*2.1. Bothrops svPLA2s Induce Inflammatory Events and Activate Defense Functions in Leukocytes*

Phospholipases A2 of GIIA are major components of *Bothrops* spp. snake venoms and play important roles in the pathophysiology of envenoming by these snakes, including the inflammatory response. Although these enzymes conserve a chemistry and catalytic structure, the natural evolution of viperid venoms introduced alterations in their primary amino acid residues, generating various other biological and toxicological effects [118]. In general, the GIIA sPLA2s found in viperid snake venoms are classified as sPLA2s, known as 'classic', containing an amino acid aspartate at position 49 (Asp49) and catalyzing the hydrolysis of the ester bond at position sn-2 of glycerophospholipids in a Ca2+ dependent manner. Meanwhile, the other type of sPLA2s is described as 'variant' and contains a lysine at the same position 49 (Lys49), with or without low catalytic activity [119]. Such a substitution affects the ability of these proteins to bind to Ca2+, an essential cofactor for the stabilization of tetrahedral intermediate, which occurs in the catalytic reaction performed by the Asp49-sPLA2s [120]. Despite the lack of enzymatic activity, sPLA2s-Lys49 homologues maintain their damaging capacity in membranes through a mechanism that is not completely understood and independent of Ca2+ [12,121,122].

It has long been demonstrated that viperid sPLA2s are potent inductors of inflammation. Although they present differences in their catalytic activity, both viperid Asp49 and Lys49 PLA2 homologues are capable of inducing local inflammation in diverse experimental models [123–125]. As such, this group of enzymes is considered to be a major component responsible for the severe local edema in envenomings by *Bothrops* spp. The inflammatory response to venom PLA2s is characterized by edema and the marked infiltration of leukocytes into the site of toxin injection. Studies on the mechanism of local edema induced by viperid sPLA2s (svPLA2s) have demonstrated an early increase in vascular permeability and a local release of inflammatory mediators, which act synergistically to cause the initiation and development of the inflammatory events. Among these mediators are vasoactive amines, including histamine, serotonin, and substance P, as well as vasodilating prostaglandins. Yet, in vivo studies employing a pharmacological approach have demonstrated that antagonists of serotonin and H1 receptors of histamine reduced the progression of edema induced by both catalytically active and inactive variants of sPLA2 isolated from *B. asper* [126], *B. neuwiedii* [127], *B. jararacussu* [128,129], or *B. insularis* [130]. In support of these reports, the release of histamine and serotonin by mast cells was observed following stimulation with bothropic sPLA2s from *B. jararacussu* [128,131]. Consistent with this evidence, the contribution of mast cells to edema formation induced by viperid PLA2s was further observed in in vitro experimental models demonstrating the ability of sPLA2s isolated from *B. pirajai*, *B. jararacussu*, and *B. atrox* snake venoms to degranulate mast cells [125,128,131,132]. It is well known that upon activation, mast cells secrete and synthesize an array of inflammatory mediators, which trigger the earliest events of inflammation [133,134]. Moreover, the contribution of the catalytic activity for the edematogenic effect of the enzymatic active Asp49 from bothropic PLA2s was suggested by studies, revealing that the chemical modification of this sPLA2 by p-bromophenacyl bromide inhibited edema formation induced by these viperid PLA2s [126–128]. In addition, the role of lipid mediators, such as PAF and eicosanoids, for hyperalgesia induced by catalytic active venom PLA2s was highlighted by studies using a pharmacological approach [135]. These authors suggested that the enzymatic hydrolysis of membrane phospholipids played a role in these events by directly releasing the precursors of lipid mediators, such as lyso-PAF and AA.

As mentioned previously, leukocytes are central components of inflammation. An important cellular component exists in the inflammatory response to *Bothrops* sPLA2s. As such, the stimulatory effect of piratoxin-I, bothropstoxin-I, and -II from *B. pirajai* and *B. jararacussu*, respectively, on neutrophil chemotaxis was demonstrated in an in vitro experimental model [136]. This effect was revealed to involve the interaction of these sPLA2s with surface heparan binding sites of neutrophils, followed by the release of chemotactic mediator leukotriene B4 (LTB4) and PAF, and is independent of enzyme activity. Furthermore, the ability of these venom PLA2s to recruit an endogenous PLA2 through the activation of GTP-binding protein and PKC was added to the mechanisms by which they cause neutrophil migration [137]. Moreover, studies conducted using in vivo experimental models have demonstrated the ability of *Bothrops* sPLA2s to induce a marked influx of polymorphonuclear and mononuclear cells into the site of their injection, as demonstrated for both catalytic active and non-catalytic venom PLA2s, such as MT-III and MT-II from *B. asper* snake venom [123,138]. A similar effect was reported by other authors, investigating various sPLA2s isolated from different *Bothrops* spp. snake venoms, such as Bothropstoxin (BthTX)-I and BthTX-II; *B. jararacussu* [131], BnSP-7, a catalytically inactive PLA2 from *B. pauloensis* [139], BatroxPLA2 from *B. atrox* [140] and BJ-PLA2-I from *B. jararaca* [141] in in vivo experimental models. The sPLA2-induced leukocyte migration was linked to the upregulation of adhesion molecules, such as l-selectin, LFA-1, and CD18, which in turn was associated with the release of inflammatory cytokines IL-1β, IL-6, and TNF-α with chemotactic activity by resident leukocytes, primarily macrophages [123]. Cytokines, chemokines, and leukotriene B4 are among the major mediators regulating the expression of adhesion molecules and chemotaxis of leukocytes [142–144]. Consistent with this information, increased serum levels of IL-6, IL-1, and TNF-α induced by Bbil-TX from *B. bilineata* snake venom were observed in a mouse experimental model [145]. In addition, there are reports that two Lys49 PLA2s isolated from *B. mattogrossensis* (BmaTX-I and BmaTX-II) venom were able to induce the release of IL-1β by murine neutrophils in culture [146] and that BatroxPLA2, an acidic sPLA2 from *B. atrox* venom, induced the release of IL-6, PGE2, and LTB4 from murine macrophages in culture [140]. In this context, the involvement of inflammasomes in the production of IL-1β induced by *Bothrops* sPLA2s was recently investigated. The participation of NLP3 inflammasome via the activation of caspase 1 in the production of IL-1β induced by BthTX-I, a Lys49-PLA2 from *B. jararacussu* venom, injected into mouse gastrocnemius muscle was reported [147]. In addition, the participation of inflammasomes in BthTX-I-induced production of IL-1β was demonstrated in peritoneal macrophages. This effect was demonstrated to be dependent on caspase 1/11, ASC, and NLRP3 and was associated with the release of ATP and activation of P2X7 receptors [148]. Despite the importance of cytokines, chemokines, and eicosanoids in orchestrating the events of inflammation and the potent proinflammatory effects triggered by viperid sPLA2s, including those from *Bothrops* genus, a complete picture of the inflammatory mediators released by immunocompetent cells upon stimulus by *Bothrops* sPLA2s has yet to be further investigated. Moreover, the mechanisms involved in the production and release of these mediators and the possible crosstalk between them remain to be better clarified. Regarding the mechanisms involved in the biosynthesis of lipid mediators induced by *Bothrops* sPLA2s, the progress made is presented in this review as a separate item (Section 2.2).

It is well recognized that the activation of innate effector functions, such as phagocytosis, and the production of microbicidal substances in leukocytes are critical for host defense and tissue repair. Regarding phagocytosis, studies have demonstrated the activity of *Bothrops* sPLA2s to induce phagocytosis following the activation of distinct receptors in immune-competent cells. In this sense, it was demonstrated that MT-II and MT-III, isolated from *B. asper* snake venom, can directly stimulate phagocytosis by macrophages in culture. MT-II significantly increased phagocytosis mediated by all classes of receptors, whereas MT-III increased phagocytosis via only mannose and beta-glucan receptors. This suggests that although the catalytic activity of *Bothrops* sPLA2s is not an essential requirement for enhancing macrophage phagocytosis, it may drive the class of phago-

cytosis receptors involved in this process. Molecular regions distinct from the catalytic network are likely involved in this effect [138]. In addition, the signaling pathways mediating zymosan phagocytosis, induced by both MT-II and MT-III, were investigated, with a focus on lipid second messengers. This study demonstrated that whereas the effect of MT-III, catalytically active, was dependent on the activation of endogenous iPLA2, the effect of MT-II was dependent on both endogenous iPLA2 and cPLA2. Likewise, COX-2 and 5-LO-derived metabolites in addition to PAF were involved in the signaling events required for phagocytosis induced by both venom sPLA2s [138]. In line with these data, BaltTX-I, devoid of catalytic activity and isolated from *B. alternatus* snake venom, was reported to activate the phagocytosis of serum-opsonized zymosan by murine macrophages, indicating the involvement of complement receptors. In addition, the participation of PKC was demonstrated. Nonetheless, BaltTX-II, a catalytically active sPLA2 isolated from the same venom did not stimulate phagocytosis in macrophages, lending support to previous findings that the catalytic activity of *Bothrops* sPLA2s is not essential for the stimulation of phagocytosis via complement receptor [149]. In addition, the sPLA2s isolated from Panamanian *B. asper* snake venom, pMTX-III (catalytically active Asp49) and pMTX-II and -IV, two enzymatically inactive Lys49 isoforms, were described to induce phagocytosis via mannose receptor and superoxide production in macrophages [150]. The mechanisms underlying the differences between the catalytic and non-catalytic active *Bothrops* PLA2s, regarding the activation of phagocytosis in macrophages and the participation of distinct receptors in their effects, require further clarification.

Concomitantly with phagocytosis, there is an increase in the oxidative metabolism, also referred to as respiratory burst, in leukocytes. In this context, the literature reveals that viperid sPLA2s can trigger the respiratory burst in immunocompetent cells. In the first study describing the ability of *Bothrops* sPLA2 to induce the release of microbicidal agents, the authors demonstrated that MT-II and MT-III, isolated from *B. asper* snake venom, induced the release of H2O2 by macrophages, with MT-III being the more potent stimulator [151]. In agreement with this evidence, it has been demonstrated that BaltTX-I and BaltTX-II from *B. alternatus* snake venom induced superoxide production by macrophages in culture in a process mediated by PKC [149]. In addition, other authors have revealed that the three sPLA2s from *B. atrox* venom, namely BaTX-I, a Lys49 variant devoid of catalytic activity; BaTX-II, a catalytically active Asp49; and BaPLA2, an acidic Asp49 sPLA2 induced the release of the superoxide anion by the J774A.1 lineage macrophages in culture [152]. BaTX-I was the only sPLA2 able to stimulate complement receptor-mediated phagocytosis, but all studied sPLA2s could increase the macrophage lysosomal volume [152]. These data demonstrate the ability of *Bothrops* PLA2s to trigger the respiratory burst, which is an essential process for the elimination of harmful agents. Although the structural determinants of such an effect were not investigated, it is likely that neither the enzymatic activity nor the basic or acidic characteristic of PLA2 is essential for the activation of the respiratory burst.

An additional defensive strategy important for host defense is the neutrophil extracellular trap, or 'NET'. The formation of NET (NETosis) occurs through the release of nuclear DNA, forming a sticky 'net' of extracellular fibers that can halt the dissemination of pathogens and toxins [153,154]. Despite its importance in the inflammatory response, little attention has been paid to the involvement of this defense mechanism in the effects of viperid sPLA2s. Yet, a report indicates that BaTX-II, an Asp49 PLA2 isolated from *B. atrox* snake venom, can activate human neutrophils in culture to produce hydrogen peroxide via the PI3K signaling pathway. Furthermore, this sPLA2 stimulated neutrophils to secrete MPO, NETs, and inflammatory mediators, including IL-1β, IL-8, and LTB4 [155]. Therefore, the activation of neutrophilic functions, including toxin trapping and inactivation, is likewise involved in the inflammatory response to *Bothrops* sPLA2s. Further studies are necessary to amplify the knowledge regarding the participation of NETs in inflammation induced by *Bothrops* spp. sPLA2s. Interestingly, in contrast to the reported ability of *Bothrops* sPLA2s to activate distinct inflammatory functions in leukocytes, a report revealed that CB (Crotoxin B), a catalytically active sPLA2 isolated from *Crotalus durissus terrificus*, which is a subunit of crotoxin complex [156,157], could, per se, display inhibitory effects in macrophage functions, including spreading and phagocytosis [158]. Such an inhibitory effect suggests an anti-inflammatory activity for this particular viperid sPLA2 [159]. In agreement with this idea, CB was reported to reduce the release of inflammatory cytokines, including IL-6 and TNF-α, and increase the release of PGE2 and lipoxin A4, both immunomodulatory lipid mediators, in dendritic cells [160]. A summary of the inflammatory activities of svPLA2s is illustrated in Figure 1. In Table 1, the svPLA2s-induced inflammatory responses are summarized according to the amino acid residue at position 49 and basic and acidic characteristics.

**Figure 1.** Scheme of inflammatory activities of svPLA2s. The svPLA2s induce inflammatory events, characterized by activation of innate immune cells and endothelial cells and release of several inflammatory mediators that interfere in the vascular dynamic. svPLA2s induce mast cells degranulation and activation of resident macrophages with release of inflammatory mediators such as prostaglandins (PGs), histamine, serotonin, and substance P, which lead to vasodilation, increase of vascular permeability, culminating in edema formation and pain. In addition, svPLA2s activate phagocytosis by macrophages and increase the local production of oxygen reactive species (ROS). Furthermore, svPLA2s, along with vascular alterations and produced inflammatory mediators, increase the expression of adhesion molecules such as LFA, CD-18 and L-selectin. These adhesion molecules, in turn, promote chemotaxis and leukocyte migration. The svPLA2s induce production of myeloperoxidase (MPO) and release of NETs by neutrophils. Both neutrophils and macrophages release proinflammatory mediators such as platelet-activating factor (PAF), IL-8, LTB4, IL-1β, IL-6, and TNF-α. These last three mediators are involved in the upregulation of COX-2 isoform, and release of PGs, thus amplifying the inflammatory response induced by svPLA2s.




**Table 1.** *Cont.*

nd, not described.
