Research ArticleChlorpyrifos Suppresses Neutrophil Extracellular Traps in Carp byPromoting Necroptosis and Inhibiting Respiratory BurstCaused by the PKC/MAPK Pathway
1College of Veterinary Medicine, Northeast Agricultural University, 600 Changjiang Road, Harbin 150030, China2Key Laboratory of the Provincial Education Department of Heilongjiang for Common Animal Disease Prevention and Treatment,College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China
Neutrophil extracellular traps (NETs) are reticular structures formed by myeloperoxidase (MPO), histones, and neutrophil elastase(NE) that are released from neutrophils in response to pathogenic stimuli. Chlorpyrifos (CPF) is wildly used as anorganophosphorus pesticide that causes a range of toxicological and environmental problems. Exposure to CPF can increase theproduction of neutrophils in carps, and this increase can be considered a biomarker of water pollution. To explore a relationshipbetween NETs and CPF and its mechanism of influence, we treated neutrophils from the blood of carp with 1μg/mL phorbol12-myristate 13-acetate (PMA), 0.325mg/L CPF, or 20 μM necrostatin-1 (Nec-1). The production of MPO and NETs wasreduced in the CPF+PMA group compared with that in the PMA group. CPF can cause an increase in reactive oxygen species(ROS), while inhibiting respiratory burst caused by PMA stimulation. We found that the expression levels of protein-coupledreceptor 84 (gpr84), dystroglycan (DAG), proto-oncogene serine/threonine kinase (RAF), protein kinase C (PKC), andmitogen-activated protein kinase 3 (MAPK3) in the CPF+PMA group were lower than those in the PMA group, indicating thatthe PKC-MAPK pathway was suppressed. The expression levels of cylindromatosis (CYLD), mixed lineage kinase domain-likepseudokinase (MLKL), receptor-interacting serine-threonine kinase 1 (RIP1), and receptor-interacting serine-threonine kinase 3(RIP3) were increased, and the expression levels of caspase 8 were reduced by CPF, indicating that CPF may cause necroptosis.The addition of Nec-1 restored the number of NETs in the CPF+PMA group. The results indicate that CPF reduced theproduction of NETs by inhibiting respiratory burst and increasing necroptosis. The results contribute to the understanding ofthe immunotoxicological mechanism of CPF and provide a reference for comparative medical studies.
1. Introduction
Chlorpyrifos (CPF) has been one of the most widely usedbroad-spectrum organophosphorus pesticides in the worldand a major environmental pollutant. Many countries havereported excessive residual levels of CPF in soil, water, fruits,vegetables, and aquatic animals [1]. High-performance liq-uid chromatography is a common method for the assay ofCPF concentrations [2]. CPF is a neurotoxin. It can inhibitthe activity of cholinesterase and cause the accumulation ofacetylcholine, thus leading to acute adverse effects, such as
tremor, paralysis, convulsion, and coma [3, 4]. CPF cancause kidney damage [5] and disrupt the liver metabolism[6]. The immune system is one of the most important targetorgans of CPF [7]. CPF can modulate the immune responseby stimulating the antigen-presenting ability of the head kid-ney of the carp [8]. It had been reported that the innateimmune system was disturbed by CPF in zebrafish [9]. Ratsexposed to CPF had an altered number of T cells and B cellswith variable degrees of changes relative to the control ani-mals after 45 and 90 days at all tested exposure levels [10].The exposure to CPF may also lead to immunosuppression
HindawiOxidative Medicine and Cellular LongevityVolume 2019, Article ID 1763589, 11 pageshttps://doi.org/10.1155/2019/1763589
in mice by inhibiting the production of proinflammatorycytokines [11]. After the exposure to CPF, the productionof neutrophils is increased and the immune system of thefish is damaged [12].
Neutrophils are blood leukocytes that can kill pathogensthrough chemotaxis and phagocytosis [13]. Recent studieshave shown that neutrophils capture invading pathogensthrough neutrophil extracellular traps (NETs) generated bya process termed NETosis [14, 15]. NET structure is basedon a DNA backbone decorated with antimicrobial proteinsincluding myeloperoxidase (MPO), neutrophil elastase(NE), and histones [16]. A number of pathological, physio-logical, and pharmacological stimuli can generate NETsincluding bacteria, inflammatory cytokines, and chemicaldrugs [17]. Different stimuli cause different pathways ofNET release. It has been shown that phorbol 12-myristate13-acetate (PMA), C. albicans, and group B Streptococcus(GBS) can stimulate the formation of NETs in a similar man-ner while ionophores act in a different manner [18]. Respira-tory burst plays an important role in the generation of NETs.Perazzio et al. studied patients with Behcet’s disease andfound that the disease stimulates neutrophil respiratory burstand NET production by producing soluble CD40L (sCD40L)[19]. Yu et al. found that celastrol inhibits respiratory burstand NET production by downregulating the SYK-ME-K-ERK-NFκB signaling cascade [20]. It was documented thatthe inhibition of mitogen-activated protein kinase (MAPK),extracellular regulated protein kinases 1/2 (ERK1/2), or p38can decrease NET release [21]. The production of NETsinduced by IL-8 involved the mobilization of intracellularand extracellular calcium pools and activated PKC via G pro-tein coupled receptors (GPCR) [22]. Necroptosis is a specialtype of necrosis induced by HXR-9 and enhanced by inhibi-tors of PKC signaling suggesting that PKC is closely related tonecroptosis [23]. RNLIP protected the heart of diabeticpatients by inhibiting necroptosis to activate STAT3, whichneeded the activation of PKC07 [24]. AdipoRon inducednecroptosis in the MIAPaCa-2 cells by the production ofsuperoxide via the activation of receptor-interacting serine-threonine kinase 1 (RIP1) and ERK 1/2 [25].
Thus, CPF is the main environmental pollutant in thewater bodies, and carp can be a valuable biomarker for envi-ronment water pollution. Neutrophils in carp can respond toadverse stimuli by releasing NETs. However, the mecha-nisms of NET production by carp neutrophils exposed toCPF remain unknown. In this study, neutrophils were usedas the research subject. After being exposed to CPF, neutro-phils were stimulated with PMA and an inhibitor of necrop-tosis (necrostatin-1). Then, the amount of NETs; the releaseof ROS and respiratory burst; the expression of the PKC-MAPK pathway-related genes (gpr84 (protein-coupledreceptor 84), dystroglycan (DAG), proto-oncogene serine/threonine kinase (RAF), PKC, and MAPK3); and the expres-sion of necroptosis-related genes (caspase 8, cylindromatosis(CYLD), mixed lineage kinase domain like pseudokinase(MLKL), RIP1, and receptor-interacting serine-threoninekinase 3 (RIP3)) were detected to clarify the effects andmechanisms of the exposure to CPF in the formation ofNETs in carp. The results provide a new reference for
environmental pollution protection and the regulation ofbiological innate immunity.
2. Materials and Methods
2.1. Test Chemicals. CPF (purity 99%) was purchased fromAladdin (China). Stock solutions of CPF were prepared indimethyl sulfoxide (DMSO) (purity 99%). The concentrationof DMSO was kept below 0.05% in all experiments.
2.2. Treatment of Experimental Animals. All procedures usedin this study were approved by the Institutional Animal Careand Use Committee of Northeast Agricultural University.Common carps (mean body weight, 600± 100 g) used in thisstudy were purchased from an aquarium specializing in freshwater fish and maintained in the laboratory tanks at 20± 1°Cwith continuous aeration [26].
2.3. Cell Counting Kit-8. Neutrophils isolated from the bloodof common carp were separated by the detection kits(P4190, Solarbio, China) according to the manufacturer’sprotocols. Then, the cells were suspended in modified RPMImedium (HyClone, Logan, UT, USA) containing 10% fetalbovine serum (NQBB, Australia) and seeded in 6-well plates(Corning, China). The cells were treated with various con-centrations of CPF (20mg/L, 15mg/L, 3mg/L, 1.5mg/L,0.75mg/L, 0.325mg/L, and 0.1625mg/L) [27] for 2 h at25°C in a humidified atmosphere containing 95% air and5% CO2. To determine a suitable working concentration ofCPF, cell viability was determined by a Cell Counting Kit-8(MedChemExpress, Monmouth Junction, NJ, USA) accord-ing to the manufacturer’s protocols.
2.4. Neutrophils Treatment. After separation, neutrophilswere suspended in modified RPMI medium containing 10%fetal bovine serum and seeded in 6-well plates; then, neutro-phils were incubated with 20μM necrostatin-1 (Nec-1) for2 h (Nec-1 group) orwith 0.325mg/LCPF for 2 h (CPF group)or with a combination of 20μM Nec-1 and 0.325mg/L CPF(Nec-1+CPF group). The control cells were untreated (NCgroup). Cells were then resuspended in the medium andtreated with 1μg/mL PMA (Sigma, St. Louis, MO, USA) for2 h (Nec-1+PMA, CPF+PMA, Nec-1+CPF+PMA, and PMAgroups). Cells were incubated at 25°C in a humidified atmo-sphere containing 95% air and 5% CO2 for all treatments.Nec-1 was dissolved in 1% dimethyl sulfoxide (DMSO).
2.5. Scanning Electron Microscopy (SEM). Approximately3× 106 neutrophils were seeded onto a glass coverslip pre-treated with 0.001% polylysine (Sigma, St. Louis, MO, USA)and placed in a 12-well cell culture plate for 2 h. Variousgroups of cells were treated as described above. For SEManalysis, the glass coverslips with the cells were fixed withprecooled 3% glutaraldehyde overnight and then washedwith PBS for 10min. Then, the cells were fixed with 1% citricacid precooled at 4°C for 1 hour and immersed twice in PBSfor 10min each time; samples were dehydrated with a gradedethanol series, critical-point dried for 1 h, and coated with alayer of platinum using a thin layer evaporator. Specimens
2 Oxidative Medicine and Cellular Longevity
were analyzed in a scanning electron microscope (SU-8010type, Japan).
2.6. Measurement of MPO. After treatment, each group ofcells was incubated with 500μL of 3,3′,5,5′-tetramethylben-zidine hydrochloride (TMB) (Sigma, USA) and then imme-diately incubated with 500μL of hydrogen peroxide (H2O2).After 3min of color reaction, 500μL of sulfuric acid wasadded to stop the reaction. The lysis group was incubatedwith Triton X-100 (Bio-Rad, Hercules, CA, USA). All cellswere centrifuged at 600 g for 15min; 200μL of supernatantof each group was transferred to a 96-well plate, and the opti-cal density (OD) was quantified at 405nm. The experimentwas repeated 3 times to enhance the accuracy of the results.
2.7. Fluorescent Microscopy. For fluorescence microscopy,cells were seeded on a glass coverslip and treated with Sytoxgreen (Thermo Fisher, Shanghai, China) and Hoechst33258 fluorescent dyes (Wanleibio, China) for 30minaccording to the instructions of the manufacturers; then,the cells were once washed with PBS. Specimens were ana-lyzed using a fluorescence microscope (Bothell, WA, USA).
2.8. Quantification of NETs. The treated neutrophils weresuspended in PBS and seeded in a black 96-well plate. Sytoxgreen was added to the cells for 30min. Then, fluorescencewas quantified as relative fluorescence units (RFU) at485nm excitation and 530nm emission wavelengths usinga Cytation 5 demo fluorescence spectrophotometer (BioTek,USA).
2.9. Measurement of ROS. The treated neutrophils weresuspended in PBS and seeded in a black 96-well plate. Theconversion of nonfluorescent dye chloromethyl-2,7-dichlor-ofluorescindiacetate (DCF-DA) was used to assay ROS.DCF-DA was added to the cells for 30min. Then, fluores-cence was quantified as RFU at 485nm excitation and525nm emission wavelengths by a Cytation 5 fluorescencespectrophotometer.
2.10. Quantitative Real-Time PCR Analysis of the mRNALevels. Total RNA was isolated from the neutrophils byTRIzol reagent [28] (Invitrogen, China), and reverse tran-scription was performed using a Transcriptor First-StrandcDNA synthesis kit (Roche, Mannheim, Germany) according
to the manufacturer’s instructions. The primer sequences ofthe target mRNAs were designed by Primer Premier 6.0and are shown in Table 1. β-Actin was used as an internalreference. QRT-PCR was performed using SYBR® PremixEx Taq™ (Takara, Beijing, China) with a Light Cycler®480system (Roche, Basel, Switzerland) [29]. The 2-ΔΔCt methodwas used to analyze the transcription levels of mRNA.
2.11. Western Blot Analysis. Total protein was resolved bySDS-polyacrylamide gel electrophoresis [30] and then wastransferred to nitrocellulose membranes. After blocking in5% bovine serum albumin (BSA) at 25°C for 1 h, themembranes were washed 3 times with TBST for 15min eachtime and incubated at 4°C overnight with primary antibodiesagainst MPO (1 : 500), DAG (1 : 500), RAF (1 : 500), gpr84(1 : 500), PKC (1 : 2000), MAPK3 (1 : 2000), caspase 8(1 : 500), RIP1 (1 : 500), RIP3 (1 : 500), MLKL (1 : 500), andGAPDH (1 : 5000, Santa Cruz Biotechnologies, CA, USA).Then, the membranes were washed in TBST 3 times, incu-bated with a secondary antibody at 25°C for 1 h, and washedin TBST 3 times. Finally, the protein band signals were exam-ined using an enhanced chemiluminescence system (Apply-gen Technologies Inc., Beijing, China) and analyzed byImageJ software (National Institutes of Health). GADPHwas used as a loading control.
2.12. Statistical Analysis. The statistical analysis of all datawas performed using GraphPad Prism 5.0 (San Diego, CA,USA). One-way ANOVA was used for the analysis of thedata [31]. Quantitative data are presented as the mean± SD.Samples with different letters were considered to be signifi-cantly different at P < 0 05 [32].
3. Results
3.1. The Inhibitory Effects of CPF on the Viability ofNeutrophils. The inhibitory effects of CPF on the viability ofneutrophils were detected with CCK-8. The results are shownin (Figure 1). CPF at various concentrations inhibited theviability of neutrophils. The inhibitory effects became moresignificant at higher doses of CPF showing a distinct dose-dependent relationship. At 2 h, the LC50 of CPF in neutro-phils was 18.85mg/L. We selected the concentration of0.325mg/L as the optimum concentration corresponding to
Table 1: Gene-target primers used in real-time PCR.
95% cell viability. In the subsequent experiments, the con-centration of CPF was 0.325mg/L.
3.2. SEM of Neutrophils and NETs. SEM was used to observethe morphology of neutrophils and NETs. The NC groupneutrophils showed normal morphology. When blood neu-trophils were treated with CPF, damage to the membranewas detected by scanning electron microscopy. We also seelots of NETs which seemed like nets in the PMA group; how-ever, the NETs in the CPF+PMA group were less abundant(Figure 2(a)).
3.3. MPO Parameters in PMA-Treated Neutrophils. MPO isan important component of NETs. It is an antimicrobialprotein with important function in the formation of NETs.Thus, we detected the release and the mRNA and proteinexpression levels of MPO. MPO analysis showed that in thePMA and CPF+PMA groups, MPO levels increased; how-ever, the level in the latter group was lower than that in theformer group (Figure 2(b), A). Then, we used RT-PCR(Figure 2(b), B) and western blot (Figure 3(b), A) to test theexpression of MPO. In the case of RT-PCR, the mRNAexpression levels in the PMA group were the highest followedby those in the CPF+PMA, CPF, and NC groups. Westernblot analysis confirmed this result.
3.4. Fluorescent Microscopy.We used Sytox green (a dye spe-cific for dead cells and NETs) and Hoechst 33258 (a dyespecific for live cells, dead cells, and NETs) as the fluores-cence dyes for fluorescence microscopy (Figure 4). The
percentage of dead cells in the NC, CPF, PMA, and CPF+PMA groups is shown in Figure 5(c). To investigate whyCPF inhibited the production of NETs induced by PMA,we added an inhibitor of necroptosis (Nec-1) to investigatea possible link between CPF and necroptosis. The numberof dead cells was higher, and the number of live cells waslower in the CPF group than in the NC group. However,the coaddition of Nec-1 increased the viability of the cells.The results in the Nec-1 group were similar to those in theNC group. Abundant NET structures were detected in thePMA group; however, there were less NETs in the CPF+PMA group. Fluorescence microscopy showed that numer-ous NETs reappeared in the Nec-1+CPF+PMA group, sug-gesting that CPF may inhibit production of NETs by PMAand the effect may be related to necroptosis.
3.5. The mRNA and Protein Levels of Necroptosis Markers.Caspase 8, CYLD, MLKL, RIP1, and RIP3 are critical factorsin the necroptosis pathway. RIPK3 is one of the core mole-cules of necroptosis that forms a necrotic complex withRIPK1 and activates MLKL. Ultimately, the complex can leadto cell membrane rupture, the release of cell contents, andnecroptosis. Additionally, MLKL can promote necroptosisvia the activation of the Ca2+ influx [33]. In subsequentexperiments, we detected the levels of genes related tonecroptosis with RT-PCR and western blot. We tested theexpression levels of caspase 8 and CYLD by RT-PCR. Wetested the expression levels of caspase 8, MLKL, RIP1, andRIP3 by western blot. The results confirmed our hypothesis.The levels of CYLD, RIP1, RIP3, and MLKL were increased,and the level of caspase 8 was reduced by CPF. Nec-1 can effi-ciently suppress these changes (Figure 5(a)).
3.6. Quantification of NET Formation. Microscopy can onlyillustrate the NETs in appearance; however, we demon-strated the inhibitory effect of CPF on NETs by quantifyingNETs. Figure 5(b) illustrates that PMA can induce the pro-duction of NETs but that the addition of CPF reduced theamount of released NETs. The addition of Nec-1 reducedthe inhibitory effects of CPF. This is consistent with our pre-vious test results.
3.7. ROS, mRNA, and Protein Levels of the PKC-MAPKPathway Components in Neutrophils. Respiratory burst isan essential stage in the formation of NETs and plays a deci-sive role in the process. The PKC-MAPK pathway is theupstream pathway of the respiratory burst, and the changesin the expression of the components of in the pathway willinfluence respiratory burst. Thus, we assayed the productionof ROS and the mRNA and protein levels of the genes relatedto the PKC-MAPK pathway. The production of ROS was sig-nificantly enhanced in a time-dependent manner in neutro-phils treated with all stimulators. The ROS levels in thecells treated with PMA, CPF+PMA, and Nec-1+CPF+PMAreached a peak after treatment for 45min indicating thatrespiratory burst occurred in the PMA-treated cells at45min. The ROS levels in the PMA-treated cells were thehighest followed by the Nec-1+CPF+PMA and CPF+PMAgroups. The ROS levels in the CPF group continued to
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Figure 1: The inhibitory effects of CPF on neutrophils.Neutrophils were treated with various concentrations of CPF for2 h. Quantitative data are presented as the mean± SD. Sampleswith different letters were considered significantly different(P < 0 05). The samples with the same letters were not significantlydifferent (P > 0 05). The LC50 for CPF-treated neutrophils wasapproximately 18.85mg/L. We selected 0.325mg/L as the optimumconcentration corresponding to 95% viability of neutrophils.
4 Oxidative Medicine and Cellular Longevity
increase during 2 h treatment. The NC and Nec-1 groups hadthe lowest ROS levels (Figure 3(a)). Then, we tested themRNA and protein levels of the genes of the PKC/MAPKpathway. As shown in Figure 3(b), the gene expression levelsof gpr84, DAG, RAF, PKC, and MAPK3 were increased afterthe addition of PMA. The levels of these genes followed asimilar trend. The PMA group had the highest levels, theCPF+PMA group was the second high, and the NC groupwas the lowest. The protein levels of gpr84, DAG, RAF,PKC, and MAPK3 were similar to the mRNA levels.
4. Discussion
Neutrophils can respond to various stimuli, such as PMA,IL-8, or bacteria, by producing NETs [34]. Wei et al. hadshown that sodium arsenic can induce NETs in neutrophils,and the induction was a NADPH oxidase signaling
pathway-independent process [35]. Organophosphorus pes-ticides are widely used worldwide, and their harmful effectson neutrophils are not negligible. After the examination ofworkers exposed to organophosphorus pesticides, Queirozet al. found that the killing of Candida albicans by neutro-phils from the exposed workers was reduced [36]. Afterexposure to organophosphorus insecticides, leukopenia char-acterized by neutrophil phagocytosis and decreased phago-cytic index was detected in carp [37]. In our study, wefound that CPF inhibited the production of NETs causedby PMA and increased necroptosis. The inhibition of thePKC-MAPK pathway inhibited PMA-induced respiratoryburst. After the addition of a necroptosis inhibitor, theamount of NETs has recovered.
Cell death is an irreversible process that includes apopto-sis, necrosis, and necroptosis. There is a close correlationbetween the various types of cell death. The removal of
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Figure 2: Production of NETs according to SEM and production of MPO in neutrophils after various treatments (a) Detection of NETs byscanning electron microscopy. (b) Effects of PMA or/and CPF on the production of MPO (A) and the mRNA levels of MPO (B) inneutrophils. The experiments were repeated three times. The data are presented as the mean± SD. Bars with different letters wereconsidered significantly different (P < 0 05).
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ROS can reduce cell necrosis and increase apoptosis [38].Selenium deficiency in chicken cardiomyocytes induced apo-ptosis while inhibiting autophagy through the inhibition of
Bax/Bcl-2 [39]. Mycobacterium tuberculosis killed infectedmacrophages by promoting necroptosis and inhibitingapoptosis [40]. Recent studies have shown that NETosis is a
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Figure 3: ROS levels, protein levels of MPO, and the mRNA and protein levels of the PKC-MAPK pathway components in neutrophils (a)Effects of PMA and/or CPF and/or Nec-1 on the release of ROS. (b) The protein levels of MPO and the mRNA and protein levels of the genesrelated to the PKC-MAPK pathway. The experiments were repeated three times. The data are presented as the mean± SD. The samples withdifferent letters were considered significantly different (P < 0 05). The samples with the same letters were not significantly different (P > 0 05).
6 Oxidative Medicine and Cellular Longevity
special form of cell death closely linked to other forms of celldeath. Eduardo Caberlon has discovered that gallic acid canweaken the antiapoptotic effect produced by LPS and reducethe amount of generated NETs [41]. The inhibition ofautophagy can reduce NETosis measured by chromatincondensation and can promote apoptosis [42]. ZVAD-fmk-induced necroptosis of the L929 cells depended on theautocrine production of TNFαmediated by the PKC-MAPK-s-AP-1 pathway [43]. It has been shown that caspase 8, RIP1,RIP3, MLKL, and CYLD are important for the necroptosispathway. Impaired MAPK activation was shown in theRIP1-deficient cells, and the cells were prone to necroptosis[44]. Yang et al. claimed that miR-200a-5p and RNF11 areinvolved in the RIP3-dependent necroptosis pathwaytriggered by MAPK [45]. The FOLE-induced overproductionof ROS can promote RIP1-dependent and caspase 8-licensednecroptosis [46]. The inhibition of caspase 8 inducedRIPK3-dependent necroptosis [47]. In our study, the treat-ment of blood neutrophils with CPF can increase the levelsof CYLD, RIP3, and MLKL and reduce the levels of caspase8 suggesting that CPF induced necroptosis in neutrophils.PMA-induced NET release was also reduced. When we usedNec-1, necroptosis was inhibited. At the same time, theamount of NETs was recovered. Thus, we speculate thatCPF can promote necroptosis and inhibit NETosis.
CPF can manifest its toxic effects through the generationof ROS. After treatment with various concentrations of CPF,ROS in the rat pheochromocytoma cells was increased in adose-dependent manner [48]. Betanin (a natural pigment)can decrease the CPF-induced increase in ROS formationto reduce hepatotoxicity in the primary rat hepatocytes[49]. Cotreatment with apocynin (a NADPH oxidase inhibi-tor) blunted the generation of ROS and the neurotoxicityinduced by CPF [50]. Our results are consistent with thesefindings because CPF exposure induced an increase in theROS levels. Liu et al. found that neutrophils can rapidly killanthrax and respiratory burst contributed to this efficienteffect [51]. Filifactor alocis resisted the antibacterial effect ofneutrophils by preventing respiratory burst [52]. Respiratoryburst is closely related to NETs. The formation of NETsinduced by PMA requires the generation of respiratory burst[53]. Neutrophils eradicate microorganisms by respiratoryburst and the release of NETs [54]. Plasma-free heme cancause the formation of NETs through respiratory burst[55]. The necessary condition for NETs is that ROS activatesPAD4 and PAD4 regulates the citrullination of histones andthen causes the decondensation of the chromatin [56]. ROSare not only necessary for PAD4 activation. ROS serves as asubstrate for MPO, leading to the release of NE of theMPO/NE complex, allowing NE to enter the nuclei without
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Figure 4: Detection of NETs by fluorescence microscopy. Detection of NETs using Sytox green and Hoechst 33258 dyes. The pictures weretaken by a fluorescence microscope. Hoechst 33258 can stain live cells, dead cells, and NETs (blue color). Sytox green can stain dead cells andNETs (green color).
7Oxidative Medicine and Cellular Longevity
any rupture of the granular and core membrane and cleavinghistone H4 and therefore contribute to DNA decondensation[57–62]. Additionally, histone deamination by PAD4 duringPMA-induced NETosis is still under investigation [18, 63,64]. Moreover, the formation of NETs depends on the activa-tion of PKC and ERK [65]. Menegazzo et al. found that met-formin can prevent the membrane translocation of PKC-βIIin neutrophils diminishing the response of NETosis [66].Respiratory burst is closely related to the PKC-MAPK path-way. Berberine upregulated macrophage respiratory burstactivity and PKC mRNA expression stimulated by PMA[67]. The attenuation of respiratory burst was regulated in amanner dependent on the PKC and MAPK pathways [68].Our results showed that NETs induced by PMA are relatedto respiratory burst and the PKC-MAPK pathway. Oncethe respiratory burst was suppressed by CPF, the amount ofreleased NETs decreased and the expression levels of gpr84G,
DAG, RAF, PKC, and MAPK3 were also reduced. Rajeeveet al. demonstrated that CPF can suppress the respiratoryburst and interfere with the production of NETs [69] consis-tent with our findings.
In summary, our results suggest that CPF could reducePMA-induced NET formation by necroptosis. At the sametime, CPF inhibited respiratory burst induced by PMAthrough the PKC-MAPK pathway to inhibit NET produc-tion. Thus, these results provide the basis for studies of CPFtoxicology and the physiological functions of neutrophilsand add new insight into the mechanism of NETs.
Figure 5: Detection of necroptosis, production of NETs, and the percentage of dead cells (%) according to fluorescence microscopy ofneutrophils after various treatments. (a) The mRNA (A) and protein (B) levels of the genes related to necroptosis. The experiments wererepeated three times. The data are presented as the mean± SD. Samples with different letters were considered significantly different(P < 0 05). (b) Production of NETs in neutrophils after various treatments. Neutrophils were treated with various reagents including PMA,CPF, and Nec-1; the formation of NETs was detected by a fluorescence spectrophotometer. The experiments were repeated three times.Quantitative data are presented as the mean± SD. Samples with different letters were considered significantly different (P < 0 05). Thesamples with the same letters were not significantly different (P > 0 05). (c) Percentage of dead cells (%) according to fluorescentmicroscopy. The percentages of dead cells in the NC, CPF, Nec-1, and Nec-1+CPF groups determined by fluorescence microscopy werecalculated and are shown in the figure. The percentage of dead cells in a descending order: CPF, Nec-1+CPF, NC, and Nec-1 groups. Thedata are presented as the mean± SD. Bars with different letters were considered significantly different (P < 0 05).
The data used to support the findings of this study areavailable from the corresponding author upon request.
Conflicts of Interest
The authors declare that there are no conflicts of interest.
Authors’ Contributions
Shiwen Xu and Ziwei Zhang provided ideas for the exper-iment. Qiaojian Zhang completed the experiment andwrote the manuscript. Shengchen Wang and ShufangZheng completed the parts of the figures.
Acknowledgments
The study was supported by the National Key Research andDevelopment Program of China (no. 2016YFD0500501).
Supplementary Materials
Figure S1: graphical abstract CPF reduces PMA-inducedNET formation by activating necroptosis, and Nec-1 canreduce the inhibition. At the same time, CPF inhibits respira-tory burst induced by PMA through the PKC-MAPK path-way to inhibit NET production. (Supplementary Materials)
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