Radiation from UV-A to Red Light Induces ROS-Dependent Release of Neutrophil Extracellular Traps

Abstract

Neutrophils release decondensed chromatin or extracellular traps (NETs) in response to various physiological and pharmacological stimuli. Apart from host defensive functions, NETs play an essential role in the pathogenesis of various autoimmune, inflammatory, and malignant diseases. In recent years, studies have been performed on photo-induced NET formation, mainly activated by UV radiation. Understanding the mechanisms of NET release under the influence of UV and visible light is important to control the consequences of the damaging effects of electromagnetic radiation. Raman spectroscopy was applied to record characteristic Raman frequencies of various reactive oxygen species (ROS) and low-frequency lattice vibrational modes for citrulline. NETosis was induced by irradiation with wavelength-switchable LED sources. Fluorescence microscopy was used to visualize and quantify NET release. The ability of five wavelengths of radiation, from UV-A to red light, to induce NETosis was investigated at three different energy doses. We demonstrated, for the first time, that NET formation is activated not only by UV-A but also by three spectra of visible light: blue, green, and orange, in a dose-dependent manner. Using inhibitory analysis, we established that light-induced NETosis proceeds through NADPH oxidase and PAD4. The development of new drugs designed to suppress NETosis, especially when induced by exposure to intense UV and visible light, can help to mitigate light-induced photoaging and other damaging effects of electromagnetic radiation.

Keywords: Raman spectroscopy; UV and visible light; cytochrome_b558; neutrophil extracellular traps; neutrophils; photo stimulation; reactive oxygen species.

Figure 1

Micrograph (left), Raman map (middle), and spectrum (right) of neutrophils exposed to radiation with a wavelength of 625 nm and a dose of 32 J/cm². The characteristic sharp lines of H₂O₂ can be seen on the right. The spectral gate was aligned within the interval of 872–882 cm⁻¹ with excitation at 632.8 nm. Scan area 24 × 24 μm, bars: 5 μm.

Figure 2

Raman spectrum of neutrophils exposed to radiation with a wavelength of 405 nm and a dose of 32 J/cm². The characteristic Raman frequency of 732 cm⁻¹ for HClO can be seen.

Figure 3

Raman spectrum of neutrophils exposed to radiation with a wavelength of 405 nm and a dose of 32 J/cm². The characteristic lattice vibrational modes of citrulline can be seen.

Figure 4

The release of NETs depending on dose at wavelengths of 365 and 625 nm. Freshly isolated neutrophils of healthy donors were irradiated with LED-lengths of 365 and 625 nm (UV-A and orange light spectrum, respectively) and doses 4, 16, and 32 J/cm². NET formation was registered after 3 h incubation at 37 °C and 5% CO₂ using fluorescence microscopy. The data represent the mean ± SD from five independent experiments (n = 5). Statistically significant p values are indicated as follows: * p < 0.05, and *** p < 0.001 (A). The corresponding representative images of neutrophils irradiated with LED-length of 365 and 625 nm or stimulated with PMA are shown. Bars: 50 µm (B).

Figure 5

Effects of selective inhibitors of NADPH oxidase and PAD4 on NET formation in neutrophils irradiated with five LED-lengths and the same energy dose. Neutrophils isolated from the blood of healthy donors were treated with selective inhibitors of NADPH oxidase (apocynin, 400 µM) and PAD4 (GSK484, 10 µM) for 30 min. NET formation was induced by irradiation with LED-lights of 365, 405, 530, 625, and 656 nm and the same energy dose of 32 J/cm². PMA (50 nM) was used as a positive control. After incubation for 3 h, the cells were stained with DAPI and analyzed using fluorescence microscopy. The data represent the mean ± SD from five independent experiments (n = 5). Statistically significant p values are indicated as follows: * p < 0.05, ** p < 0.01, and **** p < 0.0001. Insignificant differences are marked as “ns” (A). Representative fluorescence images of NET formation after neutrophil treatment with inhibitors and irradiation with LED-lengths are shown. Scale bars: 50 µm (B).

Figure 6

The proposed mechanism of NETosis induced by UV-A and wavelengths of visible light. (A) Cytochrome_b₅₅₈, being the catalytic electron transport part of NADPH oxidase, absorbs UV-A, blue, green, and orange light, which leads to the activation of NADPH oxidase and the following ROS generation (B). ROS activate the translocation of enzymes NE and MPO from the azurosomes of azuphilic granules to the cytoplasm and their subsequent migration to the nucleus (C), where—together with PAD4 (D)—they promote the decondensation of nuclear chromatin and NETosis. Inhibition of NADPH oxidase by apocynin and PAD4 by GSK484 leads to suppression of NETosis.