*2.3. Bothrops svPLA2s Trigger Lipid Accumulation in Immunocompetent Cells*

As mentioned, the high enzymatic activity exhibited by ophidian sPLA2 provides a microenvironment rich in free fatty acids that exert stimulating effects in immunocompetent cells, leading to the biosynthesis of lipid mediators. Moreover, excess free fatty acids exert cytotoxic effects that trigger the activation of cellular mechanisms capable of converting

free fatty acids into metabolites of lower toxicity, known as neutral lipids (triacylglycerol and cholesterol—energetic body reserve) [210]. In recent decades, numerous studies have demonstrated the activation of intracellular metabolic pathways responsible for the metabolization of free fatty acids in neutral lipids and the consequent formation of dynamic organelles called lipid droplets (LDs) [211,212]. These organelles are composed of a hydrophobic neutral lipid core surrounded by a phospholipid monolayer membrane, which contains numerous proteins related to cellular activation in addition to structural proteins, such as perilipin 2 (PLIN2), which plays an important role in LD assembly and the formation of foam macrophage [213], marker cells in metabolic diseases, such as atherosclerosis and obesity [214]. LDs are commonly present in adipocytes due to the already-established role of these cells in the supply of energy in mammalian organisms [215]. In addition to the relevance regarding lipid homeostasis, the direct relationship between increased LD formation and inflammatory processes was evidenced by numerous studies [216–218]. In this sense, mammalian group IIA sPLA2s have been identified as potential plasma biomarkers for diseases related to lipid imbalance, such as atherosclerotic cardiovascular disease and obesity [219]. Alongside a marked inflammatory reaction, these metabolic diseases are characterized by lipid accumulation in immunocompetent cells [220,221]. In line with the ability of svPLA2s to elicit an inflammatory response characterized by a high level of inflammatory mediators and free fatty acids, it was demonstrated that MT-III (Asp49 PLA2), isolated from *B. asper* venom, induced LD formation enriched by PLIN2 protein in mice peritoneal macrophages [170]. This effect was likewise observed in rat vascular smooth muscle cells isolated from the thoracic aorta stimulated by MT-III [168]. Moreover, the ability of MT-II, a Lys49 PLA2 homologue devoid of catalytic activity from *B. asper* venom, to directly activate macrophages to form LDs was reported [167]. 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 the MT-II-induced effect [167]. Similarly, BaTX-I, a catalytically inactive Lys49 variant; BaTX-II, a catalytically active Asp49; and BaPLA2, an acidic catalytically active Asp49 PLA2, isolated from *B. atrox* snake venom, increased the number of LDs on murine macrophages cell line J774A.1 [152]. The formation of LDs upon stimulus by MT-III was likewise demonstrated in human monocytes of peripheral blood [222].

Considering the above information, LD formation induced by svPLA2s in phagocytes proved to be inherent to the action of sPLA2s regardless of the catalytic activity. The capability of *Bothrops* svPLA2s to induce the formation of LDs is related to the activation of PRRs of an innate immune response, kinase proteins, and intracellular PLA2 signaling pathways involved in cellular metabolism, proliferation, and differentiation [170,205]. Hence, by using gene knockout mice cells and a pharmacological approach, the participation of TLR2, MYD 88 adaptor protein, and CD36 in LD formation in macrophages in culture has been reported [170,205]. In addition, the upregulation of CD36 receptors was observed in these cells. Considering the participation of the MYD 88 adaptor molecule, the involvement of other TLRs has yet to be investigated. In line with the reported Asp49 svPLA2 action on PRRs, the upregulation of both SRA-1, from the scavenger receptor family, and LOX-1, an LDL receptor, was demonstrated in mouse aortic smooth muscle cells (VSMCs) [168]. These findings demonstrate that the inflammatory response elicited by Asp49 svPLA2 likewise involved the upregulation of PRRs associated with lipid uptake in immunocompetent cells. This fact indicates that svPLA2s can be useful tools in studies aiming to understand the diseases associated with lipid imbalance.

In addition to providing the synthesis of mediators, TLR2 activation elicited by MT-III action was related to cytoskeleton activation [205], a critical step in the transport of structural proteins into LDs, such as PLIN2. Cytoskeletal activation involves the activation of kinase proteins [223]. Consistent with this information, the participation of kinase proteins in LD formation induced by both Asp49 and Lys49 svPLA2s from *B. asper* venom has been demonstrated through a pharmacological approach and the detection of phosphorylated kinase proteins. Yet, MT-II- and MT-III-induced LD biogenesis is dependent on the activation of PKC, PI3K, p38MAPK, and ERK1/2 signaling pathways in mice peritoneal macrophages [224]. It is well known that PKC regulates a variety of processes associated with lipid droplet biology, such as adipocyte differentiation [225], magnolol-induced lipolysis [226], cholesterol-induced targeting of caveolin to lipid droplets [227], and the expression of the PAT family [228]. Hence, the activation of the PKC signaling pathway in macrophages stimulated by the *Bothrops* PLA2s MT-II and MT-III in peritoneal macrophages may be implicated in an increase in PLIN2 protein expression, since LD formation induced by both svPLA2s has always been accompanied by an increase in PLIN2 protein expression in macrophages.

PI3K/AKT is a classical pathway involved in insulin resistance, cell growth, and lipid metabolism associated with the inhibition of cholesterol efflux leading to LD formation [229]. In this sense, the participation of the PIK3 signaling pathway in MT-III-induced LDs in macrophages and vascular smooth muscle cells stimulated with MT-III has been demonstrated [167,224]. In the case of vascular smooth muscle cells, the activation of PI3K was related to the uptake of fatty acids to LDs by macropinocytosis [167]. Furthermore, the findings that MT-III increases phagocytic activity and upregulates macrophage markers in VSMCs reinforce the importance of this class of enzymes as inducers of factors implicated in the formation of foamy cells in both mononuclear phagocytic cells and VSMCs, which are key elements in the development of metabolic diseases.

The MAPK signaling pathway has been revealed to mediate the activation of intracellular PLA2s in physiologic and inflammatory contexts [230,231]. It has been demonstrated that the ERK1/2 signaling pathway is implicated in LD formation via the activation of phospholipase D (PLD) and phosphorylation of dynein [232,233]. Consistent with this information, the critical role of ERK1/2 for LD formation induced by MT-II and MT-III in macrophages and VSMCs was reported [167,224]. Regarding p38 MAPK, the literature evidenced its importance in the development of atherosclerosis [234] and the apoptosis of foam macrophages. Macrophage death is a feature of atherosclerotic plaque linked to necrosis and plaque destabilization [235]. Interestingly, although MT-III has been demonstrated to activate apoptotic pathways, including the p38 MAPK signaling pathway, DGAT, ACAT, cPLA2, and LD formation in macrophages, no change in cell viability was observed. Further studies may clarify this lack of apoptotic effect.

It is known that the MAP kinases signaling pathway is implicated in intracellular PLA2 activation, including the Ca2+-dependent cytosolic group IVA PLA2 (cPLA2α) and the Ca2+-independent group VIA PLA2 (iPLA2) involved in both physiological and pathophysiological conditions [236,237]. The biogenesis of LDs in CHO-K1 cells submitted to an enriched environment of fatty acids demonstrated ERK, p38, and JNK signaling pathway activation, with JNK cascade being responsible for cPLA2<sup>α</sup> phosphorylation in this event. Of note, cPLA2 was likewise implicated in LD biogenesis stimulated by MT-III associated with the activation of ERK and the p38 MAPK signaling pathway [224]. Considering the ability of cPLA2 to mobilize cell membrane fatty acids [238], the aforementioned activation should amplify the action of MT-III and provide a greater substrate for the metabolization and formation of LDs. Moreover, the biogenesis of LDs induced by the svPLA2s MT-III and MT-II is dependent on the activation of iPLA2 signaling pathways [167,224]. This signaling pathway was associated with the processing of fatty acids into triacylglycerol, a relevant component in the constitution of LDs [214]. Hence, the crosstalk already evidenced between intracellular PLA2 and svPLA2 to elicit inflammatory conditions [169] might contribute to elucidating mechanisms related to the formation of LDs.

The peroxisome proliferator-activated receptors (PPARs) are transcription factors belonging to the family of nuclear receptors that regulate glucose homeostasis, inflammation, and lipid metabolism. Three proteins, encoded by distinct genes, have been identified: PPAR-α, PPAR-β/δ, and PPAR-γ, which control gene expression by binding to PPREs in the promoters [239]. The activation of PPARs is a tightly regulated process implicated in the control of lipid homeostasis, which involves the biogenesis of LDs and protein expression involved in lipid uptake, including PRRs and structural protein PLIN2, and

enzymes implicated in neutral lipid synthesis (triacylglycerol and cholesterol) [240]. PPARs have been demonstrated to increase in foam macrophages [241]. In this sense, it was revealed that MT-III induced the upregulation of the transcription factors PPAR-γ and PPAR-β/δ, in addition to the translocation of these factors to the nucleus of mouse peritoneal macrophages. The pharmacological blockage of the PPAR-β/δ transcription factor abolished the increase in PLIN2 and CD36 protein expression induced by MT-III. Moreover, the PPAR-γ blockage caused a reduction in LD formation and abolished CD36 receptor protein expression induced by MT-III. Since an increased expression of CD36 and PLIN2 is related to macrophage differentiation into foam cells [242,243], these findings suggest that MT-III induces foam cell formation by this route. In addition, MT-III caused an increase in the levels of triacylglycerol and cholesterol due to the uptake of free fatty acids. These effects were mediated by DGAT and ACAT enzymes, which are involved in the synthesis of triacylglycerol and cholesterol, respectively [244]. In agreement with this study, a significant increase in triacylglycerol and cholesterol levels was observed in human monocytes under MT-III stimulation [222]. This effect was dependent on fatty acid reacylation. Moreover, the fatty acid composition of triacylglycerol and cholesterol induced by MT-III was compatible with fatty acids released by the enzymatic action of this svPLA2 on cell membranes. According to the above information, the mechanisms triggered by MT-III, in both mice peritoneal macrophages and human monocytes, align with macrophages differentiation into foamy cells, a cell type characteristic of inflammatory metabolic diseases, such as atherosclerosis [245]. Similarly, MT-III could stimulate LD formation in VSCMs. This lipid accumulation was likewise mediated by the activation of transcription factors PPAR-γ and PPAR-β/δ and DGAT and ACAT enzymes. Moreover, it is noteworthy that VSCMs under stimulation of MT-III exhibited an increase in the protein levels of PRRs, SRA-1 (scavenger receptor type 1), and LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1). Interestingly, the blockage of these receptors did not alter the formation of LDs induced by MT-III, but the upregulation of LOX-1 was associated with an increased uptake of acetylated-low density lipoprotein (acLDL) in VSMCs stimulated by this svPLA2. This higher uptake of acLDL by VSMCs identifies new pathways involved in the accumulation of lipids triggered by a sPLA2 that is not directly linked to the reacylation of free fatty acids. In addition, lipid accumulation induced by MT-III in VSCMs was related to the expression of ATP-binding cassette transporters ABCA1 and ABCG1, responsible for the efflux of cholesterol of macrophage-derived foam cells [242]. Although the signaling pathway by which MT-III induces an increased expression of the factors implicated in lipid homeostasis has not been fully elucidated, these studies have broadened the knowledge about the actions of svPLA2s on the formation of LDs and the synthesis of lipid mediators and provided new insights into the actions of group IIA sPLA2s in diseases related to lipid imbalance. The pathways and factors involved in lipid accumulation in an innate immune cell (macrophage) upon stimulus by svPLA2s are summarized in Figure 4.

Another aspect related to the metabolism of free fatty acids is the biosynthesis of lipid mediators [246–248]. On the one hand, it is known that the synthesis of eicosanoids is closely related to the triggering of the inflammatory process induced by svPLA2s, regardless of the catalytic activity on the cell membranes [13]. In this sense, the immunofluorescence approach has demonstrated that LDs stimulated by svPLA2s synthesize PGE2 [167,249]. On the other hand, some eicosanoids are implicated in the resolution of inflammatory processes, such as PGJ2. This mediator was co-located in LDs in macrophage peritoneal mice stimulated by MT-II [161], indicating, for the first time, that the LDs not only are related to the production of inflammatory mediators but also might play a role in regulating this process.

Adipose tissue is the principal organ responsible for balancing energy metabolism in the mammalian body. An imbalance in adipose tissue functions is linked to the triggering of the inflammatory process observed in metabolic diseases, including obesity [250,251]. Recently, it has been demonstrated that MT-III activated proinflammatory mechanisms in 3T3-L1 preadipocytes, including the biosynthesis of PGE2 and PGI2, lipid mediators implicated in preadipocytes differentiation into mature adipocytes, and IL-6 and MCP-1 [169]. In these cells, PGE2 production induced by MT-III was dependent on cPLA2 activation, the upregulation of COX-2 and mPGES-1, and the engagement of the PGE2 EP4 receptor. In addition, the release of IL-6 and MCP-1 was dependent on EP4 or EP3 activation, respectively. These data indicate that the production of PGE2 is critical for the activation of proinflammatory pathways associated with cytokine production in preadipocytes stimulated by this svPLA2. Furthermore, MT-III upregulated the gene expression of the adipokines leptin and adiponectin in preadipocytes [169]. These mediators have been described as regulating appetite and satiety, glucose and lipid metabolism, inflammation, and immune functions [252–255]. Although the mechanisms related to the release of adipokines and the ability of MT-III to induce lipid accumulation in adipocytes have not yet been investigated, these data offer new directions for investigating the actions triggered by svPLA2s and mammalian GIIA sPLA2s.

**Figure 4.** Schematic representation of the mechanisms and factors involved in lipid accumulation induced by svPLA2s, in macrophages. svPLA2 acts on membrane phospholipids generating free fatty acids, which are ligands of and may activate the TLR2, CD36, and cytoplasmic transcription receptors and factors PPARs. svPLA2 induces the activation of transcription factors PPAR-γ and PPAR-δ/β and increases protein expression of PPARs and CD36. PPAR-γ, PPAR-β/δ, TLR2/MyD88, and CD36 receptors, as well as DGAT and ACAT enzymes are involved in the lipid droplets formation stimulated by MT-III. PPAR-β/δ, but not PPAR-γ, is implicated in upregulation of PLIN2 protein expression, induced by MT-III. Moreover, TLR2 and the Myd88 adaptor molecules participate in the recruitment of the PLIN2 protein via cytoskeleton activation stimulated by MT-III. In addition, LD formation induced by svPLA2 is dependent on activation of PKC, PI3K, p38MAP, ERK, cPLA2, and iPLA2 signaling pathways.
