Real-time redox adaptations in human airway epithelial cells exposed to isoprene hydroxy hydroperoxide

Abstract

While redox processes play a vital role in maintaining intracellular homeostasis by regulating critical signaling and metabolic pathways, supra-physiological or sustained oxidative stress can lead to adverse responses or cytotoxicity. Inhalation of ambient air pollutants such as particulate matter and secondary organic aerosols (SOA) induces oxidative stress in the respiratory tract through mechanisms that remain poorly understood. We investigated the effect of isoprene hydroxy hydroperoxide (ISOPOOH), an atmospheric oxidation product of vegetation-derived isoprene and a constituent of SOA, on intracellular redox homeostasis in cultured human airway epithelial cells (HAEC). We used high-resolution live cell imaging of HAEC expressing the genetically encoded ratiometric biosensors Grx1-roGFP2, iNAP1, or HyPer, to assess changes in the cytoplasmic ratio of oxidized glutathione to reduced glutathione (GSSG:GSH), and the flux of NADPH and H₂O₂, respectively. Non-cytotoxic exposure to ISOPOOH resulted in a dose-dependent increase of GSSG:GSH in HAEC that was markedly potentiated by prior glucose deprivation. ISOPOOH-induced increase in glutathione oxidation were accompanied by concomitant decreases in intracellular NADPH. Following ISOPOOH exposure, the introduction of glucose resulted in a rapid restoration of GSH and NADPH, while the glucose analog 2-deoxyglucose resulted in inefficient restoration of baseline GSH and NADPH. To elucidate bioenergetic adaptations involved in combatting ISOPOOH-induced oxidative stress we investigated the regulatory role of glucose-6-phosphate dehydrogenase (G6PD). A knockout of G6PD markedly impaired glucose-mediated recovery of GSSG:GSH but not NADPH. These findings reveal rapid redox adaptations involved in the cellular response to ISOPOOH and provide a live view of the dynamic regulation of redox homeostasis in human airway cells as they are exposed to environmental oxidants.

Keywords: Air pollution; Glutathione; Isoprene hydroxy hydroperoxide; Live cell imaging; NADPH; Oxidative stress.

Graphical abstract

Fig. 1

The availability of glucose modulates ISOPOOH-induced oxidation of glutathione in HAEC. The ratio of oxidized glutathione to reduced glutathione (GSSG:GSH) was monitored using live cell confocal microscopy of HAEC expressing Grx1-roGFP2 that were pre incubated in glucose supplied (A) medium for 30 min or glucose deficient (B) medium for 2 h prior to exposure to 1–9 μM ISOPOOH. Following the acquisition of a baseline signal, vehicle, 1–9 μM ISOPOOH, and 1 mM glucose were added at the indicated times. Emitted fluorescence intensity values are shown normalized to their respective baselines and expressed as the ratio of signal induced by 405/488 nm excitation. Data are expressed as MEAN ± SEM for n = 3–5 independent experiments in which the responses from 10 individual cells were averaged for each experiment.

Fig. 2

Exposure of HAEC to ISOPOOH dose-dependently reduces intracellular NADPH. NADPH flux was monitored using live cell confocal microscopy of HAEC expressing iNAP1 that were pre incubated in glucose deficient medium for 2 h prior to exposure to 1–9 μM ISOPOOH. Following the acquisition of a baseline signal, vehicle, 1–9 μM ISOPOOH, and 1 mM glucose were added at the indicated times. Emitted fluorescence intensity values are shown normalized to their respective baselines and expressed as the ratio of signal induced by 405/488 nm excitation. Data are expressed as MEAN ± SEM for n = 5–6 independent experiments in which the responses from 10 individual cells were averaged for each experiment.

Fig. 3

Glucose dose-dependently reverses glutathione oxidation and NADPH flux in HAEC following ISOPOOH exposure. The ratio of oxidized glutathione to reduced glutathione (GSSG:GSH) and NADPH flux was monitored using live cell confocal microscopy of HAEC expressing Grx1-roGFP2 (A) or iNAP1 (B). HAEC were pre incubated in glucose deficient medium for 2 h prior to exposure to 9 μM ISOPOOH. Following the acquisition of a baseline signal, vehicle, 9 μM ISOPOOH, and 10–90 μM glucose were added at the indicated times. Emitted fluorescence intensity values are shown normalized to their respective baselines and expressed as the ratio of signal induced by 405/488 nm excitation. Data are expressed as MEAN ± SEM for n = 5 independent experiments where the response from 10 individual cells were averaged for each experiment.

Fig. 4

Introduction of 2-deoxyglucose (2-DG) inefficiently reverses glutathione oxidation and decrements in NADPH in HAEC exposed to ISOPOOH. The ratio of oxidized glutathione to reduced glutathione (GSSG:GSH) and NADPH flux was monitored using live cell confocal microscopy of HAEC expressing Grx1-roGFP2 (A) or iNAP1 (B). HAEC were pre incubated in glucose deficient medium for 2 h prior to exposure to 9 μM ISOPOOH. Following the acquisition of a baseline signal, vehicle, 1–9 μM ISOPOOH, and 1 mM 2-DG were added at the indicated times. Emitted fluorescence intensity values are shown normalized to their respective baselines and expressed as the ratio of signal induced by 405/488 nm excitation. Data are expressed as MEAN ± SEM for n = 5–6 independent experiments in which the responses from 10 individual cells were averaged for each experiment.

Fig. 5

Dampened expression of glucose-6-phosphate dehydrogenase (G6PD) impairs spontaneous recovery of glutathione oxidation and NADPH following ISOPOOH exposure of HAEC. The ratio of oxidized glutathione to reduced glutathione (GSSG:GSH) and NADPH flux was monitored using live cell confocal microscopy of G6PD KD HAEC expressing Grx1-roGFP2 (A) or iNAP1 (B). G6PD KD HAEC were pre incubated in glucose deficient medium for 2 h prior to exposure to 1–9 μM ISOPOOH. Following the acquisition of a baseline signal, vehicle, 1–9 μM ISOPOOH, and 1 mM glucose were added at the indicated times. Emitted fluorescence intensity values are shown normalized to their respective baselines and expressed as the ratio of signal induced by 405/488 nm excitation. Data are expressed as MEAN ± SEM for n = 5–6 independent experiments where the responses from 10 individual cells were averaged for each experiment.

Fig. 6

A knockout of glucose-6-phosphate dehydrogenase (G6PD) in a BALB/c cell model impairs glucose-mediated recovery of glutathione oxidation following ISOPOOH exposure. The ratio of oxidized glutathione to reduced glutathione (GSSG:GSH) and NADPH flux was monitored using live cell confocal microscopy of G6PD knockout (A, C) or wild-type BALB/c cells (B, D), respectively. BALB/c cells expressing Grx1-roGFP2 (A, B) or iNAP1 (C, D) were pre incubated in glucose deficient medium for 2 h prior to exposure to 1–9 μM ISOPOOH. Following the acquisition of a baseline signal, vehicle, 1–9 μM ISOPOOH, and 1 mM glucose were added at the indicated times. Emitted fluorescence intensity values are shown normalized to their respective baselines and expressed as the ratio of signal induced by 405/488 nm excitation. Data are expressed as MEAN ± SEM for n = 3–5 independent experiments where the responses from 10 individual cells were averaged for each experiment.

Fig. 7

Exposure to ISOPOOH induces glutathione oxidation in fully differentiated primary human airway epithelial cells cultured at an air-liquid-interface (pHAEC-ALI). The ratio of oxidized glutathione to reduced glutathione (GSSG:GSH) was monitored using live cell confocal microscopy of pHAEC-ALI expressing Grx1-roGFP2 that were pre incubated in glucose deficient medium for 2 h prior to exposure to 10–90 μM ISOPOOH. Following the acquisition of a baseline signal, vehicle, 10–90 μM ISOPOOH, and 1 mM glucose were added at the indicated times. Emitted fluorescence intensity values are shown normalized to their respective baselines and expressed as the ratio of signal induced by 405/488 nm excitation. Data are expressed as MEAN ± SEM for n = 5–6 independent experiments where the response from 10 individual cells were averaged for each experiment.