Does Pioglitazone Lead to Neutrophil Extracellular Traps Formation in Chronic Granulomatous Disease Patients?

Abstract

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme complex responsible for reactive oxygen species (ROS) production, is defective in chronic granulomatous disease (CGD) patients. This enzyme helps in antimicrobial host defense by phagocytes. CGD patients are unable to form neutrophil extracellular traps (NETs), which are composed of granule-derived proteins from neutrophils decorated with decondensed chromatin. Mitochondria have gained attention, being a rich source of flavochrome enzymes due to the presence of several sites for superoxide production. Recently, PPARγ agonists, a mitochondrial ROS inducer, induce mitochondrial ROS formation post-treatment in murine NADPH oxidase knockout models. Mitochondrial ROS is also essential for NOX-independent NETosis. Our study for the first time detects induction of NETosis independent of NADPH oxidase post-treatment with agonists such as pioglitazone and rosiglitazone in CGD subjects. Neutrophils isolated from CGD subjects were treated with pioglitazone and rosiglitazone. After treatment, qualitative analysis of NET formation was done using confocal microscopy after staining with DAPI. Quantitative estimation of extracellular DNA was performed using Sytox green. Mitochondrial ROS production with PPARγ agonist-treated/untreated neutrophils was detected using MitoSOX red. Pioglitazone and rosiglitazone induce significant NET formation in CGD patients. Our data clearly signify the effect of PPARγ agonists in induction of NET formation in CGD cases. Apart from the proposed experimental studies regarding the detailed mechanism of action, controlled trials could provide valuable information regarding the clinical use of pioglitazone in CGD patients as curative HSCT remains challenging in developing countries.

Keywords: MitoSOX red; NOX independnent NETosis; chronic granulomatous disease; mitochondrial ROS; neutrophil extracellular traps; peroxisome proliferator-activated receptor gamma agonists; pioglitazone; reactive oxygen species.

Figure 1

(A) Flow cytometric evaluation of dihydrorhodamine-123 (DHR) assay on neutrophils in control, corresponding mother (to distinguish XL-CGD from autosomal CGD patient), and CGD patients. Unstimulated neutrophils (sky blue) showed no oxidation of dihydrorhodamine-123 (DHR) reagent to rhodamine in contrast to stimulated neutrophils (blue) by PMA. (A-1) Oxidation of DHR in patient P1 representative of autosomal recessive CGD(P1,P2,P3)/de novo X-linked CGD (P5) patient, control, and mother's sample. (A-2) Oxidation of DHR in patient P4-X-linked CGD patient, carrier mother, and control sample. (B) Flow cytometric evaluation of p47^phox and p22^phox expression on neutrophils. Median fluorescent intensities were recorded for stained (blue) and unstained (sky blue) neutrophils in control, patient, and mother's sample. (B-1) Defective p47phox component expression in patient P1, control, and mother. (B-2) Defective p22^phox component expression in patient P5, control, and mother*. (B-3) Defective p22^phox component expression in patient P4, control, and mosaic pattern in mother clearly indicating X-linked defect (gp91^phox defect) in patient P4. *Important to further confirm by molecular characterization in patient and parents.

Figure 2

Induction of NETosis by PPARγ agonists. Human neutrophils from one control (A) and representative CGD (B) patient with homozygous delGT mutation were stimulated with 14 μg/μl of pioglitazone and 15 μg/μl of rosiglitazone for specified time intervals. Cells were treated with or without GW9662 along with PPARγ agonist treatment. Cells were then fixed with 4% PFA and stained with DAPI (nuclear stain) and antibodies against MPO and observed under confocal microscopy (63×). Scale bars, 20 μm. Images are representative of three independent experiments. *NETs formation was observed in a similar way in autosomal recessive and X-linked CGD cases and no difference was observed in both categories. (B) Representative image taken from a P2 patient with homozygous delGT.

Figure 3

(A) Quantitation of NETosis using Sytox green by fluorimetry. Neutrophil extracellular trap (NETs) formation was quantified by fluorimetry after treatment of neutrophils from control and CGD cohort with PPARγ agonists only/PPARγ agonist + antagonist treatment, using 5 μM Sytox green dye. % DNA release was calculated. Graph shows mean ± SD from three independent experiments for each patient. Statistically significant comparisons were obtained by unpaired t-tests and comparisons are as follows: *Respective patient/control cohort compared to untreated cells (p < 0.0001). ^#Respective patient/control cohort compared to respective agonists only (p < 0.001). -C, controls; -P, patients; PMA, phorbol myristate; Cal, calcium ionophore; Pio, pioglitazone; Rosi, rosiglitazone. (B) Quantitation of mitochondrial ROS using MitoSOX red by fluorimetry: PPARγ agonist treatment enhances production of mitochondrial ROS by neutrophils from control and CGD patient neutrophils with or without MitoTempo (Mitochondrial ROS inhibitor)/with or without GW9662 treatment. Mitochondrial ROS was quantified by MitoSOX red and represented as geometric mean. Graph shows mean ± SD from three independent experiments for each subject. *Respective patient/control cohort compared statistically with respective untreated cells of patient/control (p < 0.05). ^#Respective patient/control cohort compared statistically with respective group agonists only (p < 0.05).