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A Lys49 Phospholipase A2, Isolated from Bothrops asper Snake Venom, Induces Lipid Droplet Formation in Macrophages Which Depends on Distinct Signaling Pathways and the C-Terminal Region

MT-II, a Lys49PLA2 homologue devoid of catalytic activity from B. asper venom, stimulates inflammatory events in macrophages. We investigated the ability of MT-II to induce formation of lipid droplets (LDs), key elements of inflammatory responses, in isolated macrophages and participation of protein kinases and intracellular PLA2s in this effect. Influence of MT-II on PLIN2 recruitment and expression was assessed, and the effects of some synthetic peptides on LD formation were further evaluated. At noncytotoxic concentrations, MT-II directly activated macrophages to form LDs. This effect was reproduced by a synthetic peptide corresponding to the C-terminal sequence 115–129 of MT-II, evidencing the critical role of C-terminus for MT-II-induced effect. Moreover, MT-II induced expression and recruitment of PLIN2. Pharmacological interventions with specific inhibitors showed that PKC, PI3K, ERK1/2, and iPLA2, but not P38MAPK or cPLA2, signaling pathways are involved in LD formation induced by MT-II. This sPLA2 homologue also induced synthesis of PGE2 that colocalized to LDs. In conclusion, MT-II is able to induce formation of LDs committed to PGE2 formation in a process dependent on C-terminal loop engagement and regulated by distinct protein kinases and iPLA2. LDs may constitute an important inflammatory mechanism triggered by MT-II in macrophages.


Introduction
Phospholipases A 2 s (PLA2; EC 3.1.1.4) constitute a family of lipolytic enzymes with key roles in several cellular processes by regulating the release of arachidonic acid and lysophospholipids from cell membrane phospholipids. Venoms from snakes of the Viperidae family contain group IIA phospholipases A 2 (PLA 2 s), which share structural and functional features with PLA 2 s found in in�ammatory exudates in mammals [1,2]. A number of Bothrops snake venom PLA 2 s have been shown to induce in�ammatory events such as edema and leukocyte in�ltration and to directly activate in�ammatory cell functions [3][4][5][6].
Basic PLA 2 s are considered the most important venom components responsible for the severe local myotoxicity and in�ammation characteristic of the envenomation induced by Bothrops genus snakes [7]. ese enzymes are further divided into two subgroups, namely, catalytically active variants, presenting a conserved aspartic acid residue at position 49 (Asp49PLA 2 s), and catalytically inactive homologues, known as Lys49PLA 2 s, which present various substitutions in residues of the Ca 2+ binding loop, as well as at position 2 BioMed Research International 49, where Lys replaces the highly conserved Asp [8,9]. Such modi�cations drastically affect the catalytic ability of these proteins rendering these homologues enzymatically inactive [10]. Interestingly, Lys49PLA 2 homologues are highly myotoxic, bactericidal, and proin�ammatory [9], evidencing that phospholipid hydrolysis is not strictly required for these activities. Studies on synthetic peptides and site-directed mutagenesis identi�ed the C-terminal region of Lys49PLA 2 s as essential for their biological activities [10,11]. us, Lys49PLA 2 homologues constitute interesting models to investigate a series of cellular effects which do not depend on membrane phospholipid hydrolysis.
In the Bothrops asper snake venom three myotoxic Lys49-PLA 2 s have been identi�ed, named MT-II, MT-IV, and M1-3-3, and reported in UNIPROT database. Besides myotoxicity, MT-II, the most studied Lys49PLA 2 homologue, has been reported to induce in�ammation in vivo [5,12] and to activate some in�ammatory functions of macrophages in vitro, increasing phagocytosis, respiratory burst, and release in�ammatory mediators [4] at noncytotoxic concentrations. However, the knowledge on the effects of this Lys49PLA 2 in macrophages functions, is still fragmentary.
Macrophages play key roles in a wide variety of processes associated with tissue maintenance, antigen presentation, in�ammation, and tissue repair [13]. Upon in�ammatory stimuli, quiescent macrophages become activated and present increased number of lipid rich cytoplasmic organelles named lipid droplets (LDs), also known as lipid bodies. ese organelles are functionally involved in biosynthesis, transport, and catabolism of lipids [14,15] as well as biosynthesis and accumulation of in�ammatory mediators, such as eicosanoids and cytokines [16,17]. Moreover, leukocyte LDs associated with in�ammatory responses have been shown to compartmentalize signaling proteins involved in cellular activation and structural proteins, mainly perilipin 2 (PLIN2), also named adipophilin (Adipose differentiationrelated protein, ADRP), which has an important role in LD assembly and formation of foam macrophages [18,19], which are markers of atherosclerotic plaques [19]. Increased numbers of lipid droplets are described in distinct populations of leukocytes during in�ammatory and infectious processes [20,21]. Recently, MT-III, a catalytically active variant Asp49PLA 2 from B. asper venom, has been shown to activate macrophages to form increased amounts of LDs [22], but no such effect has been described for the action of Lys49PLA 2 s. erefore, it is relevant to assess the effects of MT-II on macrophages in terms of LD formation. Such macrophage activation might play a relevant role in the scenario of the local pathological alterations induced by snake venom toxins. Based on these information, in the present study the ability of MT-II to induce LD formation in macrophages was evaluated and the mechanisms involved in this effect were analyzed in terms of recruitment and expression of PLIN2, participation of intracellular PLA 2 s (cPLA 2 and iPLA 2 ) and signaling protein kinases. In light of the absence of catalytic activity in MT-II, the effects of some synthetic peptides related to distinct regions of this Lys49PLA 2 molecule on lipid droplet formation were further evaluated in macrophages.

Chemicals and Reagents.
MTT and L-glutamine were obtained from USB Corporation (Cleveland, OH, USA). H7, LY294002, SB202190, PD98059, and Pyr-2 were purchased from Calbiochem-Novabiochem (La Jolla, CA, USA). Racemic mixture of BEL and anti-mouse PGE 2 was obtained from Cayman Chemical (Ann Arbor, MI, USA). Guinea pig polyclonal antibody anti-mouse PLIN2 and FITCconjugated donkey anti-guinea pig antibody were obtained from Research Diagnostics Inc. (Flanders, NJ, USA). Secondary antibodies anti-mouse and anti-guinea pig conjugated to horseradish peroxidase and nitrocellulose membrane were obtained from GE Healthcare (Buckinghamshire, UK). Gentamicin was purchased from Schering-Plough, NJ, USA). DMSO and BSA were obtained from Amresco (Solon, OH, USA). Mouse monoclonal antibody anti--actin, Nile Red, RPMI-1640, thiocarbohydrazide, OsO 4 , and EDAC were purchased from Sigma Aldrich Co. (St. Louis, MO, USA). PFA was purchased from Electron Microscopy Science (USA). Alexa Fluor 488 Goat Anti-mouse IgG was purchased from Life Technologies (Grand Island, NY, USA). DAPI and �uoromount G were purchased from Molecular Probes (Eugene, OR, USA). Donkey serum was obtained from Jackson ImmunoResearch Laboratories (PA, USA). Triton-X was obtained from Union Carbide Corporation (Danbury, USA). GA, thioglycolate, and all salts used were obtained from Merck (Darmstadt, Germany).

Animals.
Male Swiss mice (18-20 g) were obtained from Butantan Institute (São Paulo, Brazil). Animals were housed in a temperature-controlled room (22-24 ∘ C) with a 12 h light-dark cycle and fresh water and food ad libitum until used. is study was approved by the Butantan Institute Animal Experimentation Ethics Committee (reference number 760/10) in accordance with the procedures laid down by the Universities Federation for Animal Welfare.

Synthetic
Peptides. e following synthetic peptides were synthesized and used for biological assays: (a) peptide 115-129 (KKYRYYLKPLCKK) corresponding to the original sequence 115-129 of MT-II from B. asper snake venom; (b) peptide p115-W3 (KKWRWWLKPLCKK) corresponding to the triple tyrosine-to-tryptophan substitution of p115-129; (c) peptide pEM-2 (KKWRWWLKALAKK), in which the proline and cysteine residues of p115-W3 were each replaced by an alanine residue; (d) peptide 60-71 (KKDRYSYSWKDK) corresponding to a central region sequence of MT-II; (e) peptide p-Scr (FKFKYKKACKKYK) corresponding to a scrambled peptide version of the sequence 115-129 of ACL myotoxin, a Lys49 PLA 2 homologue from the venom of the snake Agkistrodon contortrix laticinctus.
Synthetic peptides were prepared in automated benchtop simultaneous multiple solid-phase synthesizer (PSSM 8 system from Shimadzu Co.) using solid-phase peptides synthesis by the Fmoc procedure [25�. Brie�y, sequential couplings of protected amino acids were performed with HOBt, TBTU and NMM on Fmoc-Lys(Boc)-Wang resin (Merck KGaA, Germany). Fmoc group cleavage was performed with 30% piperidine (v/v) in DMF. e resin-bound peptides were cleaved/deprotected with TFA/thioanisole/EDT/phenol/water (82.5 : 5: 2.5 : 5 v/v/v/v) at room temperature for 4 h. Aer �ltration, the �ltrate was concentrated under argon stream and precipitated with diethyl ether. All crude peptides were puri�ed by reversedphase chromatography (Shim-pack Prep-ODS, Shimadzu Co.) semipreparative HPLC, and the purity and identity of the peptide were con�rmed by mass spectrometry and by analytical HPLC.

Harvesting of Macrophages.
Peritoneal macrophages were harvested 4 days aer i.p. injection of 1 mL of 3% thioglycolate. Animals were killed under CO 2 atmosphere and cells were harvested by washing peritoneal cavities with 3 mL of PBS, pH 7.2, containing 10 IU/mL heparin. Aliquots of the washes were used for total cell counts in a Neubauer chamber aer dilution (1 : 20, v/v) in Turk solution (0.2% crystal violet dye in 30% acetic acid). Differential cell counts were performed on smears stained with Hema3. More than 95% of the cell population consisted of macrophages, as determined by conventional morphological criteria. e remaining wash volumes were centrifuged at 500 ×g for 6 min (4 ∘ C) and the cell pellets were used for subsequent studies aer suitable dilutions.
2.6. Cytotoxicity Assay. Cytotoxicity of MT-II towards elicited macrophages was evaluated using the MTT assay. In brief, 2 × 10 5 macrophages/well in RPMI-1640 medium supplemented with 40 g/mL gentamicin sulfate and 2 mM L-glutamine were plated in 96-well plates and incubated with 100 L of selected concentrations of MT-II (0.4-0.8 M) diluted in medium or with the same volume of medium alone (control) for 1, 6, 12, and 24 h at 37 ∘ C in a humidi�ed atmosphere (5% CO 2 ). MTT (5 mg/mL) was dissolved in PBS and �ltered for sterilization and removal of a small amount of insoluble residue present in some batches of MTT. Stock MTT solution (10% in culture medium) was added to all wells in each assay, and plates were incubated at 37 ∘ C for 3 h. One hundred L of DMSO were added to all wells and mixed thoroughly at room temperature for 30 min. Absorbances at 540 nm were then recorded in a microtiter plate reader. Results were expressed as percentage of viable cells, considering control cells incubated with medium alone as 100% viable.

Stimulation and Treatment of Macrophages.
Macrophages were plated on glass coverslips in 24-well plates at a density of 2 × 10 5 cells/coverslip and allowed to attach for 30 min at 37 ∘ C under a 5% CO 2 atmosphere. Non-adherent cells were removed by washing with PBS. Cell monolayers were cultured for 1 h in RPMI-1640 supplemented with 40 g/mL gentamicin sulfate and 2 mM L-glutamine at 37 ∘ C and 5% CO 2 and were then challenged with selected concentrations of MT-II (0.2-1.2 M) or synthetic peptides (250 g/mL) or medium (control). Where appropriate, the following inhibitors were used: 1 M SB202190, inhibitor of p38MAPK; 1 M LY294002, inhibitor of PI3 K; 6 M H7-Dihydro, inhibitor of PKC; 25 M PD98059, inhibitor of ERK1/2; 1 M Pyr-2 (Pyrrolidine-2), inhibitor of cPLA 2 and 2 M BEL (bromoenol lactone) an inhibitor of iPLA 2 . All stock solutions were prepared in DMSO and stored at −20 ∘ C. Aliquots were diluted in RPMI-1640 to the required concentration immediately before use. e �nal DMSO concentration was always lower than 1% and had no effect on lipid body numbers. All pharmacological inhibitors were added between 30 and 60 min before stimulation of macrophages with MT-II or medium (control). Cells treated with the inhibitors were analyzed for viability by the tetrazoliumbased (MTT) colorimetric assay. No signi�cant changes in cell viability were registered with any of the above agents or vehicle at the concentrations used (data not shown).

2.�. �ipid �ody Staining and �uanti�cation.
Analysis of lipid body numbers was performed in osmium-stained cells. In brief, macrophages (2 × 10 5 cells) adhered to glass coverslips were �xed in 4% PFA in 0.1 M phosphate buffer, pH 7.2, for 15 min, and stained with OsO 4 . e coverslips were then rinsed in 0.1 M phosphate buffer, stained in 1% OsO 4 (30 min), rinsed in deionized H 2 O, immersed in 1.0% thiocarbohydrazide (5 min), rinsed again in 0.1 M phosphate buffer, restained with 1% OsO 4 (3 min), rinsed with H 2 O, and then dried and mounted. e morphology of the �xed cells was observed and round osmiophilic structures were identi�ed as lipid droplets, which were then counted under phase-contrast microscopy using the 100x objective lens in 50 consecutively scanned leukocytes in each coverslip. For assays with �uorescent-labeled lipid droplets, macrophages (2 × 10 5 cells) adhered to glass coverslips were incubated with Nile Red staining solution freshly prepared in 0.1 M phosphate buffer (10 g/mL) for 20 min at room temperature and washed with phosphate buffer. Aer several washes the coverslips were mounted with �uoromount G and examined under a �uorescence microscope equipped with the appropriate �lter (�eiss LSM 510 Meta).

Electron Microscopy.
A standardized protocol for electron microscopy procedure was developed for ultrastructural analysis of lipid droplets. Macrophages (5 × 10 6 cells) incubated with either MT-II (0.8 M) or medium alone for 1 h were �xed in a diluted mixture of freshly prepared aldehydes (4% PFA/1% GA in 0.1 M phosphate buffer) containing 3.5% sucrose for 2 h at room temperature and then washed three times in 0.1 M phosphate buffer. Cells were centrifuged at 129 ×g, and cell pellets were post�xed with OsO 4 (1% in phosphate buffer at room temperature) followed by three washes with saline solution. Uranyl acetate in aqueous solution was then added for 2 h at room temperature before dehydration in a graded series of ethanol (70, 95 and 100% twice for 10 min each). For embedding, aliquots of propylene oxide were added twice for 10 minutes, followed by Spurr resin diluted in propylene oxide (1 : 1) and undiluted Spurr resin for 12 h. Polymerization was carried out for 48 h at 70 ∘ C. Samples were then incubated with 4% uranyl acetate and lead citrate for contrast. in sections were examined with LEO 906E and Zeiss EM109 transmission electron microscopes. 2.11. Western Blotting of PLIN2. Aliquots of MT-II-stimulated and -nonstimulated cells (2 × 10 6 cells) were lysed with 100 L of sample buffer (0.5 M Tris-HCl, pH 6.8, 20% SDS, 1% glycerol, 1 M -mercaptoethanol, and 0.1% bromophenol blue) and boiled for 10 min. Samples were resolved by SDS polyacrylamide gel electrophoresis (SDS-PAGE) on 10% bis-acrylamide gels overlaid with a 5% stacking gel. Proteins were then transferred to nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) using a Mini Trans-Blot (Bio-Rad Laboratories, Richmond, CA, USA). e membranes were blocked for 1 h with 5% nonfat dry milk in TTBS (20 mM Tris, 100 mM NaCl and 0.5% Tween 20) and incubated with primary antibodies against PLIN2 (1 : 2000 dilution) and -actin (1 : 3000) for 1 h. ey were then washed and incubated with the appropriate secondary antibody conjugated to horseradish peroxidase. Detection was by the enhanced chemiluminescence (ECL) method according to the manufacturer's instructions (GE Healthcare, Buckinghamshire, UK). Band densities were quanti�ed with a GS 800 Densitometer (Bio-Rad Laboratories, Richmond, CA) using the image analysis soware from Molecular Analyst (Bio-Rad Laboratories, Richmond, CA, USA).

Statistical
Analysis. Data are expressed as the mean ± standard error of mean (SEM) of at least three independent experiments. Multiple comparisons among groups were performed by one-way analysis of variance (ANOVA) followed by Tukey's test. Values of probability lower than 5% ( 0 0 ) were considered signi�cant.  of LDs was performed using a standardized procedure for TEM. As seen in Figure 3(a) control macrophages showed small, non-membrane-bound, light cytoplasmic LDs. Aer 1 h incubation, MT-II-stimulated macrophages showed light cytoplasmatic LDs that were present in markedly greater numbers than in the control cells but morphologically similar to LDs in these cells (Figure 3(b)). Also, an enlarged ER was observed in MT-II-stimulated cells in incubation period tested as showed in Figure 3(c).

Effects of Peptides Corresponding to Selected
Regions of MT-II Molecule on LD Formation. e effects of synthetic peptides derived from distinct regions of MT-II protein on LDs formation were investigated in macrophages. Experiments were carried out with macrophages stimulated with peptides corresponding to distinct regions of MT-II molecule for 3 h and then treated as necessary for lipid �xation and stained with OsO 4 . Figure 4(a) demonstrates that incubation of macrophages with the C-terminal peptide p115-129 induced a signi�cant increase ( 0.05) in the number of LDs aer 3 h of incubation in comparison with nonstimulated control cells. e effect induced by this Cterminal peptide did not differ from that observed in cells stimulated with MT-II native protein for 3 h, which caused a signi�cant increase in the number of LDs in comparison with control cells. On the other hand, neither a scrambled version of the residue sequence used as a control, p-Scr, nor the peptide comprising amino acid residues 60-71 of central region of MT-II modi�ed the basal numbers of LDs aer 3 h of incubation as compared with RPMI-treated control cells. As a positive control macrophages were incubated with MT-II native protein for 3 h. In this case, a signi�cant increase in LD number was detected in comparison with RPMI-treated control cells. All peptides tested were used in a concentration (250 g/mL) previously demonstrated in literature as effective to induce biologic effects (32), but without toxic effect on the viability of macrophages aer 3 h of exposure. Figure 4(b) demonstrates that incubation of cells with peptides p115-129, pScr, and p60-71 at a concentration of 250 g/mL did not affect macrophages viability, whereas incubation of cells with peptides pEM2 (150 g/mL) and p115-W3 (150 g/mL) signi�cantly ( 0.05) reduced the viability of macrophages making these peptides unsuitable for the present study. Although MT-II is recognized as a sPLA 2 devoid of catalytic activity, we investigated whether a possible residual enzyme activity of MT-II would lead to formation of LDs in macrophages. As shown in Figure  4

LD Formation Triggered by MT-II Is Dependent on Distinct Signaling Pathways.
To assess the role of kinases in the described actions of MT-II, we determined the effects of the speci�c inhibitors of p38, PI3 K, PKC, and ERK1/2 (SB202190, LY294002, H7-Dihydro, and PD98059, resp.) on MT-II-induced LDs in macrophages. As seen in Figure 5(a), the PI3 K and PKC inhibitors abolished the LDs formation in MT-II-stimulated macrophages compared with vehicletreated macrophages stimulated with MT-II. e ERK1/2 inhibitor, in turn, caused 49% reduction in the number of LDs in MT-II-stimulated macrophages when compared with vehicle-treated macrophages stimulated with MT-II ( Figure  5(b)). In contrast, the preincubation of macrophages with p38MAPK inhibitor did not change the number of LDs induced by MT-II, in comparison to cells stimulated with MT-II only ( Figure 5(b)). [26,27]. erefore, we investigated whether MT-II induces expression of this LD structural protein. Levels of PLIN2 protein expression were analyzed by western blotting in cells incubated and not incubated with MT-II for selected time periods. is analysis revealed increased expression of PLIN2 protein in cells stimulated with MT-II as early as 3 h of incubation, which was sustained up to 12 h. PLIN2 was minimally expressed or absent in control nonstimulated macrophages (Figures 6(a) and 6(b)).

PLIN2 Colocalizes to LDs in MT-II-Stimulated Macrophages.
To better understand the stimulatory effect of MT-II on macrophages that leads to LD formation, cells exposed to MT-II were immunostained with speci�c antibodies that recognize PLIN2 or neutral lipids from the LD core. As Red-marked, neutral lipid inclusions. As expected, no significant staining was detected in control macrophages.

Involvement of Intracellular PLA2s in MT-II-Induced LD Formation in Macrophages.
Since a cross-talk between sPLA 2 and intracellular PLA 2 s for production of prostaglandins has been described, we examined the effects of selective inhibitors of cPLA 2 (Pyr-2) or iPLA 2 (BEL) on MT-II induced formation of LDs. As shown in Figure 8, treatment of macrophages with BEL, but not with Pyr-2 compound caused 51% reduction in the number of LDs in MT-II-stimulated macrophages compared with vehicle-treated cells stimulated with MT-II. ese results indicate that iPLA 2 , but not cPLA 2 is involved in MT-II-induced LD biogenesis.

PGE 2 Colocalizes within LD Induced by MT-II-Stimulated
Macrophages. �nder in�ammatory conditions, lipid mediators are mainly produced within LBs, which compartmentalize both substrate and the enzymatic machinery required for eicosanoid production [28]. Considering that PGE 2 is the major prostaglandin produced in macrophages, we evaluated the subcellular localization of PGE 2 within MT-II-stimulated macrophages. As illustrated in Figure 9 immuno�uorescence microscopy revealed that macrophages stimulated with MT-II (0.8 M) for 3 h exhibited strong �uorescent staining (green) for PGE 2 , with a punctate cytoplasmic pattern, which was diffuse in the nonstimulated control cells. Fluorescent Nile Red-labeled LBs were also visualized 3 h aer MT-II-induced stimulation and were virtually absent in nonstimulated control macrophages. Overlapping images show that aer stimulation with MT-II, cytoplasmic-stained PGE 2 matched perfectly with Nile Red-marked, neutral lipid inclusions. As expected, no signi�cant staining was detected in control macrophages.

Discussion
Besides myotoxic activity, MT-II, a Lys49PLA 2 homologue, has been shown to activate some cellular processes in macrophages at noncytotoxic concentrations [4,5], and these effects may contribute to the overall tissue alterations caused by this toxin. �pon in�ammatory conditions, macrophages show increased numbers of cytoplasmic LDs, which have been implicated as key organelles involved in immunity and in�ammation.
In this study, we showed that MT-II, a catalytically inactive PLA 2 homologue, was able to directly induce an increase in the numbers of LDs in isolated murine macrophages. is phenomenon was time dependent and had a very fast onset and persisted up to 24 h aer stimulation. Within 1 h of MT-II stimulus the presence of weakly osmiophilic LDs, in close association with organelles such as endoplasmic reticulum, were evidenced by the ultrastructural analysis. According to the current model of LD biogenesis, these organelles arise from endoplasmic reticulum, where the enzymes that synthesize lipids reside [15,29]. erefore, endoplasmic reticulum may play a role in LDs biogenesis induced by MT-II.
Because LDs have been associated to regulated in�ammatory mediator synthesis with roles in in�ammatory and infectious conditions, and macrophages are central elements in the innate immune response it is plausible to consider that biogenesis of LDs induced by MT-II demonstrated herein represents an important mechanism by which this PLA 2 homologue displays an in�ammatory response and leads to production and release of in�ammatory mediators. Moreover, considering that basic PLA 2 s comprise around 30% of B. asper venom [30], the fact that MT-II elicited a �ey in�ammatory event in macrophages clearly indicates that this sPLA 2 homologue contributes to the local in�ammatory response triggered by the whole venom.
In addition, our data demonstrated that the absence of Ca 2+ and presence of Sr 2+ in culture medium did not alter LDs formation induced by MT-II, con�rming that the catalytic activity is not an essential requirement to enhancement of LDs biogenesis by MT-II. A number of experimental evidences suggested that a stretch of residues, located at the C-terminus of the MT-II protein molecule, and involving cationic and hydrophobic amino acids are responsible for myotoxic and cytotoxic effects of this [9,31,32] and other Lys-49sPLA 2 homologues [31,33]. Based on these and other studies, a model of Lys49PLA 2 s-membrane interaction was proposed by Lomonte et al. [9] in which the action of Lys49PLA 2 s is based on the interaction of the C-terminal positive residues with membrane anionic phospholipids. So far, in the present study we found that the synthetic peptides 115-129 corresponding to MT-II Cterminus peptide induced LD formation in macrophages similarly to the parent protein. is �nding indicates for the �rst time the speci�c region of MT-II molecule responsible for activation of macrophages, and gives support to the notion that the effects of MT-II in leukocytes are not related to the PLA 2 enzymatic activity. e membrane target(s) and the mechanisms by which this C-terminal peptide triggers macrophages activation to form LDs were not addressed in this study, although perturbation of the membrane phospholipid bilayer is likely to be involved.
Perilipin 2 is a protein ubiquitously expressed in a number of cell types, including macrophages, as a major component of intracellular LDs [34,35]. It has fatty acidbinding properties, contributes to cytoplasmic trafficking of newly synthesized lipids, and plays an important role in assembly of LDs as well as in foam cell formation [35][36][37]. PLIN2 expression can be induced by a variety of in�ammatory stimuli and has been associated with increased numbers of LD [26,27]. Consistent with its properties, PLIN2 has been considered as a marker of LDs assembly and lipid loading in in�ammatory cells, such as macrophages. Accordingly, our results showed that PLIN2 protein expression is upregulated by MT-II, given support to data demonstrating LD formation upon stimulus by this PLA 2 homologue. Furthermore, PLIN2 clustering co-localized to LDs was seen indicating that MT-II is also able to recruit this protein from its constitutive pools into LDs, and suggesting a role for PLIN2 as a nucleation site for the assembly of lipids to form new LDs under the stimulus of this Lys49PLA 2 .
LD biogenesis in leukocytes is a highly regulated process. Studies of the intracellular signaling pathways committed to this process in leukocytes have revealed that distinct pathways can trigger LD biogenesis in a stimulus-dependent manner [38]. To better understand the stimulatory effects of MT-II on LD formation, we herein used pharmacological approaches to identify the critical downstream signaling proteins involved in LD formation induced by this PLA 2 homologue and focused on major downstream signaling molecules that have previously been shown to participate in LD biogenesis which follows in�ammatory stimuli, such as PKC [39,40], PI3 K [16,26] and MAPKs (p38 MAPK and ERK1/2) [16,41]. As a marked LD formation was observed aer 3 h of incubation, the effects of pharmacological compounds were evaluated at this time interval. We found that MT-II-induced LD formation is regulated by speci�c signaling pathways and that PKC, PI3 K, ERK1/2, but not p38 MAPK are involved in the formation of LD induced by this PLA 2 homologue.
�ur �nding that macrophage activation by MT-II to form LDs is largely dependent on the PKC agrees with previous reports that PKC activation is implicated in LD formation induced by cys-fatty acid and PAF in leukocytes [39,42]. Considering that activation of PKC has been associated with increased expression of PLIN2 in macrophages [43], it is possible to suggest that in the present experimental conditions, PKC signaling pathway is important to MT-II-induced upregulation of PLIN2, and thus to the increased formation of LDs. Moreover, our observation that LDs formation induced by MT-II requires activation of the PI3 K pathway is in line with reports of participation of this signaling protein in processes related to lipid accumulation [26,44,45] and in the regulation of PLIN2 which has been largely associated to lipid accumulation into LDs, and to atherosclerosis [26,45]. Furthermore, our results implicating ERK1/2 signaling in the MT-II effect that leads to LD formation in macrophages are consistent with previous studies demonstrating the involvement of ERK1/2 in LDs biogenesis induced by cytokines and saturated fatty acids in macrophages [16,46]. Moreover, evidences of the involvement of ERK1/2 in regulation of PLIN2 expression and the growth of LDs [41] give support to our �ndings of increased protein expression of PLIN2 seen under MT-II stimulus, and is in line with the involvement of ERK1/2 in biogenesis of LDs induced by this PLA 2 homologue. Conversely, the speci�c inhibitor of p38 MAPK failed to inhibit MT-II-induced LD formation, implying that this MAPK element does not contribute to this MT-II-induced effect. Taken together, the above results evidenced that MT-II-induced LD formation is a regulated process associated to activation of selected downstream signaling pathways in macrophages. Of note, despite the lack of enzyme activity, MT-II triggers signaling pathways almost similar to those that signal increased formation of LDs induced by the catalytically active sPLA 2 MT-III in macrophages [22], thus providing an additional evidence of functional similarities between these two venom sPLA 2 variants.
It has been demonstrated that LDs are involved in production of in�ammatory mediators [28] and to act as platforms for enhanced PGE 2 synthesis during infection conditions [47,48]. Moreover, a number of enzymes and signaling proteins were shown to be associated with LDs, including the prostaglandin-forming enzymes named cyclooxygenases [47]. Our �ndings that MT-II caused an increase of PGE 2 intracellular pools, which colocalized to LDs in macrophages represent the �rst evidence that a sPLA 2 homologue is able to induce synthesis and compartmentalization of a lipid mediator in LDs. ese �ndings suggest that macrophage LD constitutes a relevant site for the synthesis and accumulation of eicosanoids under MT-II stimuli and may represent a rapid and alternative mechanism for PGE 2 production by which macrophages react to activation by this sPLA 2 homologue. Moreover, given the importance of PGE 2 in several in�ammatory settings, due to its hyperalgesic and edematogenic properties [49,50], it is reasonable to suggest that LDderived PGE 2 may have implications for the in�ammatory effects of MT-II. is hypothesis is in line with the view that LDs are dynamic organelles integrating lipid metabolism, in�ammatory mediator production, membrane tra�cking, and intracellular signaling [27,47,51].
A number of studies have demonstrated that secreted PLA 2 s crosstalk with the intracellular PLA 2 s (cPLA 2 and iPLA 2 ) to produce arachidonic acid-(AA-) derived in�ammatory mediators, such as prostaglandins, in several pathophysiological conditions [52,53]. cPLA 2 is recognized as a key regulator of stimulus-coupled cellular AA release [6,54]. e iPLA 2 in turn has no substrate speci�city for the fatty acid residue at sn-2 position, playing a minor role in eicosanoid synthesis. is intracellular enzyme, however, has a role in membrane phospholipid remodeling through deacylation/reacylation reactions [55]. In this context, cPLA 2 and iPLA 2 were demonstrated to be involved in LD biogenesis induced by stress in CHO-K1 cells [56] and by the Asp49PLA 2 MT-III in macrophages [22]. Taking the above information into account we investigated the participation of both intracellular PLA 2 isoforms in MT-II-induced LD formation. We found that treatment of macrophages with, compound Pyr-2, a speci�c inhibitor of cPLA 2 failed to inhibit MT-II-induced effect, indicating that cPLA 2 is not required for LD formation under MT-II stimulus. is �nding is in accordance with our observation that p38 MAPK is not involved in LD formation induced by MT-II, as upstream p38 MAPK is critical for phosphorylation and activation of cPLA 2 [57]. In contrast, inhibition of iPLA 2 by BEL compound signi�cantly reduced LD formation, thus implying iPLA 2 in the mechanisms involved in MT-II-induced LD formation. is �nding is supported by recent studies demonstrating participation of iPLA 2 s in the metabolism of fatty acids and triacylglycerol formation, which are involved in LD formation [56]. We believe that our results are the �rst demonstration that a sPLA 2 devoid of catalytic activity recruits an intracellular PLA 2 (iPLA 2 ) to induce a cellular event, such as LD formation in macrophages. However, the cellular steps involved in such a protein crosstalk were not addressed in the present study and deserve further studies.

Conclusions
Taken together, our data show that the venom group IIA sPLA 2 homologue MT-II directly activates murine macrophages to form LDs by a mechanism independent on enzymatic activity. is effect is related to the C-terminal loop of the MT-II molecule since a synthetic peptide corresponding to region 115-129-induced LD formation similarly to MT-II. Moreover, MT-II-induced LD formation is related to increased expression and recruitment of PLIN2 from its constitutive pools and regulated by distinct signaling pathways that include PKC, PI3 K, ERK1/2, and iPLA 2 . In addition, MT-II induced synthesis and compartmentalization of PGE 2 within LDs. erefore, LDs may represent and important platform for the synthesis and accumulation of lipid mediators under MT-II stimulus that takes place in the mechanisms whereby this Lys49PLA 2 triggers in�ammation.
Finally, considering that catalytically inactive PLA 2 s have been also described in mammalian tissues under normal and pathological conditions [58,59], this study may shed light on the possible activities of similar proteins on a more general scope, providing insights into the possible roles of human catalytically inactive PLA 2 homologues in in�ammatory conditions as it has been demonstrated for the snake venom Lys49PLA 2 .