Kurzbeschreibung
(Englisch)
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Acid regulation in immune cells is crucial because it modulates signaling and function. These cells have developped mechanisms to extrude acid, among them the highly efficient Hv1 proton channel. Hv1 regulates both cytosolic and phagosomal pH in phagocytes, sustains the activity of the NADPH oxidase for ROS production and maintains membrane potential at optimal levels that allows calcium influx. pH regulation and calcium signals regulate exocytosis, a feature of immune cells where the role of Hv1 has not been elucidated so far. Hv1 is expressed in B cells where it plays a role in BCR-signaling and function. More recently, the enrichement of a short isoform of Hv1 in malignant B cell has been reported which boosts BCR-dependent signaling, metabolism and proliferation. In this grant proposal we will assess the role of Hv1 in malignant B cells behavior and migration and investigate the impact of both short and long Hv1 isoforms on exocytosis, cytokine release and gene expression of wild type and Hv1-lacking mouse neutrophils. We will also measure submembranous pH and extracellular microenvironment pH in B cell lymphoma. To do so, we will develop innovative genetic tools to measure localized pH values in specific microenvironments, design isoform specific siRNA for Hv1 knock-down and generate a Hv1-deleted human leukocyte cell line. This work will provide new insight regarding acid-linked pathobiology of immune cells and treatment.
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Abstract
(Englisch)
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Hvl proton channels are known to play a key role in immune defense and infiammation by sustaining the activity of NOX2, the NADPH oxidase ROS generating enzyme in granulocytes. They protect the cytosol from excessive acidification by extwding protons and re-polatize the cell membrane. However, increasing number of publications recently reports that several types of malignancies hijack Hvl in order to extmde their massive acid generation resulting ftom their high metabolism. This efficient acid elimination is crucial for tumor metabolism, growth, proliferation, migration and tissue invasiveness. As a consequence, the pH gradient is inverted (more acidic in the extracellular microenvironment of the ceils) and this interferes with the passage of dwgs across cell membranes. Most of anticancer dmgs (known to be weak bases) are neutralized and end up trapped in endosomes or intracellular acidic vesicles. No known specific inhibitors of Hvl have been identified yet, besides the guanidine derivatives (2GBI) which generally have a bw affinity, block the channel from the intracellular side, and are poorly membrane permeable. To facilitate the investigaüon of Hvl‘s role in immune cells and malignancies, we deleted Hvl in a myeloid leukemia cell line; PLB-985 using the CRISPR]CAS9 technoiogy. The four clones we obtained showed, as expected, a large decrease in ROS production more than 60 % upon PMA activation, similarly to our results in Hvl-deficient neutrophils. Next we investigated the impact of Hvl on the NADPH oxidase and focused on the discrepancy reported between ROS generation and oxygen consumpDon. Our results show that PMA stimulated Hvl-deficient neutrophils show 80% decrease in oxygen consumption and 62 % decrease in proton production rate when specific conditions favonng Hvl activation were applied. This correlates weil with the 60-75 % reduction in ROS production of Hvl -deficient neutrophils. We also tested the effect of zinc, the classical inhibitor of Hvl, on the metabolism and proliferation of RAJI and CARNAVAL, two B cell lymphoma cell lines. Indeed, our results show a drastic reduction in their survival (80%) and a significantly hampered proliferation (20-25 % less proliferation than the untreated cells). Since zinc shows unspecific effects and toxicity, we tried another approach by treating these cells with specific si-RNA targeting Hvl bong and are currently trying to increase the percentage of efficiency of our silencing. The design of si-RNA against Hvl short is also underway and will be tested shortly on these same parameters. Our results reveal the impact of excessive acidification linked to the absence or blockade of Hvl channels in immune cells and tumors, besides the new tools were are currentiy developing will be most valuable for this project and beyond.
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