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.

Figure 1. K⁺ and Cl⁻ efflux in human neutrophils

LPS or PMA increased the Cl⁻ (A and B) and K⁺ (C and D) efflux in human neutrophils. The increased K⁺ or Cl⁻ efflux was significantly inhibited by DIOA, a KCC inhibitor. K⁺ efflux was determined using ⁸⁶Rb⁺ as a congener. The medium for ³⁶Cl⁻ and ⁸⁶Rb⁺ efflux assays contained 0.1 mm ouabain and 0.01 mm bumetanide to inhibit the Na⁺–K⁺ pump and NKCC activities, respectively. The detailed protocols for neutrophil stimulation and flux measuarement were described in Methods. Each value represents the mean ± SEM (n= 5). *P≤ 0.05, **P≤ 0.01. DIOA, dihydroindenyloxy alkanoic acid; LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate.

Figure 2. The KCC inhibitor, but not NKCC inhibitor, diminished the ROS production in human neutrophils

A, the representative real-time cumulated ROS recordings by the chemiluminescence assay. After pre-incubation with DIOA or 0.1% DMSO at 37°C for 45 min, human neutrophils were then stimulated by PMA (10 nm) for 10 min. Lucigenin (arrow) was injected to detect the NADPH oxidase activity in human neutrophils. The control group was neutrophils treated with 0.1% DMSO alone without PMA stimulation. B, dose–response curves showing the effect of DIOA or bumetanide on PMA-provoked ROS generation in human neutrophils. C and D, effect of DIOA (50 μm) or bumetanide (20 μm) on fMLP or LPS-stimulated ROS generation in human neutrophils. The control group was neutrophils treated with 0.1% DMSO alone without toxin stimulation. The ROS production in each experimental group was expressed relative to control group. Each value represents the mean ± SEM from at least five different samples. RLU, relative luminescence unit. *P≤ 0.05; **P≤ 0.01.

Figure 3. KCC activity regulated the NADPH oxidase of human neutrophil

A, the phosphorylation of phox units in human neutrophils. A representative immunoblot showed the phosphorylation levels of p40^phox in human neutrophils at various experimental conditions. The control group was neutrophils treated with 0.1% DMSO alone. The detailed protocols for neutrophil stimulation were described in Methods. B and C, PMA induced the membrane recruitments of phox units (p47^phox, p40^phox and p67^phox). PMA caused the puncta formation of p47^phox, especially at the membrane periphery (B). DIOA, but not bumetanide, reduced the PMA-provoked membrane recruitment of p47^phox proteins. Other oxidase components such as p40^phox and p67^phox also remained diffusely distributed in cytoplasm during latency. PMA induced the assembly of p40^phox and p67^phox into complexes that were recruited to the juxta-membrane region (C). D, the quantitative analyses for the membrane recruitment of different phox units at various experimental conditions. Each value represents the mean ± SEM from three different experiments by analysing at least 30 cells. *P≤ 0.05, **P≤ 0.01, ***P≤ 0.005. Scale bar, 2 μm.

Figure 4. KCC3 knockdown inhibited NADPH oxidase activation in HL-60 cells

A, NADPH oxidase activity as indicated by ROS generation in wild-type, shLuciferase (shLuc) shKCC1, shKCC3 and shKCC4 HL-60 cells. The ROS production in wild-type HL-60 cells without PMA stimulation was used as the control and the others were expressed relative to the control. B, a representative immunoblot of phosphorylated p40^phox in wild-type, shLuciferase (shLuc), shKCC1, shKCC3 and shKCC4 HL-60 cells from three independent experiments. C, the membrane recruitment of p47^phox was significantly diminished in shKCC3 cells in response to PMA stimulation. Representative images were from three different experiments. ***P≤ 0.005; scale bar: 2 μm; N: nucleus; arrow: membrane recruitment of p47^phox.

Figure 5. The distribution of KCC3, NADPH oxidase and phagosomes in human neutrophils

A and B, with PMA stimulation, KCC3, p40^phox and p47^phox were recruited to the juxta-membrane area, where KCC3, phox units and actin formed a complex. C, KCC3 could be re-located to phagosomes in activated neutrophils. In resting neutrophils, very few KCC3 and EEA1 (an early endosomal marker) were localized together. Activated neutrophils displayed a significant colocalization of KCC3 and EEA1. D, the quantitative analyses for the colocaliztion of KCC3 and EEA1. Each value represented the mean ± SEM from three independent experiments by analysing at least 25 cells. **P≤ 0.01; scale bar: 2 μm.

Figure 6. NADPH oxidase activity in KCC3^+/+ and KCC3^−/− murine neutrophils

A, the representative real-time cumulated ROS recordings by the chemiluminescence assay in KCC3^+/+ and KCC3^−/− murine neutrophils with or without 10 nm PMA stimulation. The dotted arrow indicated the adding of lucigenin. The control group was neutrophils treated with 0.1% DMSO alone. B, impaired ROS production in KCC3^−/− murine neutrophils. The area under the curve was calculated as the total ROS production for a 30 min period after adding lucigenin. The ROS production in KCC3^+/+ group without PMA stimulation was used as the control and the others were expressed relative to the control. Each value represented the mean ± SEM (n= 6). C, a representative immunoblot of phosphorylated p40^phox in KCC3^+/+ and KCC3^−/− neutrophils from three independent experiments. The phosphorylation level of p40^phox in the KCC3^+/+ group without PMA stimulation was used as the control and the others were expressed relative to the control.

Figure 7. KCC3-deficient mice were more susceptible to infectious challenge

A, Kaplan–Meier curves for KCC3^+/+ and KCC3^−/− mice after intraperitoneal injection with 2.36 × 10⁸ CFU S. aureus. B and C, on the 7th day, all surviving mice were killed and their gastrointestinal tracts were examined. KCC3^−/− mice showed distended intestines with stool impaction (arrows), hyperaemic peritoneum (arrowheads), pus formation (star), and caseation (dashed arrows), which were rarely observed in KCC3^+/+ mice. Scale bar: 1 cm. D, unlike the bowel of the KCC3^+/+ strain (upper panel), that of the KCC3^−/− strain showed an increased number of enlarged lymph nodes (lower panel, arrows). Scale bar: 1 cm. E, determination of the number of enlarged intestinal lymph nodes. Each bar represented the mean ± SEM (n= 9 and 10 for KCC3^−/− and KCC3^+/+ mice, respectively). F, haematoxylin–eosin stained sections of the same part of the intestine showed prominent germinal centres (arrows) with abundant internal apoptotic bodies in KCC3^−/− mice (lower panel) but not in KCC3^+/+ mice (upper panel). Scale bar: 200 μm.

Figure 8. A working model for KCC in the regulation of bactericidal activity

In the resting status of neutrophil, KCC (at least KCC3) and the membrane components of NADPH oxidase are diffusely expressed. 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. The KCC family might be one of the Cl⁻ permeation pathways providing the substrate for the generation of OCl⁻ in phagosomes.