria in neutrophil activation and cell death induction upon oxygen exposure. The first expected result of this project is a better comprehension of the adaptation of neutrophil to oxygen exposure, which is hypothesized to be essential for its antimicrobial functions and subsequent cell death (essential for the inflammation resolution). The second expected result is to exploit the novel anoxic manipulation of neutrophils in vitro to re-define their lifespan in vivo under basal conditions and to pave the way for improving their therapeutic use.