L’intensification des pratiques d’élevage s’est accompagnée de l’émergence de pathologies de production, notamment des mammites. Il s’agit d’une inflammation de la glande mammaire, d’origine bactérienne dans la majeure partie des cas. Les mammites constituent à elles seules la première source de pertes financières des cheptels bovins laitiers en France et dans le monde. Néanmoins aucun traitement prophylactique ne permet à ce jour une action préventive à long terme. Parmi les agents étiologiques majeurs des mammites, Escherichia coli (E. coli) tient son importance du fait de sa prévalence et de son impact sur les rendements et la qualité du lait. La part des facteurs de l’hôte dans la capacité à éliminer le pathogène causal est relativement avérée, tandis que le lien entre caractéristiques bactériennes et sévérité de l’infection est plus délicat à établir. Cette étude s’attache donc à déterminer si les interactions entre E. coli et la glande mammaire, en particulier les cellules épithéliales mammaires (CEM) et les neutrophiles, peuvent expliquer des degrés de sévérité variables. L’influence du lait sur ces interactions a également été investiguée. / Along with agricultural intensification of animal production, some pathologies have emerge, especially mastitis. This disease corresponds to an inflammation of the udder, and is generally provoked by bacterial infection. Mastitis on their own constitute the main source of financial impairments within dairy herds in France and worldwide. So far, there is no treatment able to prevent mastitis over time. Among major mastitis pathogens Escherichia coli (E. coli) is of great importance, because of its prevalence and its impacts on milk yield and quality. The mastitis severity has proven to be linked to host factors, but the implication of bacterial characteristics remains unknown. Thus, this study aimed at deciphering whether interactions between E. coli and the mammary gland, especially the mammary epithelial cells (MECs) and neutrophils, may explain a variability in mastitis severity. Influence of milk on these interactions was also investigated.

The motility of neutrophils and their ability to sense and to react to chemoattractants in their environment are of central importance for the innate immunity. Neutrophils are guided towards sites of inflammation following the activation of G-protein coupled chemoattractant receptors such as CXCR2 whose signaling strongly depends on the activity of Ca²⁺ permeable TRPC6 channels. It is the aim of this study to analyze data sets obtained in vitro (murine neutrophils) and in vivo (zebrafish neutrophils) with a stochastic mathematical model to gain deeper insight into the underlying mechanisms. The model is based on the analysis of trajectories of individual neutrophils. Bayesian data analysis, including the covariances of positions for fractional Brownian motion as well as for exponentially and power-law tempered model variants, allows the estimation of parameters and model selection. Our model-based analysis reveals that wildtype neutrophils show pure superdiffusive fractional Brownian motion. This so-called anomalous dynamics is characterized by temporal long-range correlations for the movement into the direction of the chemotactic CXCL1 gradient. Pure superdiffusion is absent vertically to this gradient. This points to an asymmetric ‘memory’ of the migratory machinery, which is found both in vitro and in vivo. CXCR2 blockade and TRPC6-knockout cause tempering of temporal correlations in the chemotactic gradient. This can be interpreted as a progressive loss of memory, which leads to a marked reduction of chemotaxis and search efficiency of neutrophils. In summary, our findings indicate that spatially differential regulation of anomalous dynamics appears to play a central role in guiding efficient chemotactic behavior.

Non-resolving inflammation plays a critical role during the transition from renal injury towards end-stage renal disease. The glucocorticoid-inducible protein annexin A1 has been shown to function as key regulator in the resolution phase of inflammation, but its role in immune-mediated crescentic glomerulonephritis has not been studied so far. Methods: Acute crescentic glomerulonephritis was induced in annexin A1-deficient and wildtype mice using a sheep serum against rat glomerular basement membrane constituents. Animals were sacrificed at d5 and d10 after nephritis induction. Renal leukocyte abundance was studied by immunofluorescence and flow cytometry. Alterations in gene expression were determined by RNA-Seq and gene ontology analysis. Renal levels of eicosanoids and related lipid products were measured using lipid mass spectrometry. Results: Histological analysis revealed an increased number of sclerotic glomeruli and aggravated tubulointerstitial damage in the kidneys of annexin A1-deficient mice compared to the wildtype controls. Flow cytometry analysis confirmed an increased number of CD45+ leukocytes and neutrophil granulocytes in the absence of annexin A1. Lipid mass spectrometry showed elevated levels of prostaglandins PGE2 and PGD2 and reduced levels of antiinflammatory epoxydocosapentaenoic acid regioisomers. RNA-Seq with subsequent gene ontology analysis revealed induction of gene products related to leukocyte activation and chemotaxis as well as regulation of cytokine production and secretion. Conclusion: Intrinsic annexin A1 reduces proinflammatory signals and infiltration of neutrophil granulocytes and thereby protects the kidney during crescentic glomerulonephritis. The annexin A1 signaling cascade may therefore provide novel targets for the treatment of inflammatory kidney disease.

Neutrophile sind die häufigsten weißen Blutkörperchen im menschlichen Blut. Sie bilden die erste Verteidigungslinie und töten eindringende Krankheitserreger ab. Neutrophile extrazelluläre Fallen (NETs) sind netzartige Strukturen, die aus dekondensiertem Chromatin bestehen und mit zytotoxischen Proteinen dekoriert sind. NETs können Mikroben in vitro und in vivo einfangen und abtöten, sind aber auch für verschiedene Krankheiten verantwortlich. Frühere Studien haben eine spezifische Gruppe von 20-50 Neutrophilenproteinen identifiziert, die an NETs gebunden sind und von denen einige eine mikrobizide Wirkung haben. Wie diese Proteine an die NETs binden, wie sie interagieren und wie die Bindung ihre antimikrobielle Aktivität beeinflusst, ist noch nicht bekannt. In dieser Dissertation habe ich die Verteilung von acht neutrophilen Proteinen und Nukleosomen auf NETs mit Hilfe der Superauflösungsmikroskopie untersucht. Es wurden drei unabhängige Techniken mit Auflösungen von mehr als 90 nm verwendet. Die Nukleosomen bildeten auf den NETs periodische Cluster mit deutlich größeren Abständen im Vergleich zum kondensierten Chromatin. Drei NET-Proteine waren ebenfalls in periodischen Clustern auf den NETs lokalisiert und zwei von ihnen waren stark mit Nukleosomen kolokalisiert. Alle anderen analysierten Proteine zeigten keine Muster der Bindung an NETs. Zusammengenommen zeigen diese Ergebnisse, dass die Bindung von Proteinen an NETs zumindest teilweise spezifisch ist und teilweise durch Wechselwirkungen mit Nukleosomen vermittelt wird. Die erfolgreiche Einführung der superauflösenden Mikroskopie für schwierige NET-Proben in Kombination mit einem vorgeschlagenen rekonstituierten NET-System eröffnet neue Möglichkeiten für das Verständnis der molekularen Mechanismen der NET-Bildung und der Protein-Protein-Interaktion bei der NET-vermittelten Abtötung. / Neutrophils are the most abundant human white blood cell in circulation. They are the first line of defense and kill invading pathogens. Neutrophil Extracellular Traps (NETs) are weblike structures composed of decondensed chromatin decorated with cytotoxic proteins. NETs can trap and kill microbes in vitro and in vivo, but also mediate several diseases. Previous studies identified a specific set of 20-50 neutrophil proteins bound to NETs, several with microbicidal activity. It remains unknown how these proteins bind to NETs, how they interact and how binding influences their anti-microbial activity. In this dissertation, I studied the distribution of eight neutrophil proteins and nucleosomes on NETs using super-resolution microscopy. Three independent techniques with resolutions larger than 90nm were used. Nucleosomes formed periodic clusters on NETs, with significantly larger spacing compared to condensed chromatin. Three NET proteins also localized in periodic clusters on NETs and two of them strongly co-localized with nucleosomes. All other proteins analyzed showed no patterns binding to NETs. Taken together, these findings demonstrate that, at least some, protein binding to NETs is specific and in part mediated by interactions with nucleosomes. The successful introduction of super-resolution microscopy to the challenging NET samples in combination with a proposed reconstituted NET system opens new possibilities to understand the molecular mechanisms behind NET formation and protein-protein interaction in NET mediated killing.

Les polynucléaires neutrophiles représentent 50-70% des leucocytes sanguins et possèdent un rôle majeur dans la défense de l’organisme contre les pathogènes. Le Ca2+ est un second messager qui joue un rôle primordial dans le chimiotactisme, la phagocytose, la dégranulation et la production de formes réactives de l’oxygène (FRO) afin de neutraliser l’agent pathogène. Dans ces cellules, l’influx calcique de type SOCE est essentiel pour l'homéostasie calcique. Il est peu étudié en raison du manque d’outils pharmacologiques spécifiques d’où l’importance dans un premier temps de chercher de nouvelles molécules. Les cellules T Jurkat dont le SOCE est largement caractérisé servent de modèle pour la caractérisation initiale de ces molécules. Le 2-APB est parmi les molécules les plus largement utilisées dans la caractérisation du SOCE en raison de sa double activité sur le SOCE avec une potentialisation à [1-10 μM] et une inhibition à [> 20 μM]. En revanche, ce produit manque de spécificité et agit sur d’autres cibles cellulaires comme les récepteurs à l’inositol (1,4,5)-trisphosphate (InsP3Rs). La 1ère étape est de sélectionner à partir d’analogues commerciaux du 2-APB (Methoxy-APB, Dimethoxy-APB, Cyclic-APB, Benzothienyl-APB, Thienyl-APB et MDEB), des composés plus spécifiques et également plus efficaces que la molécule mère. Deux molécules se sont distinguées : le MDEB comme uniquement potentialisant du SOCE et le Benzothienyl-APB comme un puissant inhibiteur. En revanche, tous les analogues du 2-APB inhibent les InsP3Rs à l’exception du MDEB qui semble plus spécifique du SOCE. L’effet du MDEB sur le courant calcique, ICRAC, a été étudié grâce à la technique du patch-clamp. Il augmente d’environ 4 fois l’amplitude de ICRAC par rapport à celle enregistrée dans les cellules contrôle. Par ailleurs, le MDEB ralentie l’inactivation rapide de ICRAC due au Ca2+. Sur le plan physiologique, le MDEB à des concentrations croissantes inhibe la synthèse de l’IL-2 dans les cellules Jurkat stimulées et ceci malgré son effet potentialisant du SOCE. Cette activité est liée à son effet pro-apoptotique dans les cellules Jurkat stimulées. Le MDEB et le Benzothienyl-APB caractérisés dans la 1ère partie nous ont servi d’outils potentiels afin d’étudier le SOCE des cellules PLB-985 différenciées en cellules proches de neutrophiles. Le SOCE a été induit soit par un traitement des cellules avec la thapsigargine (Tg) soit de manière physiologique avec les peptides fMLF et le WKYMVm deux chimioattractants, ligands des récepteurs aux peptides formylés FPR et FPRL1 respectivement. En plus, le SOCE induit par la Tg est modulable par le 2-APB, potentialisé par le MDEB et inhibé par le Benzothienyl-APB. La phagocytose des levures par les cellules PLB-985 différenciées ainsi que la production de FRO intraphagosomales ont été inhibées par le MDEB et le Benzothienyl-APB. Les FRO extracellulaires ont été également inhibées par Benzothienyl-APB en revanche à cause de la forte interférence du MDEB avec la technique de mesure nous n’avons pas pu étudier ses activités. En conclusion, le MDEB et le Benzothienyl-APB sont de nouveaux outils pharmacologiques potentialisant ou inhibant le SOCE des leucocytes, qui nous permettront dans l’avenir une meilleure compréhension de l'entrée calcique et ses rôles dans ces cellules. / Neutrophils represent 50-70% of human blood leukocytes; their role is to protect the body against pathogens. Calcium is a second messenger which plays an important role in chemotaxis, phagocytosis, degranulation and the production of reactive oxygen species (ROS) in order to eliminate microbes. In neutrophils, the mechanism of store-operated calcium entry (SOCE) is essential for calcium homeostasis. However, neutrophil SOCE is not well understood because of the lack of specific pharmacological tools. It is necessary to first identify and characterize new molecules using a model of Jurkat T cells in which SOCE was the best characterized. 2-APB is the most widely used molecule in SOCE characterization due to its dual activity with a potentiation at lower concentrations [1-10 μM] and an inhibition at higher concentrations [> 20 μM]. However, this molecule lacks specificity because it acts on other cellular targets such as inositol (1,4,5)-trisphosphate receptors (InsP3Rs). The first step is to select from a library of 8 commercial 2-APB analogues (Methoxy-APB, Dimethoxy-APB, Cyclic-APB, Benzothienyl-APB, Thienyl-APB and MDEB) those that are more specific and also more efficient molecules than 2-APB. Two interesting molecules were identified, MDEB as the only SOCE potentiating product currently known and the Benzothienyl-APB, which is a strong inhibitor. Like 2-APB, all these analogues inhibit InsP3Rs except MDEB, which seems to be more specific. The effect of MDEB on the calcium current, ICRAC, was also studied using the patch-clamp technique. MDEB increases ~4 times the ICRAC amplitude in comparison with control. Otherwise, MDEB slows down the fast Ca2 +-dependent inactivation of ICRAC. Functionally, MDEB at increasing concentrations inhibits IL-2 synthesis in stimulated Jurkat T cells despite its potentiating activity on SOCE. The inhibition is due to MDEB induced apoptosis in stimulated Jurkat T cells. MDEB and Benzothienyl-APB were then used as tools to study SOCE in a neutrophil-like cell model, the differentiated PLB-985 cells. SOCE was induced either by treatment of cells with thapsigargin (Tg) or physiologically with the chemotactic peptides fMLF and WKYMVm, ligands of formyl peptide receptors FPR and FPRL1 respectively. In addition, Tg-induced SOCE was modulated by 2-APB, potentiated by MDEB and inhibited by Benzothienyl-APB. The consequences of these analogues on neutrophil functions were also studied. Phagocytosis of yeasts by PLB-985 cells and intraphagosomal ROS production were inhibited by MDEB and Benzothienyl-APB. Furthermore, extracellular ROS were also inhibited by Benzothienyl-APB. However, because of the high interference of MDEB with our techniques, its activities could not be studied. In conclusion, MDEB and Benzothienyl-APB are new analogues of 2-APB potentiating and inhibiting SOCE, which allow us in the future a better understanding of leukocyte SOCE and its cellular roles.