Browsing by Subject "phagocytosis"

Now showing 1 - 3 of 3
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    A Fluorescently Tagged C-Terminal Fragment of p47phox Detects NADPH Oxidase Dynamics during Phagocytosis
    (2009-03) Li, Xing Jun; Tian, Wei; Stull, Natalie D; Grinstein, Sergio; Atkinson, Simon J; Dinauer, Mary C
    The assembly of cytosolic p47phox and p67phox with flavocytochrome b558 at the membrane is crucial for activating the leukocyte NADPH oxidase that generates superoxide for microbial killing. p47phox and p67phox are linked via a high-affinity, tail-to-tail interaction involving a proline-rich region (PRR) and a C-terminal SH3 domain (SH3b), respectively, in their C-termini. This interaction mediates p67phox translocation in neutrophils, but is not required for oxidase activity in model systems. Here we examined phagocytosis-induced NADPH oxidase assembly, showing the sequential recruitment of YFP-tagged p67phox to the phagosomal cup, and, after phagosome internalization, a probe for PI(3)P followed by a YFP-tagged fragment derived from the p47phox PRR. This fragment was recruited in a flavocytochrome b558-dependent, p67phox-specific, and PI(3)P-independent manner. These findings indicate that p47PRR fragment probes the status of the p67phox SH3b domain and suggest that the p47phox/p67phox tail-to-tail interaction is disrupted after oxidase assembly such that the p67phox-SH3b domain becomes accessible. Superoxide generation was sustained within phagosomes, indicating that this change does not correlate with loss of enzyme activity. This study defines a sequence of events during phagocytosis-induced NADPH oxidase assembly and provides experimental evidence that intermolecular interactions within this complex are dynamic and modulated after assembly on phagosomes.
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    Myo-Inositol in Fermented Sugar Matrix Improves Human Macrophage Function
    (Wiley, 2022) Ghosh, Nandini; Das, Amitava; Biswas, Nirupam; Mahajan, Sanskruti P.; Madeshiya, Amit K.; Khanna, Savita; Sen, Chandan K.; Roy, Sashwati; Surgery, School of Medicine
    Scope Reactive oxygen species production by innate immune cells plays a central role in host defense against invading pathogens at wound-site. A weakened hos-defense results in persistent infection leading to wound chronicity. Fermented Papaya Preparation (FPP), a complex sugar matrix, bolstered respiratory burst activity and improved wound healing outcomes in chronic wound patients. The objective of the current study was to identify underlying molecular factor/s responsible for augmenting macrophage host defense mechanisms following FPP supplementation. Methods and results In depth LC-MS/MS analysis of cells supplemented with FPP led to identification of myo-inositol as a key determinant of FPP activity towards improving macrophage function. Myo-inositol, in quantities that is present in FPP, significantly improved macrophage respiratory burst and phagocytosis via de novo synthesis pathway of ISYNA1. Additionally, myo-inositol transporters, HMIT and SMIT1, played a significant role in such activity. Blocking these pathways using siRNA attenuated FPP-induced improved macrophage host defense activities. FPP supplementation emerges as a novel approach to increase intracellular myo-inositol levels. Such supplementation also modified wound microenvironment in chronic wound patients to augment myo-inositol levels in wound fluid. Conclusion These observations indicate that myo-inositol in FPP influences multiple aspects of macrophage function critical for host defense against invading pathogens.
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    The phosphoinositide-binding protein p40phox activates the NADPH oxidase during FcγIIA receptor–induced phagocytosis
    (2006-07) Suh, Chang-Il; Stull, Natalie D; Li, Xing Jun; Tian, Wei; Price, Marianne O; Grinstein, Sergio; Yaffe, Michael B; Atkinson, Simon J; Dinauer, Mary C
    Superoxide produced by the phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is essential for host defense. Enzyme activation requires translocation of p67phox, p47phox, and Rac-GTP to flavocytochrome b558 in phagocyte membranes. To examine the regulation of phagocytosis-induced superoxide production, flavocytochrome b558, p47phox, p67phox, and the FcγIIA receptor were expressed from stable transgenes in COS7 cells. The resulting COSphoxFcγR cells produce high levels of superoxide when stimulated with phorbol ester and efficiently ingest immunoglobulin (Ig)G-coated erythrocytes, but phagocytosis did not activate the NADPH oxidase. COS7 cells lack p40phox, whose role in the NADPH oxidase is poorly understood. p40phox contains SH3 and phagocyte oxidase and Bem1p (PB1) domains that can mediate binding to p47phox and p67phox, respectively, along with a PX domain that binds to phosphatidylinositol-3-phosphate (PI(3)P), which is generated in phagosomal membranes. Expression of p40phox was sufficient to activate superoxide production in COSphoxFcγR phagosomes. FcγIIA-stimulated NADPH oxidase activity was abrogated by point mutations in p40phox that disrupt PI(3)P binding, or by simultaneous mutations in the SH3 and PB1 domains. Consistent with an essential role for PI(3)P in regulating the oxidase complex, phagosome NADPH oxidase activation in primary macrophages ingesting IgG-coated beads was inhibited by phosphatidylinositol 3 kinase inhibitors to a much greater extent than phagocytosis itself. Hence, this study identifies a role for p40phox and PI(3)P in coupling FcγR-mediated phagocytosis to activation of the NADPH oxidase.