A novel mechanism that regulates cellular injury by phagocytes during inflammation

Phagocytes such as macrophages and neutrophils contain multiple lysosomes, which have a variety of digestive enzymes. On stimulation, phagocytes secrete these digestive enzymes through a process called lysosomal exocytosis to lyse external pathogens or tumor cells. The research team has recognized myoferlin as a critical regulator of this process. Furthermore, they found that it plays an important role in inducing mobile injury by phagocytes during inflammation.

Lysosomes are packed using more than 50 different hydrolytic enzymes, which are able of digesting most mobile components. Notably, activated phagocytes secrete lysosomal enzymes with cytotoxic activity against exterior pathogens or tumor tissues, inducing heterolysis. Nevertheless , the molecular mechanisms that manage lysosomal exocytosis by phagocytes remain largely unknown. Within several types of tissues, transmembrane proteins possessing C2 domains, such as Munc13-4 and synaptotagmin-VII, are present on the lysosomal membrane and known to manage Ca2+-triggered lysosomal exocytosis. C2 domains mediate Ca2+-dependent joining to phosphatidylserine (PS) in the inner leaflet of the plasma membrane, which is a critical process for membrane fusion. Myoferlin is a type II transmembrane protein with 7 C2 domains in the cytoplasmic region. The very first C2 domain (C2A domain) binds PS in a calcium-dependent manner. Myoferlin was first recognized as a protein indicated in the plasma membrane of myoblasts undergoing fusion and has since already been implicated in the repair of injured plasma membranes. Injury-induced Ca2+ influx through membrane lesions triggers endocytosis and the generation of endocytosed vesicles expressing myoferlin, which fuse with the injured membrane to produce a membrane patch. This particular characteristic of myoferlin resulted in the hypothesis that it might be a regulator of calcium-dependent lysosomal exocytosis by phagocytes.

We found that myoferlin was highly expressed by macrophages and localized to lysosomes. Analysis of changed cells expressing shRNA against myoferlin demonstrated that myoferlin knockdown cells contained significantly more lysosomes and cytoplasmic vesicles filled up with debris and additional membranous materials in comparison with the control tissues. The amount of lysosomal enzyme secreted after calcium mineral stimulation was significantly reduced by myoferlin knockdown, but was restored by re-expressing myoferlin protein. Similarly, macrophages from myoferlin-/- mice also displayed accumulation of lysosomes and reduction of lysosomal enzyme secretion after various calcium stimuli. The shot of Escherichia coli BioParticles into control mice increased the amount of the lysosomal enzymes in the peritoneal fluid (ascites), which was inhibited in myoferlin-/- mice. Additionally , the peritoneal fluid had a cytotoxic result when added into culture media of tumor tissue, which was also fallen in myoferlin-/- mice, regular with the amount of lysosomal enzyme present.

This specific study reveals that myoferlin is a Ca2+-dependent limiter of lysosomal exocytosis by phagocytes. As autolysosomes stuffed with debris accumulated in the myoferlin knockdown tissue, we speculated that myoferlin on autolysosomes might promote expelling of indigestible dirt developed inside autolysosomes via exocytosis. Phagocytes have a potent cytotoxic capacity activated by antibodies against targeted cells (e. g., pathogens and tumor cells), known as antibody-dependent cell-mediated cytotoxicity (ADCC). These antibodies stimulate Fc receptors to result in signaling pathway that induce Ca2+-dependent lysosomal exocytosis. Remarkably, neutrophils have particularly strong ADCC activity, when they secrete large amounts of cytotoxic molecules, including hydrolytic enzymes, oxidative metabolites, and host defense peptides such as defensins. It would therefore be useful to study whether myoferlin also regulates the discharge of these molecules from neutrophils. Furthermore, our demonstration that myoferlin deficiency decreased the cytotoxicity of phagocytes is medically important and may help the development of book therapeutic approaches based on myoferlin-mediated lysosomal exocytosis..

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