*1.1. Phospholipases A2*

Phospholipases A2 constitute a group of enzymes with diverse biological functions, ranging from homeostasis and membrane remodeling to the generation of metabolites and second messengers involved in biological processes and signal transduction. These enzymes hydrolyze the acyl ester bond at the sn-2 position of phospholipids, generating free fatty acids, such as arachidonic acid (AA) and oleic acid, and lysophospholipids, such as lyso-PAF [21,22]. Currently, PLA2s are classified into close families, including secretory PLA2 (sPLA2), cytosolic phospholipase (cPLA2), and Ca2+-independent PLA2 (iPLA2); platelet activating factor acetylhydrolase (PAF); and lysosomal PLA2 (LPLA2), PLA/acyltransferase (PLAAT), α/β hydrolase (ABHD), adipose-PLA2 (AdPLA), and glycosylphosphatidylinositol (GPI)-specific PLA2 [23–25]. This classification of PLA2s is based on the cell location, amino acid sequence, molecular weight, presence of intramolecular disulfide bridges, calcium requirement, and catalytic activity, including the hydrolysis at the sn-2 position of glycerophospholipids [22,26]. The sPLA2 family is largely distributed in nature, and its components are classified into I, II, III, V, IX, X, XI, XII, XIII, and XIV groups. They are present in high concentrations in snake, bee, and wasp venoms and in several mammalian organs, cells, and pancreatic juice [21,27]. Among them, we highlight group I PLA2s, comprising the secreted enzymes found in the snake venoms of the Elapidae and Hydrophiidae families and in the pancreas secretion of mammals. Meanwhile, group II consists of sPLA2s found in snake venoms of the Viperidae family and in mammalian tissues and are expressed in inflammatory processes [13,28].

For a long time, the pathophysiological activities of sPLA2s were related exclusively to their enzymatic activity, capable of providing a substrate for the synthesis of second messengers and inflammatory mediators. However, alternative mechanisms of action have been associated with the ability of sPLA2 to interact with receptors or even specific domains present on cell membrane surfaces, such as heparan sulfate proteoglycans, which have already been described to be important for triggering the activation of other sPLA2s in the target cells [29–32]. The identification of sPLA2s binding proteins was initially achieved by Lambeau et al. (1989) [33], who used the sPLA2 from the *Oxyuranus scutelatus* snake venom, called OS2. Due to its prevalence in brain tissue, this protein was denominated an N (neuronal) type receptor. Then, through screening in various tissues and cells, for studies of other OS2 binding proteins, a second type of PLA2 receptor was described, present in rabbit skeletal muscles, called an M (muscle) type receptor [34]. The presence of this latter receptor was identified in several tissues and in neutrophils, monocytes, and human alveolar macrophages [35,36], but not in murine peritoneal macrophages [37]. The M-type phospholipase A2 receptor has a high degree of homology (~30%) to mannose receptors, a member of the lectin receptor family, constitutively expressed in macrophages [38,39]. Mannose receptors are involved in phagocytosis, antigen processing [29,38,40], and the production of inflammatory cytokines by macrophages [40–42]. Additionally, this receptor has been demonstrated to play a role in cell proliferation and AA release, via MAPKs (protein kinase activated by the mitogens family), induced by the group IB sPLA2s [43–45]. Furthermore, it has been revealed that group IIA sPLA2s can bind to mannose receptors and promote the release of IL-6 by human alveolar macrophages [46].

Investigations into the biological role of mammalian group IIA PLA2, also known as inflammatory PLA2, in the development of several pathologies of inflammatory and immunological origin have been described. Several studies have revealed that group IIA sPLA2s are present in high levels in rheumatoid arthritis [47–50], acute pancreatitis [51–53], septic shock [54,55], Crohn's disease and ulcerative colitis [56–58], respiratory distress syndrome [59–61], bronchial asthma and allergic rhinitis [59,62], atherosclerosis [63,64], autoimmune diseases [65], and cancer [66–69]. These observations imply that both local and systemic inflammation are associated with the release of sPLA2s in vivo, thus raising the unclear question of the role of these PLA2s in inflammatory reactions. Additionally, it was found that proinflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α), induce, in a variety of tissues, the gene transcription of sPLA2s and the subsequent increase in their secretion, thus, supporting the hypothesis of the involvement of sPLA2s in inflammation [70–73]. In addition, sPLA2s activate intracellular signaling events in cells that participate in inflammatory processes, caused by the generation of second messengers, and the phosphorylation of kinases as of MAPK [74–77]. Thus, sPLA2s represent an important target for investigations regarding the mechanisms of inflammatory events.

#### *1.2. Inflammation—General Concepts and Signaling Pathways*

Inflammation is a response of body tissues to noxious conditions for restoring homeostasis, setting the stage for the healing and reconstitution of injured tissue. The acute inflammatory response to injury involves functional alterations of microvessels that occur early after injury and develop at varying rates. The major features of these alterations include transient vasoconstriction, followed by vasodilation, then, leakage of protein-rich fluid from the microcirculation leading to edema formation, and movement of phagocytic leukocytes into the site of injury followed by local pain [78]. Immunocompetent cells, such as neutrophils and monocytes, found in blood circulation are capable of rapidly infiltrating tissues; macrophages and dendritic cells reside within tissue and play key roles in tissue surveillance and antigen presentation [79,80]. The vascular and cellular reactions are triggered and highly regulated by chemical factors, called inflammatory mediators, which include cytokines, chemokines, vasoactive amines, and eicosanoids and are produced by plasma components or are released in close proximity to the injury by endothelial cells, tissue-resident leukocytes such as mast cells, and macrophages at the early stages, followed by infiltrated leukocytes. The effects of inflammatory mediators involve the engagement of specific receptors, which then display signaling pathways responsible for the immune response [81–83]. Parallel to changes in blood flow, the margination of leukocytes begins, and leukocytes adhere to the microvascular endothelium through rolling and firm adhesion, and then moving through the vascular wall into the interstitial tissue. The various steps in the leukocyte migration are regulated by different subsets of cell-adhesion molecules expressed by both leukocyte and endothelial cells [84,85]. Thereafter, the phagocytosis of offending agents by migrated leukocytes occurs, followed by a release of lysosomal enzymes and an increase in the oxidative metabolism in leukocytes, known as respiratory burst,

resulting in the production of microbicidal agents, such as superoxide anion (O2 −) and hydrogen peroxide (H2O2) [86,87]. Four major classes of receptor-mediated phagocytosis exist: receptors of complement that recognize complement-coated particles; Fcg receptors, which are constitutively active for phagocytosis of IgG-coated particles; mannose receptors that recognize mannose and fucose on the surface of pathogens; and β-glucan receptors that recognize β-glucans-bearing ligands [87]. Furthermore, receptors for the Fc portion of immunoglobulin G and mannose/fucose residues lead to the release of proinflammatory mediators and reactive oxygen [88].

Despite the stereotyped features associated with an inflammatory response, the signal pathways involved in this response are determined by the nature of the inflammatory trigger, the sensors that detect them, the inflammatory mediators released, and the tissue affected. A number of surface and cytosolic receptors expressed in innate immune cells can sense pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), or venom-associated molecular patterns (VAMPs) with high sensitivity and specificity. The recognition of these molecules is achieved by interacting with pattern recognition receptors (PRRs) [89]. These sensing receptors include Toll-like receptors (TLRs) [90,91], C-type lectin receptors (CLRS), RIG-I-like receptors (RLRs), and nucleotidebinding domain leucine-rich repeat (NLRs) or nucleotide-binding and oligomerisation domain (NOD)-like receptors [92,93]. Among them, TLRs (TLR1-TLR10) are highly conserved transmembrane proteins that play an important role in recognizing microbial pathogens and endogenous damage molecules, thereby triggering the generation of signals for the production of proinflammatory proteins and cytokines, via cooperation of adaptor proteins (MyD88, TIRAP, TRIF, and TRAM) [94,95]. The recognition of specific molecular patterns by NLRs induces the oligomerisation of proteins in the cytosol, generating platforms called inflammasomes [96]. The inflammasome is a high molecular weight protein complex that elicits the activation of inflammatory caspases and the processing of pro-interleukin-1β (pro-IL-1β) and pro-IL-18, generating the mature biologically active cytokines and a rapid inflammatory form of cell death termed pyroptosis [97]. Several distinct inflammasomes have been identified, including NLR and the pyrin domain containing receptor 1 (NLRP1), NLRP3, and NLR; the caspase recruitment domain containing receptor 4 (NLRC4); and the AIM2-like receptors (ALR) family [98,99]. These receptors positively regulate genes related to inflammatory mediators, including cytokines and key enzymes in the biosynthetic pathways of lipid mediators known as eicosanoids [100].

The early induction of most inflammatory transcripts depends on networks of transcription factors whose activation is coupled to pathways of signal transduction. The nuclear factor-kappa B (NF-κB) is a major and the best-studied transcription factor of inflammatory response [101,102]. The binding of activated NF-κB to the nuclear promoter region of diverse inflammatory factors leads to the transcriptional activation and expression of inflammatory mediators and enzymes. Currently, the major signaling pathways involved in inflammation are mediated by the cascade phosphorylation of protein kinases, such as the mitogen activated kinases (MAPKs) encompassing ERK1/2, SAPK, c-Jun NH2-terminal or JNK and p38MAPKs, phosphatidylinositol 3 kinase (PI3K), protein kinase C (PKC), and protein tyrosine kinase (PTK). These kinases mediate distinct intracellular signaling pathways associated with cytokines production, cytokine receptors, growth factors, mobilization of intracellular Ca2+, and regulate a variety of functions of immunocompetent cells, including cell migration, phagocytosis, degranulation, respiratory burst, and programmed cell death [103–112].

An effective acute inflammatory response results in the removal of noxious factors followed by the resolution and repair stages [113]. The shift in inflammatory markers, including the switch from proinflammatory mediators to anti-inflammatory, resolutioninducing mediators (lipoxins, maresins, protectins, and resolvins), is vital for the change from inflammation to resolution [114]. This switch drives the transition from neutrophil to monocyte recruitment into the affected sites, resulting in the clearance of dead cells and other debris, assisted by the lymphatic system, and the initiation of tissue repair at

the damaged site [115]. However, if the acute events are not properly controlled, the inflammatory response becomes detrimental to the host. Yet, if the acute response does not succeed in neutralizing the injurious stimulus, the resolution phase might not be appropriately induced, and a chronic inflammatory state may ensue, leading to several inflammatory-mediated diseases [116,117].
