Selective detection of NADPH oxidase in polymorphonuclear cells by means of NAD(P)H-based fluorescence lifetime imaging

Abstract

NADPH oxidase (NOX2) is a multisubunit membrane-bound enzyme complex that, upon assembly in activated cells, catalyses the reduction of free oxygen to its superoxide anion, which further leads to reactive oxygen species (ROS) that are toxic to invading pathogens, for example, the fungus Aspergillus fumigatus. Polymorphonuclear cells (PMNs) employ both nonoxidative and oxidative mechanisms to clear this fungus from the lung. The oxidative mechanisms mainly depend on the proper assembly and function of NOX2. We identified for the first time the NAD(P)H-dependent enzymes involved in such oxidative mechanisms by means of biexponential NAD(P)H-fluorescence lifetime imaging (FLIM). A specific fluorescence lifetime of 3670 +/- 140 picoseconds as compared to 1870 picoseconds for NAD(P)H bound to mitochondrial enzymes could be associated with NADPH bound to oxidative enzymes in activated PMNs. Due to its predominance in PMNs and due to the use of selective activators and inhibitors, we strongly believe that this specific lifetime mainly originates from NOX2. Our experiments also revealed the high site specificity of the NOX2 assembly and, thus, of the ROS production as well as the dynamic nature of these phenomena. On the example of NADPH oxidase, we demonstrate the potential of NAD(P)H-based FLIM in selectively investigating enzymes during their cellular function.

Figure 1

(a) Time series of 2D fluorescence images obtained by means of steady-state TPLSM representing a neutrophil granulocyte (PMN) phagocyting one of three conidia of Aspergillus fumigatus. The fluorescence of the CTO stained PMN (red) was observed at 590 nm, while that of the FITC stained fungus (green) was observed at 535 nm. (λ _exc = 800  nm, time step = 30 seconds, time lapse = 10 minutes) (b) 3D fluorescence image of a PMN and phagocytosed conidia. TPLSM results reveal that the fungal spores are readily internalised by the PMNs. The z-step between two consecutive images is 1 μm.

Figure 2

(a) 3D and 2D images obtained by means of steady-state TPLSM showing the endogenous fluorescence of PMNs. The endogenous fluorescence intensity is low in the nuclei and high in small organelles around the nucleus supposed to be mitochondria. (b) Endogenous fluorescence (green, left) and Rhodamine 123 fluorescence (red, centre) are shown to be colocalised in living PMNs (yellow, right). Since Rhodamine 123 accumulates in the mitochondria of living cells, we can conclude that the bright structures in the autofluorescence image are mithocondria. (c) Fluorescence decay curves in different PMNs 10 minutes after NaCN addition (30 μmol/L). While in some cells (left diagram) the decay is monoexponential, that is, only free NAD(P)H, in other cells (right diagram), a biexponential behaviour of the decay has been retrieved, that is, both free and enzyme-bound NAD(P)H is monitored. These results indicate that under the given experimental conditions (λ _exc = 760  nm and fluorescence detection at 460 nm), the endogenous cellular fluorescence mainly originates from the coenzymes NADH and NADPH.

Figure 3

(a) Fluorescence lifetime images of free (τ ₁ image) and enzyme-bound NAD(P)H (τ ₂ image), respectively, as they resulted from the biexponential FLIM experiments performed on PMNs suspended in PBS + 1% FCS. The distributions of τ ₁ and τ ₂, respectively, in these images are shown in the left diagram. The fluorescence lifetime images were obtained by overlapping intensity images in gray scale with the spatially resolved fluorescence lifetime information in colour scale. (b) τ ₁ image (green) and τ ₂ image (red) obtained in FLIM experiments on a mixed suspension of PMNs and aspergillus conidia, as well as, the overlapp of the τ ₁ and τ ₂ images. The overlapped image confirms that conidia contain only free NAD(P)H, that is, their metabolic activity is very low, whereas PMNs contain both free and enzyme-bound NAD(P)H.

Figure 4

Fluorescence lifetime images of enzyme-bound NAD(P)H (τ ₂ images) in naive PMNs (a), in PMNs after addition of PMA (NADPH oxidase activator) (b), in PMNs after addition of AEBSF (NADPH oxidase inhibitor) (c), and in PMNs after addition of AEBSF followed by PMA as a negative control of the stimulation (d). Note that the fluorescence lifetime after addition of PMA (Figure 4(b))—especially in the membrane regions (Figure 4(h))—is longer than in the other images (Figures 4(a), 4(c), 4(d)). The increased average fluorescence lifetime after the stimulation with PMA is confirmed by the corresponding τ ₂-histograms in (e). Statistics over regions with increased τ ₂ in PMNs treated with PMA allowed a quantification of the fluorescence lifetime corresponding to the NADPH bound to NADPH oxidase (Figure 4(f)). The τ ₂-distribution in each region of increased fluorescence lifetime in PMNs treated with PMA was evaluated in order to identify the specific fluorescence lifetime peak of NADPH bound to NADPH oxidase as shown in (g). Figure 4(h) depicts the overlapp of τ ₂-maps of PMNs activated with PMA (green) and of regions of NOX2-specific fluorescence lifetime (3670±170 picoseconds) in the same cells (white). All fluorescence lifetime images were obtained by overlapping intensity images in gray scale with the spatially resolved fluorescence lifetime information in colour scale.

Figure 5

Fluorescence lifetime images of enzyme-bound NAD(P)H (τ ₂ images) in PMNs interacting with aspergillus conidia (a) or with two clusters of aspergillus hyphae (c). Also inhere the fluorescence lifetime images were obtained by overlapping intensity images in gray scale with the spatially resolved fluorescence lifetime information in colour scale. Hence both free and phagocytosed conidia in (a) appear in gray tones indicating the absence of enzyme-bound NAD(P)H, whereas PMNs appear in colour due to the fluorescence lifetime of the enzyme-bound NAD(P)H. The increase of the fluorescence lifetime of bound NAD(P)H at the membrane of the phagosome in a PMN (enlarged detail in (a)) can be easily detected in this way. Also at the contact regions between PMNs and aspergillus hyphae (enlarged detail in (c)), the fluorescence lifetime of enzyme-bound NAD(P)H is increased. In order to quantify the increase in fluorescence lifetime of bound NAD(P)H in PMNs interacting with A. fumigatus, we performed statistics on the τ ₂-distributions of 90 contact regions between PMNs and hyphae and of 80 phagosome membrane regions. The results are depicted in (b) for PMNs interacting with conidia and in (d) for PMNs interacting with hyphae. Note that the average values of both distributions of increased τ ₂ amount to approximately 3600 picoseconds and are similar to the value determined in PMNs treated with PMA, confirming the fact that this fluorescence lifetime is specific for NADPH bound to NADPH oxidase. Furthermore, the well-defined sites of this specific lifetime in PMNs indicate the location of ROS production in these cells.

Figure 6

Time series of fluorescence lifetime images of enzyme-bound NAD(P)H (τ ₂ images) of PMNs interacting with an aspergillus hypha. Note that the cells which are not in contact with the hypha do not show any increase in fluorescence lifetime, while the PMN which contacts the hypha shows the specific 3600 picoseconds lifetime of NADPH bound to NOX2. The time point 0 was arbitrarily chosen.