*2.2. Influence of Bothrops svPLA2s on Pathways of Arachidonic Acid Metabolism*

It is well established that sPLA2s play key modulatory roles in numerous cellular processes in physiological and pathological conditions by regulating the release of AA from membrane phospholipids [27,171]. It has long been recognized that the AA-derived lipid mediators are potent mediators of inflammation [83]. The AA is rapidly metabolized by several enzyme complexes, including cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 (CYP450). These enzymatic pathways promote the synthesis of oxygenated and bioactive products, generically called eicosanoids, which include prostaglandins (PG), leukotrienes (LT), hydroperoxyeicosatetraenoic acids (HPETEs), hydroxyeicosatetraenoic acids (HETEs), epoxides (EETs), and lipoxins (LX) [172–178]. A summary of the cascades involved in biosynthesis of eicosanoids is shown in Figure 2.

It is important to emphasize that COX-1 is a constitutive isoform present in most tissues and is responsible for generating PGs for diverse physiological functions [179–182]. In contrast, COX-2 is upregulated by inflammatory cytokines and growth factors [183,184] and is constitutively expressed in some tissues [185,186].

Regarding the production of inflammatory eicosanoids by *Bothrops* svPLA2, studies have demonstrated that the intraperitoneal injection of MT-III [187] and MT-II [162] in mice induced an early and transient release of PGD2, followed by a rapid and sustained release of PGE2. Likewise, in mice injected with BatroxPLA2 [140], from *B. atrox* snake venom, and BJ-PLA2-I from *B. jararaca* [141], an early release of PGE2 was observed. The in vivo experimental models of previous studies have revealed that *B. asper* sPLA2s induce the release of other eicosanoids, such as thromboxane A2 (TXA2) and LTB4 [123]. Moreover, an Asp49 svPLA2 from *B. atrox* venom [140] stimulated the production of LTB4, lipoxin, and PGE2.

PGE2 and PGD2 are important modulators of vasodilation, and PGE2 can potentiate an increase in vascular permeability, promoted by mediators of this phenomenon, with a consequent formation of edema [188,189]. Studies using pharmacological treatment with non-steroidal anti-inflammatory compounds were crucial in demonstrating the participation of these COXs-derived lipid mediators on edema [126,190] and hyperalgesia [135], induced by *B. asper* sPLA2s. In addition, studies demonstrating that MT-III and MT-II upregulated COX-2 protein expression in peritoneal leukocytes without altering the constitutive expression of COX-1 evidenced the ability of these venom PLA2s to influence downstream cyclooxygenase isozymes and suggested this as a mechanism by which these sPLA2s induced the production of prostaglandins [162,163]. Moreover, these findings suggested that the catalytic activity of these bothropic PLA2s did not contribute to the induction of PG biosynthesis, since MT-II, devoid of catalytic activity, caused the same effect.

In this regard, studies have demonstrated that the IκB phosphorylation inhibitor TPCK effectively prevented both MT-II- or MT-III-induced COX-2 expression, suggesting that the activation of NF-κB was critical for the induction of COX-2 expression by these bothropic svPLA2s. The involvement of NF-κB as the mechanism underlying this venom sPLA2sinduced upregulation of COX-2 expression was further confirmed by results that revealed

the inhibition of the NF-κB nuclear translocation site, markedly reduced svPLA2s-induced COX-2 expression and, as a consequence, reduced PGE2 production by macrophages in culture [162,164].

**Figure 2.** Scheme of arachidonic acid metabolism by several enzymatic pathways leading to production of bioactive lipid mediators. Abbreviations: (PLA2) phospholipase A2, (Lyso-PAF) lysophospholipid-platelet-activating factor, (COX-1) cyclooxygenase-1, (COX-2) cyclooxygenase-2, (5-LO) 5-lipoxygenase, (15-LO) 15-lipoxygenase, (12-LO) 12-lipoxygenase, cytochrome P450 (CYP450), (PGG2) prostaglandin G2, (PGH2) prostaglandin H2, (TXA2) thromboxane A2, (PGE2) prostaglandin E2, (PGD2) prostaglandin D2, (PGJ2) prostaglandin J2, (PGF2α) prostaglandin F2 alpha, (PGI2) prostacyclin, (15-HPETE) 15-hydroperoxyeicosatetraenoic, (15-HETE) 15-hydroxyeicosatetraenoic acid, (12-HPETE) 12-hydroperoxyeicosatetraenoic, (12-HETE) 12-hydroxyeicosatetraenoic acid, (5-HPETE) 5-hydroperoxyeicosatetraenoic, (5-HETE) 5-hydroxyeicosatetraenoic acid, (LTA4) leukotriene A4, (LTB4) leukotriene B4, (LTC4) leukotriene C4, (LTD4) leukotriene D4, (LTE4) leukotriene E4, (LXA4) lipoxin A4, (LXB4), lipoxin B4, (19-HETE) 19-hydroxyeicosatetraenoic acid, (20-HETE) 20-hydroxyeicosatetraenoic acid, (EETs) epoxyeicosatrienoic acids.

Studies employing mouse resident peritoneal macrophages or neutrophils in culture revealed that viperids sPLA2s induced a marked release of PGE2 in cell supernatants, accompanied by the release of AA [162,165,166,191]. These data support the results in vivo and serve as evidence that immune innate leukocytes, such as resident macrophages and neutrophils, are important sources of PGs under in vivo stimuli by sPLA2 from *Bothrops* spp. snake venoms. Interestingly, studies have demonstrated that the incubation of resident peritoneal macrophages with MT-II or MT-III significantly increased the concentration of AA [162,164]. Although the release of AA induced by the Asp49 sPLA2 was approximately 20 times greater than that induced by MT-II, it demonstrated that the catalytic activity of viperid sPLA2s was not an essential requirement for inducing COX-2 expression and PGE2 production. According to Kini and Evans (1995) [192], in mechanisms independent

on catalytic activity, as in the case of MT-II, the interaction of sPLA2s to acceptor regions can cause the biological effect or interfere with the interaction of target proteins with their physiological ligands. Furthermore, some effects may result from combinations of both enzymatic and non-enzymatic mechanisms [192], leading to the activation of several signaling pathways; this should be considered when interpreting the effects of group IIA Lys49 svPLA2s. In this context, since crosstalk among sPLA2, cPLA2, and iPLA2s has been demonstrated to occur in several physiological and inflammatory conditions [193–196], the contribution of prey/victim cPLA2 and/or iPLA2 to the increased production of PGs and upregulation of COX-2 protein expression induced by svPLA2 variants was evaluated in diverse in vitro experimental models [162,165,187]. Thus, the pharmacological treatment of cells with the cPLA2 inhibitor but not the iPLA2 inhibitor decreased the release of AA and the production of PGE2 and PGD2 induced by svPLA2. In contrast, these pretreatments did not modify the MT-III-induced COX-2 expression but reduced the COX-2 expression induced by MT-II. These results demonstrate that cPLA2 is required for distinct actions of MT-II in the PG biosynthetic pathway in macrophages [162,187]. This is consistent with the reported functional cooperation between intracellular PLA2s and GIIA sPLA2 for PG biosynthetic responses in several other cell systems [171,197–199]. The role of cPLA2 as a key enzyme in supplying AA for COX-2-dependent PGs production is well established [171,200,201]. Taken together, the available data demonstrate that the Asp49 svPLA2s are functionally coupled with cPLA2, since prior activation of cPLA2 is required for MT-III to act with downstream enzymes for PG biosynthesis in macrophages and neutrophils [165,187]. Interestingly, the association of Lys49 sPLA2 with cPLA2, in addition to being important for the supply of AA for the production of PG, appears to modulate the transcription and protein expression of COX-2 inflammatory isoform. The mechanisms involved in the coupling between the venom GIIA sPLA2s and mammalian cPLA2 have yet to be investigated. One possibility is that GIIA svPLA2s activate cPLA2 by distinct signaling cascades that mimic the transducing mechanism conveyed by physiological activators of cPLA2, such as MAPKs, since this enzyme family is likewise important for the activation of NF-κB [202]. In line with this concept, the Asp49 svPLA2, MT-III, in addition to Lys49 PLA2, MT-II, from *B. asper* snake venom, were revealed to stimulate the phosphorylation of protein kinases, including the MAPKs, such as p38MAPK and ERK1/2 [167,169,171]. Moreover, other protein kinases, including PI3K, PKC, and PTK, were reported to be phosphorylated in macrophages stimulated by both MT-III and MT-II [162,164]. In this regard, studies have demonstrated that MT-III (Asp49 sPLA2) stimulates PKC and p38MAPK pathways to positively modulate PGE2 production and COX-2 expression via NF-κB, while MT-II (Lys49 PLA2) displays similar effects by activating PKC, ERK1/2, and PTK in murine peritoneal macrophages [167,169,171]. Since PTK is involved in the activation of MAPKs, which, in turn, are essential for cPLA2 activation, this signaling protein might be involved in the activation of cPLA2 by MT-II [202–204]. Furthermore, another pathway implicated in the release of PGE2 and the expression of COX-2 induced by MT-III was demonstrated to be independent of NF-κB activation. This pathway involved the activation of ERK1/2 by the 12-HETE pathway, the main product of 12-LO [166]. The involvement of another transcription factor in these MT-III-induced effects was suggested by the authors. Together, these findings reveal the variety and complexity of the mechanisms involved in the effects of svPLA2 leading to the generation of lipid mediators. The signaling molecules and pathways acting in an innate immune cell (macrophage) upon stimulus either by Asp49 or Lys49 svPLA2 are summarized in Figure 3.

Although the participation of the M-type PLA2 receptor or another type of interaction of *Bothrops* sPLA2 with membrane sites for the stimulation of signal transduction pathways has not yet been demonstrated, a study revealed for the first time the involvement of the TLRs in the inflammatory response induced by MT-III (Asp49-PLA2) from *B. asper* in macrophages [205]. The involvement of TLR2 and MyD88 adapter molecules was demonstrated to be critical in producing PGs, COX-2 protein expression, and cytokines IL-1β and IL-10 induced by this svPLA2. An indirect mechanism for the activation of TLRs through the release of DAMPs was suggested by the authors, since the analysis of the fatty acids released by the hydrolysis of membrane phospholipids by MT-III revealed high levels of oleic and palmitic acids. In this context, it is known that arachidonic, oleic, and palmitic acids produced by membrane cleavage by sPLA2s are important bioactive mediators involved in the induction and release of COX-2 and PGE2, respectively, through the activation of intracellular signaling mechanisms in several cell types [206–209].

**Figure 3.** Schematic representation of signaling pathways stimulated by Asp49 and Lys49 PLA2s from *B. asper* snake venom to produce prostaglandins in macrophages. Asp49 PLA2 induces AA and fatty acids release from macrophage membrane. Free fatty acids can act as DAMPs and activate TLR2 or other TLRs (still unknown), via activation of adapter protein MyD88, leading to COX-2 protein expression and release of PGs, Asp49PLA2 upregulates the 12-LO pathway, culminating the release of 12-HETE. 12-HETE, in turn, activates the ERK1/2 pathway, leading to COX-2 protein expression and PG release, independent on NF-κB translocation. Asp49PLA2 also activates the signaling protein PI3K leading to COX-2 expression and production of PGs independent on NF-κB activation. Asp49 PLA2 also activates PKC and p38 MAPK pathways promoting COX-2 expression and production of PGs via NF-κB activation. In addition, Asp49PLA2 provides AA for activation of COX-1 activity which is followed by production of proinflammatory PGs. Asp49 and Lys49 PLA2s both produce PGs by pathways independent on iPLA2. Although both sPLA2 from bothropic venom produce PGs via crosstalk with cPLA2, only Lys49PLA2 induces COX-2 expression dependent on this cytosolic PLA2. Lys49PLA2 activates signaling pathways mediated by p38 MAPK, PTK, PKC, and ERK1/2. All these kinase pathways, except for p38 MAPK, are involved in NF-κB activation and COX-2 protein expression and PG production. Full arrows indicate actions already studied and demonstrated. Dotted arrows indicate hypothesized or unknown effects.
