K+-Cl- cotransport mediates the bactericidal activity of neutrophils by regulating NADPH oxidase activation

Research output: Contribution to journalArticle

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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 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.

Original languageEnglish
Pages (from-to)3231-3243
Number of pages13
JournalJournal of Physiology
Volume590
Issue number14
DOIs
Publication statusPublished - 2012 Jul 1

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NADP
Oxidoreductases
Neutrophils
Phagosomes
Phagocytosis
Superoxides
Phosphorylation
Hypochlorous Acid
Innate Immunity
Oxidants
Survivors
Staphylococcus aureus
Cell Membrane
potassium-chloride symporters
Acids
Membranes
Mortality

All Science Journal Classification (ASJC) codes

  • Physiology

Cite this

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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 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 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.",
author = "Yuan-Ting Sun and Chi-Chang Shieh and Eric Delpire and Meng-Ru Shen",
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K+-Cl- cotransport mediates the bactericidal activity of neutrophils by regulating NADPH oxidase activation. / Sun, Yuan-Ting; Shieh, Chi-Chang; Delpire, Eric; Shen, Meng-Ru.

In: Journal of Physiology, Vol. 590, No. 14, 01.07.2012, p. 3231-3243.

Research output: Contribution to journalArticle

TY - JOUR

T1 - K+-Cl- cotransport mediates the bactericidal activity of neutrophils by regulating NADPH oxidase activation

AU - Sun, Yuan-Ting

AU - Shieh, Chi-Chang

AU - Delpire, Eric

AU - Shen, Meng-Ru

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N2 - 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 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.

AB - 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 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.

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