title: K⁺-Cl⁻ cotransport mediates the bactericidal activity of neutrophils by regulating NADPH oxidase activation.
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 aCl− 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 36Cl− or 86Rb+, 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.
authors.affiliation: Institute of Clinical Medicine, National Cheng Kung University Hospital, Tainan 704, Taiwan.
authors.forename: Yuan-Ting
authors.initials: YT
authors.lastname: Sun
authors.forename: Chi-Chang
authors.initials: CC
authors.lastname: Shieh
authors.forename: Eric
authors.initials: E
authors.lastname: Delpire
authors.forename: Meng-Ru
authors.initials: MR
authors.lastname: Shen
mesh_terms: D000328:Adult; D000818:Animals; D001770:Blood Bactericidal Activity / Q000276:immunology*; D002264:Carboxylic Acids / Q000494:pharmacology; D045744:Cell Line, Tumor; D002712:Chlorides / Q000378:metabolism; D004789:Enzyme Activation; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D006997:Hypochlorous Acid / Q000378:metabolism; D007113:Immunity, Innate; D007192:Indenes / Q000494:pharmacology; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D019255:NADPH Oxidases / Q000378:metabolism*; D018375:Neutrophil Activation / Q000276:immunology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010587:Phagocytosis; D034741:RNA, Small Interfering; D013203:Staphylococcal Infections / Q000276:immunology; D013481:Superoxides / Q000378:metabolism; D027981:Symporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000096922:K Cl- Cotransporters
chemical_list: D002264:Carboxylic Acids; D002712:Chlorides; D006997:Hypochlorous Acid; D007192:Indenes; D019255:NADPH Oxidases; D034741:RNA, Small Interfering; D013481:Superoxides; D027981:Symporters; C059990:((dihydroindenyl)oxy)alkanoic acid; C492280:Slc12a6 protein, mouse; D000096922:K Cl- Cotransporters