Mécanismes moléculaires régulant la polarisation des lymphocytes B

In secondary lymphoid organs, B cells acquire antigens that are tethered at the surface of neighboring cells. Engagement of the B cell receptor (BCR) with such immobilized antigens leads to the formation of an immune synapse and the subsequent polarization of B cells. This includes the repositioning of the centrosome towards the immune synapse as well as the recruitment and local secretion of lysosomes required for efficient antigen extraction, processing and presentation onto class II major histocompatibility complex (MHC-II) molecules to primed CD4+ T cells. Pioneer work performed in the lab has highlighted the first molecular players involved in this process. However, the precise mechanism governing centrosome polarization remains to be fully elucidated. The work performed during this thesis aimed at identifying new regulators supporting centrosome polarization in B lymphocytes upon BCR engagement with immobilized antigens. In addition, in view of the emerging role played by the tissue microenvironment in shaping B cell activation and functions we investigated whether extracellular Galectin-8 modulates the ability of B cells to polarize, extract and present immobilized antigens. We show here that, in resting lymphocytes, centrosome-associated Arp2/3 (actin related protein-2/3) locally nucleates F-actin, which is needed for centrosome tethering to the nucleus via the LINC (linker of nucleoskeleton and cytoskeleton) complex. Upon lymphocyte activation, Arp2/3 is partially depleted from the centrosome as a result of its HS1-dependent recruitment to the immune synapse. This leads to a reduction in F-actin nucleation at the centrosome and thereby allows its detachment from the nucleus and polarization to the synapse. In addition, we show that extracellular Galectin-8 favors lysosome recruitment and secretion at the immune synapse, hence providing B cells with an enhanced capacity to extract and present immobilized antigens. Our findings highlight unexpected mechanisms that tune B cell polarity in response to antigenic stimulation and raise exciting questions concerning the coordinated regulation of these mechanisms to provide B cells with the capacity to efficiently extract, process and present surface-tethered antigens.Dans les organes lymphoïdes secondaires, les lymphocytes B acquièrent des antigènes immobilisés à la surface de cellules voisines. L’engagement du BCR (récepteur des cellules B) avec de tels antigènes induit la formation d’une synapse immunologique et la polarisation des lymphocytes B. Cette polarisation inclut le repositionnement du centrosome à la synapse immunologique ainsi que le recrutement et la sécrétion locale des lysosomes qui sont nécessaires à l’extraction, l’apprêtement et la présentation des antigènes sur les molécules du complexe majeur d’histocomptabilité de classe II (CMH-II) aux lymphocytes T CD4+ pré-activés. Des travaux précurseurs menés dans le laboratoire ont permis de mettre en évidence les premiers acteurs moléculaires impliqués dans ce processus. Cependant, le mécanisme précis gouvernant la polarisation du centrosome demeure encore aujourd’hui inconnu. Le travail réalisé pendant cette thèse avait pour objectif d’identifier de nouveaux régulateurs contrôlant la polarisation du centrosome dans les lymphocytes B après engagement du BCR avec des antigènes immobilisés. De plus, au regard du rôle grandissant joué par le microenvironnement tissulaire dans l’activation des lymphocytes B ainsi que dans la modulation de leurs fonctions, nous avons étudié l’effet de la protéine extracellulaire Galectine-8 sur la régulation de la capacité des lymphocytes B à se polariser et à extraire et présenter des antigènes immobilisés. Le travail présenté dans ce manuscrit montre que la présence du complexe Arp2/3 au centrosome des lymphocytes B non activés permet la nucléation locale de filaments d’actine qui permettent, grâce à leur interaction avec le complexe LINC, de lier le centrosome au noyau. L’activation des lymphocytes B induit la déplétion partielle du complexe Arp2/3 du centrosome qui est recruté à la synapse immunologique par la protéine HS1. Ceci induit une diminution de la nucléation d’actine au centrosome entraînant la séparation entre le centrosome et le noyau et permettant la polarisation du centrosome vers la synapse. De plus, nous montrons que la présence de la protéine Galectine-8 dans le milieu extracellulaire favorise le recrutement et la sécrétion des lysosomes à la synapse immunologique, conférant aux lymphocytes B une meilleure capacité à extraire et présenter des antigènes immobilisés. Nos résultats mettent en évidence des mécanismes inattendus régulant la polarisation des lymphocytes B en réponse à une stimulation antigénique et soulèvent des questions intéressantes concernant la régulation coordonnée de ces mécanismes qui confèrent aux lymphocytes B la capacité d’extraire, d’apprêter et de présenter des antigènes immobilisés efficacement

Actin filaments regulate microtubule growth at the centrosome

International audienceThe centrosome is the main microtubule-organizing centre. It also organizes a local network of actin filaments. However, the precise function of the actin network at the centrosome is not well understood. Here, we show that increasing densities of actin filaments at the centrosome of lymphocytes are correlated with reduced amounts of microtubules. Furthermore, lymphocyte activation resulted in disassembly of centrosomal actin and an increase in microtubule number. To further investigate the direct crosstalk between actin and microtubules at the centrosome, we performed in vitro reconstitution assays based on (i) purified centrosomes and (ii) on the co-micropatterning of microtubule seeds and actin filaments. These two assays demonstrated that actin filaments constitute a physical barrier blocking elongation of nascent microtubules. Finally, we showed that cell adhesion and cell spreading lead to lower densities of centrosomal actin, thus resulting in higher microtubule growth. We therefore propose a novel mechanism, by which the number of centrosomal microtubules is regulated by cell adhesion and actin-network architecture

Inhibitors of the Neisseria meningitidis PilF ATPase provoke type IV pilus disassembly

International audienceDespite the availability of antibiotics and vaccines, Neisseria meningitidis remains a major cause of meningitis and sepsis in humans. Due to its extracellular lifestyle, bacterial adhesion to host cells constitutes an attractive therapeutic target. Here, we present a high-throughput microscopy-based approach that allowed the identification of compounds able to decrease type IV pilus-mediated interaction of bacteria with endothelial cells in the absence of bacterial or host cell toxicity. Compounds specifically inhibit the PilF ATPase enzymatic activity that powers type IV pilus extension but remain inefficient on the ATPase that promotes pilus retraction, thus leading to rapid pilus disappearance from the bacterial surface and loss of pili-mediated functions. Structure activity relationship of the most active compound identifies specific moieties required for the activity of this compound and highlights its specificity. This study therefore provides compounds targeting pilus biogenesis, thereby inhibiting bacterial adhesion, and paves the way for a novel therapeutic option for meningococcal infections

Colonization of dermal arterioles by Neisseria meningitidis provides a safe haven from neutrophils

International audienceAbstract The human pathogen Neisseria meningitidis can cause meningitis and fatal systemic disease. The bacteria colonize blood vessels and rapidly cause vascular damage, despite a neutrophil-rich inflammatory infiltrate. Here, we use a humanized mouse model to show that vascular colonization leads to the recruitment of neutrophils, which partially reduce bacterial burden and vascular damage. This partial effect is due to the ability of bacteria to colonize capillaries, venules and arterioles, as observed in human samples. In venules, potent neutrophil recruitment allows efficient bacterial phagocytosis. In contrast, in infected capillaries and arterioles, adhesion molecules such as E-Selectin are not expressed on the endothelium, and intravascular neutrophil recruitment is minimal. Our results indicate that the colonization of capillaries and arterioles by N. meningitidis creates an intravascular niche that precludes the action of neutrophils, resulting in immune escape and progression of the infection

Actin nucleation at the centrosome controls lymphocyte polarity.

International audienceCell polarity is required for the functional specialization of many cell types including lymphocytes. A hallmark of cell polarity is the reorientation of the centrosome that allows repositioning of organelles and vesicles in an asymmetric fashion. The mechanisms underlying centrosome polarization are not fully understood. Here we found that in resting lymphocytes, centrosome-associated Arp2/3 locally nucleates F-actin, which is needed for centrosome tethering to the nucleus via the LINC complex. Upon lymphocyte activation, Arp2/3 is partially depleted from the centrosome as a result of its recruitment to the immune synapse. This leads to a reduction in F-actin nucleation at the centrosome and thereby allows its detachment from the nucleus and polarization to the synapse. Therefore, F-actin nucleation at the centrosome-regulated by the availability of the Arp2/3 complex-determines its capacity to polarize in response to external stimuli

Polarity protein Par3 controls B-cell receptor dynamics and antigen extraction at the immune synapse

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Galectin-8 Favors the Presentation of Surface-Tethered Antigens by Stabilizing the B Cell Immune Synapse

International audienceComplete activation of B cells relies on their capacity to extract tethered antigens from immune synapses by either exerting mechanical forces or promoting their proteolytic degradation through lysosome secretion. Whether antigen extraction can also be tuned by local cues originating from the lymphoid microenvironment has not been investigated. We here show that the expression of Galectin-8-a glycan-binding protein found in the extracellular milieu, which regulates interactions between cells and matrix proteins-is increased within lymph nodes under inflammatory conditions where it enhances B cell arrest phases upon antigen recognition in vivo and promotes synapse formation during BCR recognition of immobilized antigens. Galectin-8 triggers a faster recruitment and secretion of lysosomes toward the B cell-antigen contact site, resulting in efficient extraction of immobilized antigens through a proteolytic mechanism. Thus, extracellular cues can determine how B cells sense and extract tethered antigens and thereby tune B cell responses in vivo

LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

<p>Abstract</p> <p>Background</p> <p>Patients with metastatic melanoma have a poor median rate of survival. It is therefore necessary to increase our knowledge about melanoma cell dissemination which includes extravasation, where cancer cells cross the endothelial barrier. Extravasation is well understood during travelling of white blood cells, and involves integrins such as LFA-1 (composed of two chains, CD11a and CD18) expressed by T cells, while ICAM-1 is induced during inflammation by endothelial cells. Although melanoma cell lines cross endothelial cell barriers, they do not express LFA-1. We therefore hypothesized that melanoma-endothelial cell co-culture might induce the LFA-1/ICAM ligand/receptor couple during melanoma transmigration.</p> <p>Methods</p> <p>A transwell approach has been used as well as blocking antibodies against CD11a, CD18 and ICAM-1. Data were analyzed with an epifluorescence microscope. Fluorescence intensity was quantified with the ImageJ software.</p> <p>Results</p> <p>We show here that HUVEC-conditioned medium induce cell-surface expression of LFA-1 on melanoma cell lines. Similarly melanoma-conditioned medium activates ICAM-1 expression in endothelial cells. Accordingly blocking antibodies of ICAM-1, CD11a or CD18 strongly decrease melanoma transmigration. We therefore demonstrate that melanoma cells can cross endothelial monolayers in vitro due to the induction of ICAM-1 and LFA-1 occurring during the co-culture of melanoma and endothelial cells. Our data further suggest a role of LFA-1 and ICAM-1 in the formation of melanoma cell clumps enhancing tumor cell transmigration.</p> <p>Conclusion</p> <p>Melanoma-endothelial cell co-culture induces LFA-1 and ICAM-1 expression, thereby favoring in vitro melanoma trans-migration.</p