Low-Dose Ionizing γ-Radiation Elicits the Extrusion of Neutrophil Extracellular Traps.

PURPOSE: Patients with cancer frequently undergo radiotherapy in their clinical management with unintended irradiation of blood vessels and copiously irrigated organs in which polymorphonuclear leukocytes circulate. Following the observation that such low doses of ionizing radiation are able to induce neutrophils to extrude neutrophil extracellular traps (NET), we have investigated the mechanisms, consequences, and occurrence of such phenomena in patients undergoing radiotherapy. EXPERIMENTAL DESIGN: NETosis was analyzed in cultures of neutrophils isolated from healthy donors, patients with cancer, and cancer-bearing mice under confocal microscopy. Cocultures of radiation-induced NETs, immune effector lymphocytes, and tumor cells were used to study the effects of irradiation-induced NETs on immune cytotoxicity. Radiation-induced NETs were intravenously injected to mice for assessing their effects on metastasis. Circulating NETs in irradiated patients with cancer were measured using ELISA methods for detecting MPO-DNA complexes and citrullinated histone 3. RESULTS: Irradiation of neutrophils with very low γ-radiation doses (0.5-1 Gy) elicits NET formation in a manner dependent on oxidative stress, NADPH oxidase activity, and autocrine IL8. Radiation-induced NETs interfere with NK cell and T-cell cytotoxicity. As a consequence, preinjection of irradiation-induced NETs increases the number of successful metastases in mouse tumor models. Increases in circulating NETs were readily detected in two prospective series of patients following the first fraction of their radiotherapy courses. CONCLUSIONS: NETosis is induced by low-dose ionizing irradiation in a neutrophil-intrinsic fashion, and radiation-induced NETs are able to interfere with immune-mediated cytotoxicity. Radiation-induced NETs foster metastasis in mouse models and can be detected in the circulation of patients undergoing conventional radiotherapy treatments. See related commentary by Mowery and Luke, p. 3965.

Inhibiting Neutrophil Extracellular Traps Protects against Ultraviolet B-Induced Skin Damage: Effects of Hochu-ekki-to and DNase I.

UV-B radiation induces sunburn, and neutrophils are pivotal in this inflammation. In this study, we examined the potential involvement of neutrophil extracellular traps (NETs) in ultraviolet B (UVB)-induced skin inflammation, correlating the skin inflammation-mitigating effects of Hochu-ekki-to on UV-B irradiation and NETs. To elucidate NET distribution in the dorsal skin, male ICR mice, exposed to UVB irradiation, were immunohistologically analyzed to detect citrullinated histone H3 (citH3) and peptidylarginine deiminase 4 (PAD4). Reactive oxygen species (ROS) production in the bloodstream was analyzed. To establish the involvement of NET-released DNA in this inflammatory response, mice were UV-B irradiated following the intraperitoneal administration of DNase I. In vitro experiments were performed to scrutinize the impact of Hochu-ekki-to on A23187-induced NETs in neutrophil-like HL-60 cells. UV-B irradiation induced dorsal skin inflammation, coinciding with a significant increase in citH3 and PAD4 expression. Administration of DNase I attenuated UV-B-induced skin inflammation, whereas Hochu-ekki-to administration considerably suppressed the inflammation, correlating with diminished levels of citH3 and PAD4 in the dorsal skin. UV-B irradiation conspicuously augmented ROS and hydrogen peroxide (H2O2) production in the blood. Hochu-ekki-to significantly inhibited ROS and H2O2 generation. In vitro experiments demonstrated that Hochu-ekki-to notably inhibited A23187-induced NETs in differentiated neutrophil-like cells. Hence, NETs have been implicated in UV-B-induced skin inflammation, and their inhibition reduces cutaneous inflammation. Additionally, Hochu-ekki-to mitigated skin inflammation by impeding neutrophil infiltration and NETs in the dorsal skin of mice.

Blue and Long-Wave Ultraviolet Light Induce in vitro Neutrophil Extracellular Trap (NET) Formation.

Neutrophil Extracellular Traps (NETs) are produced by neutrophilic granulocytes and consist of decondensed chromatin decorated with antimicrobial peptides. They defend the organism against intruders and are released upon various stimuli including pathogens, mediators of inflammation, or chemical triggers. NET formation is also involved in inflammatory, cardiovascular, malignant diseases, and autoimmune disorders like rheumatoid arthritis, psoriasis, or systemic lupus erythematosus (SLE). In many autoimmune diseases like SLE or dermatomyositis, light of the ultraviolet-visible (UV-VIS) spectrum is well-known to trigger and aggravate disease severity. However, the underlying connection between NET formation, light exposure, and disease exacerbation remains elusive. We studied the effect of UVA (375 nm), blue (470 nm) and green (565 nm) light on NETosis in human neutrophils ex vivo. Our results show a dose- and wavelength-dependent induction of NETosis. Light-induced NETosis depended on the generation of extracellular reactive oxygen species (ROS) induced by riboflavin excitation and its subsequent reaction with tryptophan. The light-induced NETosis required both neutrophil elastase (NE) as well as myeloperoxidase (MPO) activation and induced histone citrullination. These findings suggest that NET formation as a response to light could be the hitherto missing link between elevated susceptibility to NET formation in autoimmune patients and photosensitivity for example in SLE and dermatomyositis patients. This novel connection could provide a clue for a deeper understanding of light-sensitive diseases in general and for the development of new pharmacological strategies to avoid disease exacerbation upon light exposure.

Nanosecond pulsed electric fields induce extracellular release of chromosomal DNA and histone citrullination in neutrophil-differentiated HL-60 cells.

Nanosecond pulsed electric fields (nsPEFs) have gained attention as a novel physical stimulus for life sciences. Although cancer therapy is currently their promising application, nsPEFs have further potential owing to their ability to elicit various cellular responses. This study aimed to explore stimulatory actions of nsPEFs, and we used HL-60 cells that were differentiated into neutrophils under cultured conditions. Exposure of neutrophil-differentiated HL-60 cells to nsPEFs led to the extracellular release of chromosomal DNA, which appears to be equivalent to neutrophil extracellular traps (NETs) that serve as a host defense mechanism against pathogens. Fluorometric measurement of extracellular DNA showed that DNA extrusion was rapidly induced after nsPEF exposure and increased over time. Western blot analysis demonstrated that nsPEFs induced histone citrullination that is the hydrolytic conversion of arginine to citrulline on histones and facilitates chromatin decondensation. DNA extrusion and histone citrullination by nsPEFs were cell type-specific and Ca2+-dependent events. Taken together, these observations suggest that nsPEFs drive the mechanism for neutrophil-specific immune response without infection, highlighting a novel aspect of nsPEFs as a physical stimulus.

Near infrared laser irradiation induces NETosis via oxidative stress and autophagy.

NETosis is a novel immune defense strategy in which neutrophil activation results in the formation of extracellular DNA/protein network which is able to kill microbial populations. NETosis can be induced in vitro by lipopolysaccharide (LPS) or phorbol myristate acetate (PMA). Due to the importance of NETosis in different physiological and pathological processes, photobiostimulation effect on this neutrophil activation mechanism has been investigated. Human granulocytes, isolated from venous blood of healthy donors, were stimulated with a diode laser emitting at 980 nm with an energy intensity ranging from 0 to 75 joules. After 3 h of laser stimulation, granulocytes were fixed and colored with crystal violet in order to assess the NETosis morphology while extracellular DNA produced has been quantified using Sytox Green fluorescent dye. To evaluate ROS production and autophagy role in photobiostimulation-induced NETosis, granulocytes were pre-treated with ROS scavengers (vitamin C, sodium pyruvate, L-NAME, sodium azide), and an autophagy inhibitor (wortmannin). Laser stimulation induced an energy-dependent neutrophil extracellular trap (NET) production in human granulocytes starting from 50-J laser intensity. ROS scavengers and the autophagy inhibitor were able to abrogate both morphological features of NETosis and extracellular DNA production without modifying the basal level of NETosis. Photobiostimulation induced an increase in NET production due to an increase in ROS levels and autophagy activation.

Effect of Continuous-Wave Low-Intensity Ultrasound in Inflammatory Resolution of Arthritis-Associated Synovitis.

BACKGROUND: Low-intensity ultrasound (LIUS) can reduce pain and improve function in arthritic joints. Neutrophils are first-line actors in host defense that recruit macrophages. Dead neutrophils are removed during resolution of inflammation. Delayed neutrophil clearance can lead to extended inflammation or even chronic autoimmune disease. Although neutrophil extracellular traps (NETs) in arthritic tissue are involved in the pathogenesis of arthritis, their functional role has not been clarified. OBJECTIVES: This study aimed to investigate the effect of LIUS on synovial inflammation and its resolution via neutrophil clearance. METHODS: Synovitis was induced by intra-articular injection of complete Freund's adjuvant (CFA) into the left knee joint of 58 adult male Sprague-Dawley rats. Low-intensity ultrasound (1 MHz, 200 mW/cm(2)) was applied for 10 minutes daily. Neutrophil clearance was assessed with the expression of myeloperoxidase (MPO). In addition, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and NET formation in the synovium were observed. In neutrophil and macrophage cultures from peripheral blood, the effect of NET clearance by LIUS was investigated. RESULTS: In CFA-induced synovitis, MPO-positive neutrophils peaked after 2 to 3 days, filling the inflammatory core. Monocytes and macrophages in the periphery later infiltrated the core and were reduced thereafter. Low-intensity ultrasound reduced synovial hyperplasia and induced earlier MPO clearance. Neutrophils in the core of the inflamed synovium exhibited NET formation, which LIUS increased. Low-intensity also induced NETs in peripheral polymorphonuclear cells in an intensity-dependent manner and potentiated phorbol myristate acetate (PMA)-induced NETosis. The PMA-induced NETs were cleared by macrophages; clearance was enhanced by LIUS. LIMITATIONS: The effect of LIUS on CFA-induced inflammation was observed only during the acute phase. Although the effect of LIUS on NETosis in the in vitro neutrophil culture system was clear, the in vivo NETosis cannot be quantified. CONCLUSIONS: Neutrophil extracellular traps act in inflammatory synovitis, and LIUS enhanced the NETs and resulted in neutrophil clearance by enhancing the phagocytosis of macrophages, which might be a factor underlying the therapeutic effect of LIUS in arthritic synovium.