Sodium fluoride exposure triggered the formation of neutrophil extracellular traps

Jing-Jing Wang a, b, Zheng-Kai Wei a, Zhen Han b, Zi-Yi Liu b, Yong Zhang b, Xing-Yi Zhu a, Xiao-Wen Li a, Kai Wang a, Zheng-Tao Yang a

Abstract

In recent years, numerous studies paid more attention to the molecular mechanisms associated with fluoride toxicity. However, the detailed mechanisms of fluoride immunotoxicity in bovine neutrophils remain unclear. Neutrophil extracellular traps (NETs) is a novel immune mechanism of neutrophils. We hypothesized that sodium fluoride (NaF) can trigger NETs activation and release, and investigate the related molecular mechanisms during the process. We exposed peripheral blood neutrophils to 1 mM NaF for 120 min in bovine neutrophils. The results showed that NaF exposure triggered NET-like structures decorated with histones and granule proteins. Quantitative measurement of NETs content correlated positively with the concentration of NaF. Mechanistically, NaF exposure increased reactive oxygen species (ROS) levels and phosphorylation levels of ERK, p38, whereas inhibiting the activities of superoxide dismutase (SOD) and catalase (CAT) compared with control neutrophils. NETs formation is induced by NaF and this effect was inhibited by the inhibitors diphenyleneiodonium chloride (DPI), U0126 and SB202190. Our findings described the potential importance of NaF-triggered NETs related molecules, which might help to extend the current understanding of NaF immunotoxicity.

Keywords:
Sodium fluoride
Reactive oxygen species
Neutrophil extracellular traps
Immunotoxicity

1. Introduction

Fluorine is an essential trace element and is widely dispersed in the environment, occurring naturally in the air, water, soil, plants and animals, as well as cosmetic and industrial products (Tylenda, 2003). It is well known that fluoride has an important role for the integrity of bone and teeth, especially dental caries prevention. Appropriate fluoride exposure and usage has a positive impact on health, but chronic long-term or excessive exposure of fluoride may lead to dental fluorosis, skeletal fluorosis, non-skeletal manifestations, or combinations of these maladies. Moreover, fluoride toxicity targets to not only bone and teeth, but also soft tissues, several studies have reported the harmful effects of fluoride on the respiratory (Fina et al., 2014), heart (Tuluce et al., 2017; Wang et al., 2017a), kidney (Dharmaratne, 2015), and may potentially lead to cancer. Excessive fluoride exposure also lead to dysfunctions of cardiovascular system (Oyagbemi et al., 2018), reproductive system (Wang et al., 2017b), nervous system (Li et al., 2019), immune system (De la Fuente et al., 2016) in humans and animals. At higher concentration, NaF inhibited myotube formation, increased skeletal muscle catabolism, reactive oxygen species and inflammatory cytokine in C2C12 cells (Shenoy et al., 2019). Chronic exposure to NaF triggers oxidative changes and leads to oxidative biochemistry misbalance (Miranda et al., 2018). NaF exposure exerts reproduction toxicity on porcine oocyte maturation (Liang et al., 2017). Moreover, NaF exposure induced epididymal toxicity by decreasing the antioxidant activity of epididymis (Sun et al., 2018). NaFinduced immunotoxicity has also been described in adult male albino rat (Das et al., 2006). Here, the present study targets to explore the effects of NaF on immune system. In order to investigate the molecular mechanism of NaF immunotoxicity, a model of NaFexposed neutrophils was established.
Neutrophils are important components of innate immune system that defense against infection through chemotaxis and phagocytosis. Recently, another mechanism of neutrophils’ antiinfection called “neutrophil extracellular traps (NETs)” has been found, which can release chromatin decorated with specific proteins to trap and kill microorganisms (Brinkmann et al., 2004). The release of NETs helps neutrophils immobilize, catch and eliminate pathogens (e.g. bacterium, fungi or viruses) (Kenny et al., 2017; Sivanandham et al., 2018). However, overproduction and/or inadequate removal of NETs have been associated with tissue damage and some immune diseases. Several studies have demonstrated that NETs serve some harmful functions in noninfectious inflammatory diseases, systemic lupus erythematosus (SLE), diabetes (Arampatzioglou et al., 2018; Carestia et al., 2016a), thrombosis (Bertin et al., 2018; Nakazawa et al., 2018), and cancer (Albrengues et al., 2018). For instance, NETs contributed to increased production of inflammatory mediators in SLE (Lood et al., 2016). NETs are abundant in circulating blood in Type 2 diabetes mellitus (T2DM) patients and are associated with inflammation and hypofibrinolysis (Bryk et al., 2019). There is also evidence that NETs play a vital role in sustaining inflammatory signals in ulcerative colitis (Dinallo et al., 2019). We have showed that NaF induced the formation of NETs, but their contribution to the NaF immunotoxicity remains to be elucidated, especially neutrophils. In the present study, we set out to identify the role of reactive stress and NETs in NaF immunotoxicity in neutrophils.

2. Material and methods summary

2.1. Antibodies, staining reagents, and inhibitors

NaF (Sigma-Aldrich), 2,7-dichlorodihydrofluorescein diacetate (DCF-DA, Sigma-Aldrich), zymosan (Sigma-Aldrich), DPI (SigmaAldrich), U0126 (Sigma-Aldrich), SB202190 (Sigma-Aldrich), Sytox Orange nucleic acid stain (Invitrogen), Pico Green (Invitrogen), Annexin-V-FLUOS staining Kit (Roche), Histone H3 antibody (LSC353149; Life Span BioSciences, Inc), Myeloperoxidase antibody (Orb16003; Biorbyt), Anti-neutrophil elastase antibody (ab68672; Abcam), anti-p38 (Bs3566; Bioworld), anti-ERK (Bs3627), anti-pp38 (Cell Signaling Technology Inc, USA), anti-p-ERK (Cell Signaling Technology Inc, USA) were used.

2.2. Neutrophils isolation and stimulation

Neutrophils were isolated by bovine PMN isolation kit (TianJin HaoYang Biological Manufacture CO. China) following the manufacturer’s instruction. Purified neutrophils were re-suspended and seeded at a density of 2 105 cells in 96-well plates for subsequent experiments. For immunofluorescence stainings, neutrophils were seeded on poly-L-lelysine pre-treated coverslips. The cells were treated with inhibitors 20 min before induction of NET formation. All NaF stimulations were done at 0.25 mM, 0.5 mM and 1 mM. Subsequently, NETs formation, ROS production and LDH release were measured.

2.3. Formation of NETs

To assess NETs formation, immunofluorescence method was used as described previously (Wang et al., 2019). Briefly, neutrophils were seeded on poly-L-lelysine pre-treated coverslips and fixed with 4% paraformaldehyde, rinsed twice in PBS, permeabilized with Triton X-100 in PBS for 20 min. Cells were next blocked in PBS containing 5% goat serum for 120 min and incubated in antibody solution (H3, MPO and NE) overnight at 4 C. Next, cells were incubated in the presence of secondary antibody goat-antirabbit conjugated to Alexa 488 for 120 min, rinsed twice in PBS, stained with 5 mM sytox orange (dissolved in PBS) for 5 min. Images were obtained using scanning confocal microscope (Olympus FluoView FV1000). In a complementary strategy, we used QuantiT™ PicoGreen dsDNA reagent (a fluorescent nucleic acid stain) to quantify the formation of NETs as previously described (Wang et al., 2019). Briefly, the cells were seeded into 96-well plates in RPMI 1640 medium (phenol-red-free) and incubated with NaF (0.25, 0.5, or 1 mM) for 120 min. Each well was added the aqueous working solution of the Quant-iT™ PicoGreen reagent. Subsequently, the fluorescence was measured by an Infiniti M200 fluorescence plate reader (Tecan, Austria).

2.4. Detection of ROS and antioxidant enzymes

ROS production in neutrophils was measured by DCF-DA. The cells were incubated with NaF (0.25, 0.5, or 1 mM) for 120 min and removed the medium. Next, the cells were cultured in medium containing DCF-DA (10 mM) for 20 min. Wash the cells three times with PBS, The generation of intracellular ROS was detected at 485 nm of excitation and 525 nm of emission by an Infiniti M200 fluorescence plate reader (Tecan, Austria). SOD and CAT activities were measured using detection kits (Nanjing Jiancheng Bioengineering Institute, China). Briefly, cells were incubated with NaF (0.25, 0.5, or 1 mM) for 120 min and harvested. Then SOD and CAT activities were determined by biochemical methods following the manufacturer’s instructions.

2.5. Western blotting

Neutrophils were incubated with NaF (0.25, 0.5, or 1 mM) for 120 min. The presence of protein was determined by western blotting, as previously described (Wang et al., 2019). In brief, the cells were harvested and lysed with M-PER™ mammalian protein extraction reagent (Thermo Fisher Scientific) and protein concentrations were measured by a bicinchoninic acid (BCA) protein assay reagent kit (Pierce). Total protein was subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE). Then, separated proteins were transferred to a polyvinylidene fluoride (PVDF) transfer membrane (Merck Millipore, Billerica, MA, U.S.A.). The following primary antibodies were used in this study, including anti-phosphor-p38 monoclonal antibody (1:1000), anti-phosphorERK monoclonal antibody (1:1000), and horseradish peroxidase (HRP)-linked secondary antibodies. Finally, the signal was detected using ECL Plus Western Blotting Detection System (ProteinSimple, San Jose, CA, U.S.A.).

2.6. Cell apoptosis assay

Neutrophils were seeded into 6-well plates and incubated with (0.25, 0.5, or 1 mM) for 120 min. The quantification of apoptosis was assessed as described previously (Wang et al., 2019).

2.7. Lactate dehydrogenase (LDH) assay

Neutrophils were seeded into 96-well plates and incubated with NaF (0.25, 0.5, or 1 mM) for 120 min. Next, the plate was centrifuged at 400g for 5 min and the supernatants transferred to another plate and measured by a LDH cytotoxicity assay kit (Beyotime Biotechnology, China) according manufacturer’ instruction.

2.8. Statistical analysis

Data from five cows were expressed as the mean ± standard error of the mean (SEM). All data were analyzed using GraphPad Prism 5 (version 5.0, GraphPad InStat Software, San Diego, CA, U.S.A.). Statistical significance was calculated by one-way analysis of variance (ANOVA) followed by Tukey’s test. P-values < 0.05 were considered to be statistically significant.

3. Results

3.1. NaF triggered NETs formation

When neutrophils form NETs, decondensed chromatin mixed with granule-derived antimicrobial peptides is expelled into the extracellular region to form web-like structures (Brinkmann et al., 2004). We visualized the process by an immunofluorescence microscopy, the typical NETs structures co-localized with histones, MPO and NE were obviously observed (Fig. 1). Moreover, QuantiT™ PicoGreen dsDNA reagent (a fluorescent nucleic acid stain) was used to quantify NETs DNA fiber extruction. The assays showed that NaF induced an increase in extracellular DNA concentration in a dose dependent manner (Fig. 2).

3.2. NaF caused imbalance between ROS and antioxidant system

A number of studies indicate that oxidative stress is associated with fluoride toxicity. We detected ROS generation using by DCFDA, a fluorescent indicator of ROS. As shown in Fig. 3, NaF led to an increased production of ROS. SOD and CAT are important antioxidant enzymes, contributing to ROS elimination. Next, we measured the activities of SOD and CAT. NaF significantly reduced the activities of SOD and CAT (Fig. 4).

3.3. NaF activated ERK and p38 signaling pathways

We next asked whether ERK and p38 signaling pathways are involved in NaF-induced NETs formation. The western blot analysis revealed that NaF obviously elevated the phosphorylation of ERK and p38 signaling proteins (Fig. 5).

3.4. NaF-triggered NETosis is dependent on ROS, ERK and p38 signaling pathways

To elucidate the underlying mechanism in NaF-induced NETs formation, we used NADPH oxidase inhibitor DPI (50 mM), ERK inhibitor U0126 (10 mM), or p38 inhibitor SB202190 (10 mM), and measured NETs formation. The results showed that NaF-induced NETs formation is significantly inhibited in the presence of the three specific inhibitors. These results suggest that NaF-induced NETs formation is mediated by ROS-dependent pathway and the activation of ERK and p38 (Fig. 6).

3.5. NaF induced less cell apoptosis

The neutrophils apoptosis were measured by Annexin-V/PI staining. As shown in Fig. 7. At 1 mM NaF, the number of early and late apoptotic cells had a little bit of an increase. This suggested that NaF induced NETs formation is accompanied by less apoptosis. 3.6. NaF had no effect on LDH activity. Finally, we assessed the release of LDH during NaF-induced NET formation. The results showed that NaF exposure for 120 min did not result in the release of LDH (Fig. 8).

4. Discussion

Fluorine is an essential trace element that is widely distributed in drinking water, soil and atmosphere. Accumulation of excess fluoride in the natural environment has been shown to be a risk factor for plants, animals, and humans. Fluorides are inorganic and organic fluorine compounds that are mainly used as an effective prophylactic for dental caries (Griffin et al., 2007). Moderate levels of fluorides ingestion are beneficial to bone health. However, experimental studies have demonstrated that excessive fluoride exposure or long-term low fluoride intake cause metabolic, structural, and functional damage in many cells and tissues. Particularly, previous studies demonstrated that high dose NaF may affect immune system function, inducing immunotoxicity (Das et al., 2006; De la Fuente et al., 2016; Wei et al., 2019). Neutrophils are abundant in circulation system and serve as the main component of immune system against invading pathogens and tissue injury. Besides, neutrophils can depolymerize chromatin and combine various intracellular antibacterial particles to form NETs, thereby enhancing their own efficiency of phagocytosis. In addition to wellknown role as a novel immune strategy against pathogens, the components of NETs have potential cytotoxic effects. Overproduction of NETs, or their inadequate removal, can lead to tissue damage or even initiation of diseases. Herein, we observed that the process NaF exposure induced NETs formation was accompanied by increased ROS production and decreased activities of antioxidant enzyme. For further investigated the molecular pathways leading to NETs. We found that ROS, ERK and p38 inhibition reduced NETs formation, suggesting that ROS, ERK and p38 were necessary for NETs formation. Our data supporting the concept that oxidative stress and NETs formation are relevant for NaF immunotoxicity in neutrophils.
Evidence has reported that oxidative stress is associated with fluoride toxicity and tissue damage (Ameeramja et al., 2018; Campos-Pereira et al., 2017). Enzymatic ROS scavenging mechanisms include the enzymes SOD, CAT, and glutathione peroxidase (GSH-Px), nonenzymatic antioxidants, and compounds containing thiol groups. The enzymatic antioxidant system has been identified as first line of defense against ROS, including SODs which dismutate superoxide to H2O2, and CAT subsequently detoxify H2O2 (Pisoschi and Pop, 2015). Imbalance between ROS and antioxidant system leads to the occurrence of oxidative stress. In the study, our results showed that NaF exposure increased ROS production and decreased the activities of SOD and CAT, suggesting an unbalance in oxidant-antioxidant status. To some extent, the results suggest a role for oxidative stress in NaF immunotoxicity.
NETs have been studied for many years, the knowledge concerning this structure has been identified. Here, we found that NaF triggered NETs formation and detected the typical NETs components with extracellular DNA, histones, MPO and NE. The quantitation of NETs showed that NaF-induced NETs formation is a time dependent process, the peak release observed at 120 min. A larger number of stimuli can induce NETs formation, including bacteria (Ma et al., 2018), virus (Barr et al., 2018), fungi or fungaltoxin (Hoyer et al., 2018; Wang et al., 2019), heavy metal (Wei et al., 2018), protozoa (Yang et al., 2017), and activated platelets (Carestia et al., 2016b). However, the molecular events controlling NETs formation after NaF exposure remain unclear. Studies have shown that ROS production is key for NETs release (Dahlgren and Karlsson, 2019; Stojkov and Amini, 2017). We showed that DPI, a NADPH oxidase inhibitor, reduced NaF-triggered NETs formation. These data are consistent with results of previous studies demonstrating that ROS are required for NETs release (Wang et al., 2019). It is reported that ERK and p38 MAPK signaling pathways have been revealed a
pivotal role in apicomplexan mediated-NETosis (Abi Abdallah et al., 2012; Munoz Caro et al., 2014). In the study, NaF exposure was found to activate ERK and p38 pathway. Functional inhibition of these kinases resulted in a significant diminishment of NaFtriggered NET formation to some degree, suggesting that both ERK and p38 were essential for NaF-induced NETs formation.
Apoptosis is a highly regulated cell death program that plays a vital role on fluoride toxicity (Barbier et al., 2010; Zhang et al., 2016). Several studies have suggested that fluoride induced apoptosis in vitro and in vivo (Lu et al., 2017; Ribeiro et al., 2017; Ying et al., 2017). In the study, we investigated whether apoptosis occurs during NETs formation. The results showed that NaF (1 mM) treatment resulted in apoptosis but to a lesser extent. Moreover, NaF exposure had no effect on LDH release.
In brief, our results revealed that NaF-induce NETs formation was mainly dependent on ROS, ERK and p38 signaling pathways. We indicate a possible involvement of ROS production and NETs in NaF immunotoxicity, which extends the understanding of the immunotoxicity of NaF.

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