Cytosolic phospholipase A2-α participates in lipid body formation and PGE2 release in human neutrophils stimulated with an l-amino acid oxidase from Calloselasma rhodostoma venom

Cr-LAAO, an l-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, has been demonstrated as a potent stimulus for neutrophil activation and inflammatory mediator production. However, the mechanisms involved in Cr-LAAO induced neutrophil activation has not been well characterized. Here we investigated the mechanisms involved in Cr-LAAO-induced lipid body (also known as lipid droplet) biogenesis and eicosanoid formation in human neutrophils. Using microarray analysis, we show for the first time that Cr-LAAO plays a role in the up-regulation of the expression of genes involved in lipid signalling and metabolism. Those include different members of phospholipase A2, mostly cytosolic phospholipase A2-α (cPLA2-α); and enzymes involved in prostaglandin synthesis including cyclooxygenases 2 (COX-2), and prostaglandin E synthase (PTGES). In addition, genes involved in lipid droplet formation, including perilipin 2 and 3 (PLIN 2 and 3) and diacylglycerol acyltransferase 1 (DGAT1), were also upregulated. Furthermore, increased phosphorylation of cPLA2-α, lipid droplet biogenesis and PGE2 synthesis were observed in human neutrophils stimulated with Cr-LAAO. Treatment with cPLA2-α inhibitor (CAY10650) or DGAT-1 inhibitor (A922500) suppressed lipid droplets formation and PGE2 secretion. In conclusion, we demonstrate for the first time the effects of Cr-LAAO to regulate neutrophil lipid metabolism and signalling.


Results
Microarray and gene analysis expression. Microarray-based gene expression analysis allows the detection of approximately 22,000 genes, among which genes related to the COX pathway and lipid body were selected. The data were expressed in a heatmap as up-regulation (red) when the expression was higher in Cr-LAAO-stimulated neutrophils versus the negative control, and as down-regulation (green) when the expression was higher in the negative control than in the stimulated neutrophils. PLA 2 s-expressed genes were divided into cytosolic and secreted forms. The cytosolic forms were selected for this study, while the secreted forms are presented in Table S1. Group IV PLA 2 (PLA2G4) subtypes (A, B, C, D), and PLA 2 activating protein (PLAA) were predominantly up-regulated in all samples stimulated with Cr-LAAO. The PLA2G4 E and F subtypes, as well as group VI PLA 2 (PLA2G6) did not show a consistent expression in all samples. COX-1 (PTGS1), COX-2 (PTGS2), prostaglandin reductase 2 (PTGR2), prostaglandin E synthase 1 (PTGES) and 2 (PTGES2), and prostaglandin E receptor subtype EP4 (PTGER4) genes were also up-regulated in all samples stimulated with Cr-LAAO. Genes PTGES3, PTGR1, and PTGER1, 2, and 3 did not present equivalent expression in all samples analysed. Regarding genes involved in lipid body structure, we evaluated the expression of perilipines 1 to 5, but only PLIN2 and PLIN3 were up-regulated in all samples stimulated with Cr-LAAO. In addition, DGAT1 and DGAT2 enzymes that have major roles in LB formation were also up-regulated. To confirm the microarray gene expression profile, qRT-PCR was performed for PTGS1, PTGS2, PLAA, PTGES, PLA2G4A, and PLIN2 genes. The results showed a statistically significant increase in gene expression in the LPS-and Cr-LAAO-stimulated neutrophil samples compared to the negative control for all genes tested, except for PTGS1, concurring with the up-regulated genes observed in the microarray assay (Fig. 1).
To confirm the action of the cPLA2-α inhibitor (CAY10650), protein expression analysis for cPLA 2 and p-cPLA 2 -α was performed on neutrophils pretreated with 12 nM of CAY10650. Results showed that there was expression of cPLA2-α in the native form but there was no expression of the phosphorylated cPLA2-α (p-cPLA2-α) in cells pretreated with both concentrations of the inhibitor. In comparison, western blotting was also performed for cPLA2 and p-cPLA2-α in neutrophils pretreated with AACOCF3 (cPLA2-α inhibitor) at 20 µM. Results showed that as well as with CAY10650, it was observed cPLA2-α expression in the native form this was not observed the p-cPLA2-α expression in neutrophils pretreated with the inhibitor (Fig. 2) confirming that both CAY10650 and AACOCF3 inhibit cPLA2-α.
In order to show the presence of COX-2 in the cell cytoplasm, immunofluorescence experiments were conducted. Cells stimulated with Cr-LAAO or LPS had a higher fluorescence intensity after COX-2 labelling when compared to negative control (RPMI), confirming the results obtained with protein expression (Fig. 3). cPLA 2 -α activation are involved in increased lipid body formation. cPLA 2 -α (CAY10650) and diacylglycerol acyltransferase 1 (DGAT1) (A922500) inhibitors were used to evaluate the mechanisms of Cr-LAAO-induced LB biogenesis. LB quantification demonstrated an increase in neutrophils stimulated for 2 h with Cr-LAAO or LPS (positive control) when compared to RPMI (negative control). However, when the specific inhibitors were used, together or separately, the LB levels were reduced to a baseline similar to the negative control group, demonstrating that the cPLA 2 pathway was activated and participated in LB biogenesis in these cells in a mechanism that also involves DGAT-1 dependent lipid remodelling. In addition, the LBs formation in the culture medium without fetal bovine serum (FBS) was evaluated. It was observed that even in the absence of FBS there is an increase in LBs formation in human neutrophils stimulated with Cr-LAAO, while the negative control showed baseline levels of LBs formation in human neutrophils both in presence or absence of FBS (Fig. 4).
Pharmacological treatment. cPLA 2 -α (CAY10650) and DGAT1 (A922500) inhibitors were used to characterize the mechanisms involved in the PGE 2 release pathway. Cr-LAAO, similarly to LPS, stimulated PGE 2 release compared to the negative control (RPMI) (Fig. 5). When neutrophils were treated with the DGAT inhibitor, we observed a reduction in PGE 2 cell release after stimulation with Cr-LAAO and LPS, compared to cells without this treatment and with negative control cells. The treatment of neutrophils with a cPLA 2 -α inhibitor also showed reduced PGE 2 in cells without treatment and in negative control cells. When both inhibitors were administered simultaneously, neutrophils incubated with Cr-LAAO or LPS showed reduced PGE 2 release, demonstrating that Cr-LAAO and LPS used cPLA 2 and lipid bodies to generate PGE 2 . We observed a greater effect of CAY10650 on inhibition of PGE 2 release when used alone than when added in combination with the DAGT1 inhibitor (Fig. 5).
When considered as a whole, the data obtained in this study and previous results published in the literature, we proposed a mechanism of action of Cr-LAAO on human neutrophils for PGE 2 production. Firstly, Cr-LAAO interacts with the cellular membrane by an unknown mechanism, leading to PKC activation, which stimulates the p38 MAPK phosphorylation, and cPLA 2 activation. Activated cPLA 2 cleaves membrane phospholipids to form arachidonic acid (AA). AA can be catalyzed by COX-1 or COX-2, forming PGH 2 , metabolized by PTGES to form PGE 2 for release. Activation of cPLA 2 and DGAT may lead to increased numbers of lipid bodies in neutrophils. Cr-LAAO can utilize lipid bodies content to release PGE 2 . Thus, in the presence of cPLA 2 (CAY10650) and DGAT (A922500) inhibitors, there is a decrease in the level of PGE 2 released (Fig. 6).

Discussion
In leukocytes, cell membranes are permeable to hydrogen peroxide, which can activate leukocyte functions. In neutrophils, hydrogen peroxide regulates both the extension of pseudopodia and the orientation and direction of the cell 32 . Moreover, there is evidence demonstrating that hydrogen peroxide controls the recruitment of leukocytes to the inflammatory site 33 . Klyubin et al. 34 were the first to propose that hydrogen peroxide acts as a chemotactic in leukocytes 34 .
LAAO from snake venoms have been described to perform several biological functions, including proinflammatory activities 27 . According to previous studies, Cr-LAAO activates isolated human neutrophils and stimulates ROS production (hydrogen peroxide and superoxide anion), chemotaxis, phagocytosis, cytokine release such as IL-8, IL-6, TNF-α, and LTB 4 , as well as the release of PGE 2 , NETs, and MPO, in addition to activating p38MAPK and PI3K 28,29 . Moreover, Costa et al. 35 recently reported the inflammatory responses induced by Cr-LAAO. The authors showed that this enzyme induced neutrophil recruitment and IL-6, IL-1β, LTB 4 , and PGE 2 release by murine macrophages. Paloschi et al. 30 showed that Cr-LAAO induces NADPH oxidase complex and PKC-α activation, which contributes to the ROS production (hydrogen peroxide and superoxide anion) observed earlier.
Studies from our laboratory showed that isolated human neutrophils stimulated with Cr-LAAO at several concentrations (6, 12.5, 25, 50, and 100 µg/mL) remained alive until 12 h of incubation, using MTT and trypan blue viability methods. Based on these results, we adopted a concentration equal to 50 μg/mL, an incubation period of 1 h, and a temperature of 37 °C in a humid atmosphere of 5% CO 2 28,29 . Moreover, this period of time www.nature.com/scientificreports/ www.nature.com/scientificreports/ was defined based on PGE 2 production by neutrophils under Cr-LAAO action, as previously reported by Pontes et al. 29 . However, the pathways responsible for this effect were not identified. Microarray technology allows the investigation of thousands of genes simultaneously, substantially increasing the analytical capacity of molecular processes 36 . A study employing microarray assays with the LAAO of Ophiophagus hannah (OH-LAAO) on human breast adenocarcinoma cells (MCF-7) showed the expression of 178 genes after treatment with the enzyme, of which 27 were expressed due to the cytotoxic action of LAAO in relation to apoptosis, autophagy, cell cycle, DNA replication, oxidative stress, proteolysis, and intracellular signalling 37 . In another study, Guo et al. 38 used microarray analysis to screen differentially expressed genes related to molecules involved in the TGF-β signalling pathway in human hepatocellular carcinoma (HepG2) cells in response to the action of LAAO from Agkistrodon blomhoffii ussensis (Akbu-LAAO) 38 . Pontes et al. 29 have previously demonstrated PGE 2 release by human neutrophils stimulated with Cr-LAAO; therefore, we decided to perform a microarray analysis on 38 genes related to COX signalling pathway and LBs, which may be involved in PGE 2 production and release. These selected genes corroborate previous data obtained by Pontes et al. 29 and complement the cellular activation mechanism of this enzyme on human neutrophils.
The COX pathway is initially activated by PLA 2 s, which perform various functions in the maintenance of homeostasis through membrane phospholipid cleavage to produce lipid mediators 39 . Cytosolic PLA 2 (cPLA 2 ) is a type of PLA 2 that is activated upon phosphorylation (p-cPLA 2 ) 39 . PLA2G4A is one of the genes encoding cPLA 2 -α, the cytosolic form prominently expressed in neutrophils 40 . cPLA 2 -α, translocates from the cytoplasm to the intracellular membrane in response to calcium to stimulate the release of arachidonic acid from these membranes. cPLA 2 -α is phosphorylated by MAPKs, which increases its catalytic activity 39,41 . Once activated, cPLA 2 -α releases arachidonic acid from the membrane phospholipids, which triggers the activity of enzymes that oxygenate arachidonic acid (AA), generating various eicosanoids 42 . During this process, AA is formed and subsequently metabolised by COXs, which depending on the stimulus, can be driven by COX-1 (constitutively active) or COX-2 (induced), resulting in PGE 2 as the end product 9,10 . Studies have shown that the expression of the PLAA gene is necessary for the production of AA, leading to an increase in the production of PGE 2 through the activation of cPLA 2 , iPLA, and COX-2, in response to stimuli such as TNF-α and LPS [43][44][45] .
In the present study, we observed an association between PLAA and cPLA 2 gene expression and the COX-2 accumulation for PGE 2 release. Stimulation of neutrophils with Cr-LAAO resulted in the expression of cPLA 2 and accumulation of its phosphorylated form (p-cPLA 2 ), demonstrating enzyme activation. Gene and protein expression for COX-1 showed that there was an expression of this constitutive protein in all groups. However, with respect to COX-2, there was a greater expression in neutrophils stimulated with Cr-LAAO and LPS when compared to the negative control, demonstrating COX-2 accumulation in the cytosol. COX-2 presence in the cytoplasm was confirmed with immunofluorescence imaging, demonstrated with an increase in the fluorescence level of the labeled enzyme in Cr-LAAO-stimulated neutrophil cytosol. In addition, the expression of PTGES, the protein responsible for the conversion of PGH 2 to PGE 2 , was evaluated, showing the presence of this protein in neutrophils stimulated with Cr-LAAO. This is the first study to elucidate the mechanism underlying the cyclooxygenase pathway in human neutrophils stimulated with Cr-LAAO, which can be important for the comprehension and management of the local aspects observed in snakebites.
Diverse enzymes that lead to eicosanoid biosynthesis may be associated with LBs under activation conditions, including cPLA 2 -α 46,47 . cPLA 2 -α can remodel phospholipids of the endoplasmic reticulum and increase LBs involving deacylation/reacylation reactions [48][49][50] . Gubern et al. 51 demonstrated that cPLA 2 -α inhibition reduces the levels of LBs in CHO-K1 cells. The authors also showed that knocking down cPLA 2 -α expression with short interfering RNA is similar to pharmacological inhibition in terms of enzyme activity and LBs biogenesis 51 . The protein expression results showed that there was no expression of p-cPLA2-α in the cells pretreated with CAY10650. Similarly, this result was observed when AACOCF3, another effective cPLA2-α inhibitor 52-55 , was used. Meanwhile I take this opportunity to bring to the knowledge of that AACOCF3 stimulated the LBs formation in neutrophils as demonstrated by Bozza et al. 56 .
LB biogenesis during inflammation is regulated both by the stimulus that the cell is submitted to and by specific signalling pathways. Naïve leukocytes, including neutrophils, have few LBs in their cytoplasm. However, when cells are activated, there is a significant increase in the number and size of LBs 31 . Nose et al. 57 demonstrated that perilipin 3 (PLIN3) plays a crucial role in the formation of LBs in neutrophils for the release of PGE 2 . In our study, we found a positive regulation of PLIN3 and PLIN2 in human neutrophils after stimulation with Cr-LAAO. Among the proteins that comprise the LBs, PLIN2 is one of the main structural proteins found in all cell types 58 , which was a relevant factor in this study to examine PLIN2. Chen et al. 59 showed that the increase in PLIN2 mRNA level is directly linked to increased mRNA levels of proinflammatory cytokines, such as TNF-α and IL-6. This finding corroborates the results obtained by Pontes et al. 28,29 which showed that Cr-LAAO induces TNF-α and IL-6 release by neutrophils. Gene expression results obtained in the current study showed that there was an increase in PLIN2 expression in neutrophils stimulated with Cr-LAAO, supporting previous findings.
Lipid bodies are intracellular organelles that mainly store triacylglycerols (TGs) and sterol esters (SEs) as a bioenergy source. Harris et al. 60 showed that DGAT1 and DGAT2 are responsible for the synthesis of almost all TGs in adipocytes, because in the absence of DGAT, adipocytes lack TGs and LBs, but in macrophages, they are not absolutely necessary for the formation of LBs. DGAT1 is proposed to possess dual topology, contributing to triacylglyceride (TAG) synthesis on both sides of the endoplasmic reticulum membrane and esterifying only the pre-formed fatty acids. Studies suggest that DGAT2 translocates to LBs and is associated with other structural proteins to synthesis TGs from endogenous and exogenous fatty acids 56,61 . In this study, microarray data demonstrated for the first time the signalling dependent up-regulation of DGAT1 and DGAT2 in human neutrophils. In addition, an increase in the amount of LBs present in the neutrophil cytoplasm stimulated with Cr-LAAO was verified. Treatment with a DGAT1 inhibitor (A922500) diminished the biogenesis of LBs to baseline numbers in neutrophils stimulated with Cr-LAAO. This data is in agreement with previous results that demonstrate that TAG is the main component in LB and consistent remodeling of AA pools from human neutrophil 57 .
Previous studies have shown that AA-rich LBs rapidly associate with phagosomes, suggesting that AA derived from LBs functions as an activator of the NADPH oxidase complex in phagosomes 62,63 . According to Paloschi et al. 30 , NADPH oxidase is activated by Cr-LAAO in human neutrophils. Additionally, as reported by Pontes Figure 5. Prostaglandin E 2 release by neutrophils pretreated with DGAT and cPLA2 inhibitor. PGE 2 quantification in the supernatant of neutrophils (2 × 10 5 ) pre-treated or not with CAY10650 (cPLA 2 ) and A922500 (DGAT) inhibitors and followed by stimulation with Cr-LAAO (50 μg/mL), LPS (1 μg/mL; positive control) or RPMI (negative control) for 1 h at 37 °C and 5% CO 2 . This figure was created using images from Servier Medical Art Commons Attribution 3.0 Unported License (https ://smart .servi er.com) (A). Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License. PGE 2 concentrations were quantitated by specific EIA in supernatant collected after incubation with RPMI or LPS or Cr-LAAO. The results were expressed as pg/mL of PGE 2 produced and represent the mean ± S.E.M of 3 donors (B). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to negative control, and # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 compared to without pre-treatment with inhibitor (Data were presented with ANOVA followed by Tukey post-test). Values are mean S.E.M. from 3 donors. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to negative control, and # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 compared to without pre-treatment with inhibitor (Data were presented with ANOVA followed by Tukey post-test). www.nature.com/scientificreports/ et al. 29 , these cells phagocytose more zymosan particles under Cr-LAAO stimulation. Therefore, the present data support that LB biogenesis could be mediated by the activation of DGAT and cPLA 2 in neutrophils stimulated with Cr-LAAO, and it is possible that the increase in LBs may participate in the activation of NADPH oxidase, as shown previously by Paloschi et al. 30 .
Other studies conducted with snake venoms and isolated toxins have demonstrated the production of LBs in macrophages. De Carvalho et al. 64 investigated the venom capacity of Crotalus durissus ruruima and an isolated phospholipase A 2 (CBr) to activate macrophages. The researchers focused on lipid droplet formation and the synthesis of lipid mediators involved in these effects, demonstrating PLIN2 recruitment, as well as the expression and presence of PGE 2 in LBs as sites for prostatenoid synthesis. The isolated phospholipase A 2 from Crotalus durissus terrificus venom, which has an anti-inflammatory effect, caused increased LB and PLIN2 recruitment in macrophages 65 . The increase in LB formation in macrophages from J774 cell line has also been observed upon stimulation with two basic myotoxic phospholipase A 2 : BaTX-I, a catalytically inactive Lys-49 variant, and BaTX-II, a catalytically active Asp-49, and of one acidic myotoxic PLA 2 , the BaPLA 2 , a catalytically active Asp-49, all of them isolated from Bothrops atrox snake venom 66 . MT-II, a Lys49-PLA 2 from Bothrops asper venom devoid of catalytic activity, induces the formation of LBs and the synthesis of PGE 2 , as well as its localisation in LBs 67 . In addition, a study on the function of MT-III, a sPLA 2 from B. asper, in the biogenesis of LBs in murine macrophages, demonstrated the expression of PLIN2, which depends on the PKC, PI3K, p38MAPK, ERK1/2, cPLA 2 , and iPLA 2 signalling pathways, but not in the PTK, COX-1, or COX-2 pathways 68 . In human neutrophils, the relation between cPLA 2 and LB biogenesis is unknown. In the present study we showed that Cr-LAAO could activate cPLA 2 in neutrophils to induce an increase in the LBs formation in the cell. This accumulation of LBs may be directly related to the release of PGE 2 , a product of COX-2 activation, which has been shown to accumulate in the cytosol of activated neutrophils, together with PLIN2 recruitment. In addition to this finding, we have seen in our previous studies the presence of important signalling proteins for this process, such as PKC, PI3K and p38MAPK [28][29][30] .
Collectively, the data of the present study demonstrate for first time that Cr-LAAO induces the regulation of lipid signalling and metabolism in human neutrophils, leading to increased LB biogenesis and PGE 2 production. Moreover, Paloschi et al. 30 and Pontes et al. 29 demonstrated that Cr-LAAO induces the activation of PKC and p38-MAPK, respectively; and here, we demonstrated that Cr-LAAO induces the cPLA 2 activation which present an important role in the mechanisms of LBs biogenesis in human neutrophils (Fig. 6). The inhibition of Figure 6. Suggestion of mechanism of activation of the cyclooxygenase pathway stimulated by Cr-LAAO. The representative scheme shows Cr-LAAO action on neutrophils. Firstly, Cr-LAAO interacts with the cellular membrane by an unknown mechanism (01), leading to PKC activation (02), which stimulates the p38 MAPK phosphorylation (03), and cPLA 2 phosphorylation and activation (04). Activated cPLA 2 cleaves membrane phospholipids to form arachidonic acid (AA) (05). AA can be catalyzed by COX-1 or COX-2 (06), forming PGH2, metabolized by PTGES (07) to form PGE 2 (08) for release (09). Activation of cPLA 2 (10) and DGAT (11) may lead to increased numbers of LBs in neutrophils because LB is rich in arachidonic acid (AA) for PGE 2 synthesis. Cr-LAAO can utilize LBs content to release PGE 2 (12). Thus, in the presence of cPLA 2 -α (CAY10650) (13) and DGAT (A922500) (14) inhibitors, there is a decrease in the PGE 2  www.nature.com/scientificreports/ LB formation by DGAT-1 inhibitor also lead to decrease the PGE 2 production suggesting that LB participate in the increase of the PGE 2 synthesis in Cr-LAAO-stimulated neutrophils. However, the data shown here do not rule out the possibility that LB formation and PGE 2 release occur simultaneously and independently of each other. Although the mechanism through which Cr-LAAO interacts with the cellular membrane for neutrophil activation is still not fully understood, Cr-LAAO regulates lipid metabolism and signalling in human neutrophils through the expression and activation of enzymes and structural proteins, including cPLA 2 , DGAT-1, COX-2, PGES and PLIN2 and PLIN3, that participates in the amplification of the inflammatory process by triggering intracellular signalling cascade that culminate in lipid body formation and increased PGE 2 synthesis.  www.nature.com/scientificreports/ SYBR Green Supermix kit (Bio-Rad) on Rotor-Gene Q (QIAGEN), with primers pre-designed for mRNA gene expression (DNA Express Biotechnology) analysis (Table S2). The relative fold change quantification of each gene was calculated by the 2 ΔΔCt method 69 using the reference gene hemoglobin subunit beta (HBB) for normalization.

Chemicals and reagents. Crystallized
Western blot. For this assay, 1 × 10 7 isolated and stimulated human neutrophils for 1 h according to items above pre-treated with CAY10650 (cPLA 2 -α inhibitor, 12 nM for 30 min 70 ) or AACOCF3 (cPLA 2 inhibitor, 20 µM for 30 min 71 ) or the same vehicle used to dissolve the inhibitors in RPMI (control). For β-actin, cPLA 2 -α, p-cPLA 2 -α, COX-1, COX-2 and PTGES determinations, total protein extracts were prepared, resolved by 10% SDS/PAGE and transferred onto a PVDF membrane (Hybond, Amersham Pharmacia Biotech). Immunoblotting was performed using monoclonal antibodies to the referent proteins (Fig. S1). Blots were developed with 3,30-diaminobenzidine tablets and hydrogen peroxide (Sigma-Fast) 29 . The relative immunoreactivity bands of three independent experiments were quantified by densitometry using Image Studio Lite Ver 5.2 (LI-COR, Lincoln, Nebraska, EUA). The mean densitometry values of tested proteins were divided by the mean densitometry values of respective β-actin values to show the relative expression of each protein as a ration mean of the protein/β-actin 30 . Immunofluorescence. For immunofluorescence microscopy, 2 × 10 5 isolated and stimulated human neutrophils as mentioned above for 1 h were seeded on 70% alcohol-washed coverslips and treated with Poly-l-Lysine (Sigma Aldrich) and placed in 24-well plates. The cells were fixed with 4% paraformaldehyde at room temperature for 15 min. Next, cells were permeabilized with acetone PA for 5 min at room temperature and the cells were incubated with the anti-COX-2 primary antibody (Cayman Chemical) overnight, followed by incubation with FITC-conjugated Fab anti-mouse secondary antibody (Sigma Aldrich) 1 h and staining with Alexa Fluor 647 phalloidin (Invitrogen), according to the manufacturer's instructions. After DAPI staining, the coverslips were mounted with Fluoromout G (Sigma Aldrich) and analyzed under a Nikon Eclipse 80i microscope with a 100 × magnification oil immersion objective. The images were collected using constant automatic gain among the samples to quantify the differences in absolute levels of fluorescence intensity different conditions. Ten fields of view of each condition were collected impartially. The acquired images were subsequently analyzed using ImageJ software (National Institutes of Health) to quantify the absolute total fluorescence intensity. The calculated fluorescence intensity of the fields of view was plotted as mean normalized intensity for the total number of cells 72 .
Lipid bodies quantification. The 0.3% Oil Red O (ORO) solution was prepared in isopropanol P.A., diluted in distilled water (3:2) and filtered through a paper filter to avoid precipitates. Isolated and stimulated neutrophils (2 × 10 5 ) for 2 h in the presence or absence of inhibitors CAY10650 (cPLA 2 -α inhibitor, 12 nM for 30 min 70 ) and A922500 (DGAT1 inhibitor; 5 µM for 30 min 73 ), were seeded on 70% alcohol-washed coverslips, treated with Poly-l-Lysine (Sigma Aldrich) and placed in 24-well plates. The supernatant was removed and the cells were fixed with 4% paraformaldehyde at room temperature for 15 min. The coverslips were stained with ORO (300 μL/well) for 2 min, washed with 30% isopropanol and subsequently with distilled water. The coverslips were mounted with Fluoroshild with DAPI (Sigma Aldrich) and analyzed under a Nikon Eclipse 80i microscope with a 100 × magnification oil immersion objective. LBs were quantified from fifty consecutive cells and the results were expressed of mean normalized for the total number of cells 74 .