Pioglitazone restores phagocyte mitochondrial oxidants and bactericidal capacity in chronic granulomatous disease

Abstract

Background: Deficient production of reactive oxygen species (ROS) by the phagocyte nicotinamide adenine dinucleotide (NADPH) oxidase in patients with chronic granulomatous disease (CGD) results in susceptibility to certain pathogens secondary to impaired oxidative killing and mobilization of other phagocyte defenses. Peroxisome proliferator-activated receptor (PPAR) γ agonists, including pioglitazone, approved for type 2 diabetes therapy alter cellular metabolism and can heighten ROS production. It was hypothesized that pioglitazone treatment of gp91(phox-/-) mice, a murine model of human CGD, would enhance phagocyte oxidant production and killing of Staphylococcus aureus, a significant pathogen in patients with this disorder.

Objectives: We sought to determine whether pioglitazone treatment of gp91(phox-/-) mice enhanced phagocyte oxidant production and host defense.

Methods: Wild-type and gp91(phox-/-) mice were treated with the PPARγ agonist pioglitazone, and phagocyte ROS and killing of S aureus were investigated.

Results: As demonstrated by 3 different ROS-sensing probes, short-term treatment of gp91(phox-/-) mice with pioglitazone enhanced stimulated ROS production in neutrophils and monocytes from blood and neutrophils and inflammatory macrophages recruited to tissues. Mitochondria were identified as the source of ROS. Findings were replicated in human monocytes from patients with CGD after ex vivo pioglitazone treatment. Importantly, although mitochondrial (mt)ROS were deficient in gp91(phox-/-) phagocytes, their restoration with treatment significantly enabled killing of S aureus both ex vivo and in vivo.

Conclusions: Together, the data support the hypothesis that signaling from the NADPH oxidase under normal circumstances governs phagocyte mtROS production and that such signaling is lacking in the absence of a functioning phagocyte oxidase. PPARγ agonism appears to bypass the need for the NADPH oxidase for enhanced mtROS production and partially restores host defense in CGD.

Keywords: Chronic granulomatous disease; mitochondria; oxidants; phagocytes; thioglitazones.

Conflict of interest statement

Conflict of interest: The authors have no conflict of interest to declare.

Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1. Pio pretreatment of mice enhances PMA-stimulated ROS production by gp91phox−/− neutrophils and monocytes

ROS measured by DHR in blood leukocytes from vehicle(V)- or Pio(P)-treated WT (WV, WP) and gp91phox−/− mice (CV, CP): A) Representative dot-plots, gray, no PMA; black, with PMA are shown. (B) Representative histograms for neutrophils and monocytes: gray, no PMA; black, with PMA. Aggregate data for neutrophils (C) and monocytes (D) are shown as percent of cells exhibiting enhanced fluorescence and change in geometric mean fluorescence with PMA. N = 8 mice/group.

Figure 2. Pio pretreatment enhances superoxide production by recruited phagocytes from gp91phox−/− mice

Ten hours after intraperitoneal zymosan injection, phagocytes were harvested from vehicle- or Pio-treated WT (WV, WP) or gp91phox−/− (CV, CP) mice. Phagocytes were stimulated with PMA in the presence and absence of inhibitors, and oxidants measured by the reduction of cytochrome c. N = 9 mice/group.

Figure 3. Pio pretreatment of gp91phox−/− mice enhances production of mtROS in recruited phagocytes

Recruited phagocytes as in Fig. 2 from vehicle- or Pio-treated WT (WV, WP) and gp91phox−/− (CV, CP) mice, were stained with MitoTracker Green, MitoSOX Red and DAPI, and analyzed by confocal microscopy (63X). Last panel: high-resolution images of a single cell (white box). Arrows denote ingested zymosan. Representative images are shown. N=3 mice/group.

Figure 4. Pio treatment enhances production of mtROS by neutrophils (A) and macrophages (B) harvested from inflamed peritonea of WT and gp91phox−/− mice

Recruited phagocytes from mice (WV, WP, CV, CP) as in Fig. 2 and 3 were treated with or without DPI or MitoTEMPO (15 min), stained with MitoSOX and then stimulated with or without PMA, and analyzed by flow cytometry. Representative histograms without PMA, left; line depicts relative fluorescence of CV neutrophils or macrophages. Aggregate data, right. N=8 mice/group. p ≤ 0.02 for comparisons with *WV without PMA, αCV without PMA, #respective genotype/treatment group without PMA, and δrespective genotype/treatment group with PMA alone.

Figure 5. Pio treatment of gp91phox−/− mice enhances production of mtROS by stimulated blood neutrophils (A) and monocytes (B)

Blood leukocytes from WT (WV, WP) and gp91phox−/− (CV, CP) mice as in Fig. 1 were treated with and without DPI or MitoTEMPO for 15 min, stained with MitoSOX Red, stimulated or not with PMA and analyzed by flow cytometry. Representative histograms, left, and aggregate data, right. N=8 mice/group. p ≤ 0.02 for comparisons with γWV with PMA, πCV with PMA and δrespective genotype/treatment group with PMA alone.

Figure 6. Ex vivo treatment of human CGD monocytes with Pio enhances stimulated mtROS production

Human monocytes from X-linked CGD and normal subjects were isolated from heparinized blood, plated, and treated with 10 μM Pio for 2 days. The cells were then stained with MitoSOX Red and stimulated with PMA in the presence or absence of MitoTEMPO and analyzed by flow cytometry; Representative histograms (A) and aggregate data normalized to each individual untreated control (B) are shown. N=9 and 7 for normal subjects (NL) and CGD patients, respectively.

Figure 7. Recruited phagocytes from Pio-treated gp91phox−/− mice show enhanced killing of S. aureus ex vivo

Recruited, peritoneal phagocytes from WV, WP, CV, CP mice as in Fig. 2 were treated with or without SOD or MitoTEMPO for 30 min, co-incubated with S. aureus (1×106 CFU) at 37°C for 1h. The percent of bacteria killed was determined. p≤0.02 *compared to phagocytes from WV and δcompared to phagocytes from respective treatment group in the absence of inhibitors. N=6 mice/group.

Figure 8. Pio treatment of gp91phox−/− mice enhances clearance of S. aureus in vivo

Vehicle or Pio-treated WT (WV, WP) and gp91phox−/− (CV, CP) mice were injected intraperitoneally with S. aureus, and peritonea lavaged at 24h and 48h. A). Bacterial numbers in lavage were determined. p ≤ 0.02 for comparisons with *WV and δCV. B–C). CFU data are depicted on a linear scale for clarity. N=16 and 11 mice/group at 24h and 48h, respectively.