Whereas many phagocytosis steps involve ionic fluxes, the underlying ion channels remain poorly defined. As reported in mice, the calcium conducting TRPV2 channel impacts the phagocytic process. Macrophage phagocytosis is critical for defense against pathogens. In cystic fibrosis (CF), macrophages have lost their capacity to act as suppressor cells and thus play a significant role in the initiating stages leading to chronic inflammation/infection. In a previous study, we demonstrated that impaired function of CF macrophages is due to a deficient phagocytosis. The aim of the present study was to investigate TRPV2 role in the phagocytosis capacity of healthy primary human macrophage by studying its activity, its membrane localization and its recruitment in lipid rafts. In primary human macrophages, we showed that P. aeruginosa recruits TRPV2 channels at the cell surface and induced a calcium influx required for bacterial phagocytosis. We presently demonstrate that to be functional and play a role in phagocytosis, TRPV2 might require a preferential localization in lipid rafts. Furthermore, CF macrophage displays a perturbed calcium homeostasis due to a defect in TRPV2. In this context, deregulated TRPV2-signaling in CF macrophages could explain their defective phagocytosis capacity that contribute to the maintenance of chronic infection.

Cystic fibrosis (CF) is an inherited disease that is characterised by susceptibility to bacterial infections and chronic lung inflammation. Recently, it was suggested that macrophages contribute to impaired host defence and excessive inflammatory responses in CF. Indeed, dysfunction attributed to CF macrophages includes decreased bacterial killing and exaggerated inflammatory responses. However, the mechanisms behind such defects have only been partially defined. MicroRNAs (miRNAs) have emerged as key regulators of several macrophage functions, including their activation, differentiation and polarisation. The goal of this study was to investigate whether miRNA dysregulation underlies the functional abnormalities of CF macrophages. MiRNA profiling of macrophages was performed, with 22 miRNAs identified as differentially expressed between CF and non-CF individuals. Among these, miR-146a was associated with significant enrichment of validated target genes involved in responses to microorganisms and inflammation. As miR-146a dysregulation has been reported in several human inflammatory diseases, we analysed the impact of increased miR-146a expression on inflammatory responses of CF macrophages. These data show that inhibition of miR-146a in lipopolysaccharide-stimulated CF macrophages results in increased interleukin-6 production, which suggests that miR-146a overexpression in CF is functional, to restrict inflammatory responses.

Anomalies in constitutive calcium entry (CCE) have been commonly attributed to cell dysfunction in pathological conditions such as cancer. Calcium influxes of this type rely on channels, such as transient receptor potential (TRP) channels, to be constitutively opened and strongly depend on membrane potential and a calcium driving force. We developed an optogenetic approach based on the expression of the halorhodopsin chloride pump to study CCE in non-excitable cells. Using C2C12 cells, we found that halorhodopsin can be used to achieve a finely tuned control of membrane polarization. Escalating the membrane polarization by incremental changes in light led to a concomitant increase in CCE through transient receptor potential vanilloid 2 (TRPV2) channels. Moreover, light-induced calcium entry through TRPV2 channels promoted cell migration. Our study shows for the first time that by modulating CCE and related physiological responses, such as cell motility, halorhodopsin serves as a potentially powerful tool that could open new avenues for the study of CCE and associated cellular behaviors.

Les macrophages sont en première ligne de la défense innée et jouent un rôle important dans l’initiation de la réponse immunitaire puisque régulant l’inflammation et permettant la clairance des pathogènes. Dans la mucoviscidose, ces phénomènes sont exacerbés et deviennent chronique sans pouvoir être résolus. Les objectifs de cette thèse ont été de déterminer des cibles potentielles responsables des altérations du macrophage dans la mucoviscidose. Dans le contexte inflammatoire, la forme soluble du CD14 (sCD14), dont la sécrétion est augmentée par les macrophages de patients atteints de la mucoviscidose, est caractérisé comme un DAMP puisqu’il contribue à l’entretien de l’inflammation au niveau tissulaire. Dans le contexte infectieux, l’activité de TRPV2, impliqué dans la phagocytose, est altérée. Dans la mucoviscidose, l’inflammation et l’infection sont aussi intimement liées par l’intermédiaire d’une altération des microstructures de la membrane plasmique impliquée dans la production du sCD14 et dans le processus de phagocytose. En conclusion, les modifications des fonctions du macrophage affaiblissent la défense innée des patients atteints de mucoviscidose et peuvent être impliqués dans la progression de la maladie. Par conséquent, les interventions visant à réduire l’inflammation et l’infection pourraient être bénéfiques afin de préserver la fonction pulmonaire des patients. Ainsi, les approches thérapeutiques visant à corriger les dysfonctions du macrophage de patients atteints de mucoviscidose pourrait fournir une meilleure résolution de l'infection et l'inflammation.