**3. Discussion**

Inflammation is the hallmark of envenomation by snakes in the Viperidae family. The activation of the inflammatory process and its cascade of events play an important role in the pathogenesis of envenomation, in the clinical picture, and in the outcome of the accident. In this sense, the envenomation by *Bothrops asper* [35,36], *B. atrox* [37,38], and *B. jararaca* [39,40], which, among other characteristic changes such as hemostatic and cardiovascular disorders, induce a prominent inflammatory response, which has been associated not only with local damage but also with systemic disturbances caused by Viperidae venoms [11,29]. Therefore, given the importance in understanding the inflammatory response induced by the BaV as a tool for the development of complementary therapies, or the improvement of *B. arietans* antivenoms, we show that this venom was capable of inducing, in vitro, the production of several inflammatory mediators, including cytokines, chemokines, and lipid mediators.

Our previous in vivo results demonstrated that BaV induces inflammation with the participation of diverse endogenous inflammatory mediators produced by residents or derivatives of recently migrated blood cells [30]. Among these cells, macrophages became candidates for target cells in the present study of cell inflammation induced by BaV and Kn-Ba, since macrophages play a key role in inflammation [41] and also participate in the inflammatory response related to other snake envenomations [22,23,42]. In this sense, studies have shown that in the absence of macrophages, the recruitment of immune cells to the site of inflammation, especially neutrophils, is impaired [22,23].

Macrophages express, on their cell surface, receptors named pattern-recognizing receptors (PRRs), which recognize the molecular domains such as pathogen-associated molecular patterns (PAMPs) expressed by pathogens, virus, fungus, bacteria; and damage-associated molecular patterns (DAMPs), which are expressed by host-damaged molecules [43–45]. In addition, the PRRs can also recognize venom-associated molecular patterns, also known as VAMPs. The term VAMP was coined by Brazilian researchers, who showed that mice deficient in TLR (Toll-like receptor)-2 and TLR-4, two important PRRs, as well as the adapter molecule CD14, produce fewer inflammatory mediators in response to the *Tityus serrulatus* scorpion venom, including decreased levels of IL-6 and TNF-α [46].

In view of the important role of macrophages in the inflammatory process, we used THP-1 macrophages to evaluate the in vitro pro-inflammatory properties of BaV and Kn-Ba. To support the homogeneity of the experiments, the human monocytes of the THP-1 lineage were differentiated in vitro into macrophages with PMA, as described in the literature [33,47]. There are different protocols using PMA for the differentiation of THP-1 monocytes to macrophages, but in some cases the differentiation profile may be not comparable to primary monocyte-derived macrophages [48]. To overcome this inconvenience, macrophages treated with PMA were kept at rest for 4 days in culture media without PMA, which was shown to be a good alternative to induce a differentiation pattern similar to primary monocyte-derived macrophages [33]. This differentiation was evaluated by morphological analyzes such as adhesion to the support surface, spreading and emission of pseudopods, and loss of proliferation. In addition to the morphological alterations, the differentiated macrophages, but no undifferentiated THP-1 monocytes, expressed CD11b, an important surface marker of monocyte-to-macrophage maturation [31–33]. CD11b forms the molecular complex CD11b-CD18, known as CR3 receptor, which is expressed in macrophages and in other leucocytes and binds to several ligands, such as iC3b adhered to pathogens. These promote phagocytosis [49] and expanding the macrophages' biological activities [33]. After differentiation, the THP-1 macrophages were used as target cells in the studies with BaV and its purified serine protease, Kn-Ba.

In sequence, the effects of BaV and Kn-Ba as inflammation-inducers were demonstrated by the production of the cytokines TNF, IL-6, IL-1β; and the chemokines IP-10, MCP-1, RANTES, and IL-8; besides the lipid mediator PGE2.

The role of purified SVSP on the inflammation induced by snake venoms has been extensively studied [50–52]. However, although SVSPs presents a high degree of similarity between their amino acids sequences, their functions may differ [53], so that not all are involved with the inflammatory response. In this sense, two SVSPs purified from *Bothrops pirajai* venom, named BpirSP27 and BpirSP41, seem to be not involved in the inflammatory events related to *B. pirajai* envenomation, such as edema, pain, and leukocyte recruitment [54]. In contrast, it was showed that SVSPs from the venoms of *Bothrops alternatus* and *B. moojeni* are able to promote edema and pain, two classic signs of inflammation [50]. SVSPs from *Bothrops asper* can also activate endogenous matrix metalloproteases [51], while SVSPs from *Crotalus durissus terrificus* venom induced edema and the increased expression of COX-2 and PGE2 production [52]. In agreement with these finds, here we show that Kn-Ba induces the production of inflammatory cytokines and chemokines by THP-1-derived human macrophages, indicating its participation in BaV-induced inflammation.

Our results show differences between the type and concentration of some inflammatory mediators produced in response to treatments with BaV or purified Kn-Ba. In this sense, it can be suggested that the mediators produced only after stimuli with BaV, but not with Kn-Ba, as in the case of PGE2, were generated by other components present in the venom. However, here we also show that some mediators, more precisely IL-6, IP-10, and MCP-1, were produced only after stimulation with Kn-Ba. This is a complex issue involving both the composition of the venom itself and the immune response triggered against the components of the whole venom. It is well known that BaV is a complex mixture of proteins (±90–95%), which includes, besides enzymes such as SVSP and SVMPs, non-enzymatic proteins as Kunitz-type proteases inhibitors [14–17]. Therefore, it can be suggested that the activity of some proteases could be hindered by endogenous inhibitors present in the venom itself. Thus, it is possible that purified Kn-Ba inducing the production of different inflammatory mediators, in a greater degree than BaV, may be due to the absence of these inhibitors. In addition, the different components of BaV can act in a different way in immune cells, making the inflammatory response diverse from the one developed only against purified Kn-Ba.

Among the inflammatory mediators produced in response to BaV and Kn-Ba, the cytokine IL-1β is noteworthy. According to literature data, after the activation of macrophages via TLRs, a cascade of intracellular signaling begins. This cascade may culminate in the activation of an important transcription factor named NF-κB, which is responsible for the expression of diverse inflammatory genes, including TNF-α, IL-6, iNOS, and also pro IL-1β [45,55,56]. Pro IL-1β is a zymogen whose activation and later secretion depends on the inflammasomes' activation, a multiprotein complex formed due to cell activation by the recognition of patterns via cytosolic PRRs of the NLR family (NOD-like receptors) [57,58]. An important subfamily of NLRs, called NALP, is involved in the induction of the inflammatory response via cytokines of the IL-1 family, including IL-1β, IL-8, and IL-33. Recognition via NALP promotes the formation of inflammasomes, which are responsible for the activation of inflammatory caspases 1, 4, and 5 in humans, and caspases-1, 11, and 12 in mice, which can convert pro-IL-1β into mature IL-1β [59,60]. IL-1β is a highly inflammatory cytokine produced during various inflammatory conditions, which mediates innate and adaptative immune responses by promoting acute phase response and recruiting inflammatory cells [61,62]. The overproduction of IL-1β is harmful and can trigger autoimmune diseases [63,64].

Here, we show that both BaV and Kn-Ba induced the production and secretion of IL-1β in the supernatant of macrophage cultures, suggesting the participation of inflammasomes in the inflammatory process related to the *Bitis arietans* envenomation. In addition, as higher concentrations of this cytokine were detected after treatment with BaV, our results indicate there are other components present in BaV, besides Kn-Ba, which may be involved in the possible inflammasome activation.

IL-1β can act synergistically with other cytokines produced by human macrophages to amplify *Bitis-arietans*-related inflammations, such as with TNF-α, activating the endothelium and inducing vasodilation and increased vascular permeability, and, with IL-6, activating hepatocytes and inducing the production of acute phase proteins, which can activate the complement system and act as opsonins, facilitating phagocytosis by macrophages and neutrophils [45].

Summing up, our study showed that BaV and Kn-Ba are able to induce the activation and the production of inflammatory mediators in THP-1-derived human macrophages, and this is the first suggestion that the inflammasomes may play a role in *Bitis arietans* envenomation.
