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K + –Cl − cotransport mediates the bactericidal activity of neutrophils by regulating NADPH oxidase activation
Author(s) -
Sun YuanTing,
Shieh ChiChang,
Delpire Eric,
Shen MengRu
Publication year - 2012
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2011.225300
Subject(s) - nadph oxidase , phagosome , superoxide , nicotinamide adenine dinucleotide phosphate , phagocytosis , chemistry , hypochlorous acid , oxidase test , biochemistry , innate immune system , myeloperoxidase , biology , microbiology and biotechnology , reactive oxygen species , intracellular , enzyme , receptor , inflammation , immunology
Key points• Neutrophilic phagocytosis is essential for innate immunity against the bacterial infection. • During phagocytosis, the generation of bactericidal hypochlorous acid requires superoxide produced by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to kill the internalized pathogens. • We show that NADPH oxidase complexes are associated with K + –Cl − cotransporter (KCC) at the plasma membrane of activated neutrophils and are internalized to form phagosomes, where KCC activity and expression level affect the production of oxidants. • This study supports the notion that KCC, in particular KCC3, is involved in the early stages of the host's defence against microorganisms.Abstract Neutrophilic phagocytosis is an essential component of innate immunity. During phagocytosis, the generation of bactericidal hypochlorous acid (HOCl) requires the substrates, Cl − and superoxide produced by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to kill the internalized pathogens. Here we show that the neutrophilic K + –Cl − cotransporter (KCC) constitutes a Cl − permeation pathway and mediates the bactericidal activity by regulating NADPH oxidase activation. Dihydroindenyloxy alkanoic acid (DIOA), a KCC inhibitor, suppressed the toxin‐ or chemical‐induced efflux of 36 Cl − or 86 Rb + , and diminished the production of superoxide in human and murine neutrophils. Inhibition of KCC activity or knockdown of KCC expression, in particular KCC3, reduced the phosphorylation as well as the membrane recruitment of oxidase components. Activated neutrophils displayed a significant colocalization of KCC3 and early endosomal marker, indicating that KCC3 could be localized on the phagosomes once neutrophils are activated. The NADPH oxidase activity and the phosphorylation level of oxidase component were 50% lower in the neutrophils isolated from KCC3 −/− mice than in the neutrophils isolated from KCC3 +/+ mice. Mortality rate after intraperitoneal challenge with Staphylococcus aureus was higher in KCC3 −/− mice, and the bacterial clearance was impaired in the survivors. We conclude that, in activated neutrophil, NADPH oxidase complexes are associated with KCC3 at the plasma membrane and are internalized to form phagosomes, where KCC activity and expression level affect the production of oxidants.