The large majority of ophidic accidents in Brazil are caused by Bothrops jararaca (Bj) snakes (1). Pathogenesis of Bothrops envenomation involves the combined action of venom metalloproteinases, phospholipases, and serine proteinases, as well as the release of endogenous mediators originated either from plasma or from inflammatory cells (2–4).

The inflammatory response resulting from tissue damage and the direct action of toxins is characterized by a local increase in blood flow, exudation of plasma proteins, and migration and activation of leukocytes from the vessels to the site of injury. Since many of these inflammatory effects are due to the release of chemical mediators from injured tissue, mast cells have been considered important in the onset and/or amplification of acute inflammatory responses (5–7).

Metalloproteinases and phospholipases from Bothrops jararaca venom (BjV) can induce mast cells to release potent biologically active mediators present in their granules (histamines, proteoglycans, and proteases), promoting inflammation with an increase in vascular permeability, the expression and/or activation of adhesion molecules with a rapid local recruitment of neutrophils, pain, clotting and fibrinolysis systems abnormalities, and shock (4, 8–14).

In order to investigate the role of mast cells in BjV-triggered inflammatory reaction and the interference of host genetic background in this effect, we have used mouse lines phenotypically selected for maximum (AIRmax) or minimum (AIRmin) acute inflammatory response (AIR) (15, 16). At the end of the bidirectional selective process for the production of AIRmax and AIRmin lines, the gene alleles endowed with opposite effects in the regulation of the maximum and minimum acute inflammatory responses were considered to be accumulated in AIRmax and AIRmin mice, respectively (16). Therefore, these mouse lines are normal and represent the individuals who are situated at the extremes of high or low response that are found in heterogeneous populations.

As a result of their divergent inflammatory response, we have demonstrated that AIRmax and AIRmin mice differ in their susceptibility to bacterial infections (17), tumorigenesis (18, 19), and autoimmune diseases (20, 21). With regard to systemic and local responses induced by BjV, both lines are equally susceptible to venom-induced lethality, showing similar LD₅₀. However, edema, leukocyte influx, prostaglandin E₂ (PGE₂) secretion, hydrogen peroxide (H₂O₂) production, Chemokine (C-X-C motif) ligand 2 (CXCL2)/macrophage inflammatory protein (MIP)-2 expression, and interleukin (IL)-1β and IL-6 release are more intense in AIRmax than those in AIRmin mice (22, 23).

Based on previous literature showing the role of mast cells in response to venom or its components, our aim in this study was to evaluate the participation of these cells in BjV-induced inflammatory parameters such as hyperalgesia, cellular migration, and reactive oxygen species (ROS) production in mice selected for maximum and minimum inflammatory response.

To certify that CROM inhibits mast cell degranulation in our model, we analyzed the mesenteric tissue of AIRmax and AIRmin mice pretreated with CROM and injected with PBS, BjV, or C48/80, a compound known to degranulate mast cells. Despite the difference observed between PBS-injected AIRmax and AIRmin, the induction of mast cell degranulation by both C48/80 and BjV was similar in both strains. However, CROM inhibited 49.8% and 22.6% of C48/80-induced mast cell degranulation in AIRmax and AIRmin mice, respectively. Similarly, pretreatment with CROM inhibited 48% and 30.6% of BjV-induced degranulation in AIRmax and AIRmin, respectively (Figure 2A).

A visual illustration of CROM effect can be found in Figure 2B, where we show degranulated mesenteric mast cells collected from AIRmax mice after C48/80 injection. We observed a similar aspect in cells collected from mice injected with BjV, and this was not different from what we observed in AIRmin mice (image not shown).

The injection of BjV (1 µg/hind paw) induced significant hyperalgesia in both lines (Figures 3A, B); however, it was more intense in AIRmax compared to AIRmin mice (Figure 3C). Hyperalgesia peaked at 3 h in AIRmax and between 30 min and 1 h in AIRmin mice. Pretreatment with CROM partially inhibited BjV-induced hyperalgesia for up to 6 h in AIRmax and 1 h in AIRmin, with similar residual hyperalgesia in both lines (Figures 3A, B). Pain levels were unchanged in control groups at all time points.

The difference in nociceptive response to BjV between AIRmax and AIRmin mice was abolished by pretreatment with CROM, represented at the third hour (Figure 3C). These results suggest a significant contribution of mast cells in the differences in pain threshold observed between AIRmin and AIRmax mice after injection of BjV.

Flow cytometry was used to analyze the cellular profile infiltrated into the peritoneal cavity after BjV inoculation. The gate strategy for the analysis is represented in Figure 4A. The numbers of peritoneal mast cells (FcϵRI⁺ cKIT⁺) in PBS-treated mice were higher in AIRmax than in AIRmin mice and decreased after BjV injection. Degranulated cells could not be detected by flow cytometry. On the other hand, pretreatment with CROM increased mast cell numbers in BjV-injected AIRmax but not in AIRmin mice likely due to the inhibition of their degranulation (Figure 4B). Pretreatment with CROM did not alter the number of mast cells in the peritoneal cavity of PBS controls.

The peritoneal macrophage population (CD11b⁺ GR-1^low F4/80⁺ MHC-II⁺) in control mice was higher in AIRmax mice compared to AIRmin mice. This population was not significantly altered by any treatment in both mouse lines (Figure 4C). On the other hand, infiltrated neutrophil population (CD11b⁺ GR-1⁺) was increased in AIRmax mice after BjV inoculation but not in AIRmin and was partially inhibited by CROM (Figure 4D).

Since macrophages and neutrophil’s function can be modulated by mast cells, we quantified PMA-stimulated ROS production as a method to evaluate the effect of BjV on peritoneal cell activation. AIRmax cells produced higher levels of ROS than AIRmin cells 24 h after i.p. venom injection. CROM pretreatment inhibited ROS production in AIRmax but not in AIRmin, suggesting the involvement of mast cells in BjV-induced peritoneal cell activation (Figure 4E).

In this work, we have investigated the role of mast cells in some important parameters of the inflammatory response triggered by BjV in mice genetically selected for inflammation, AIRmax and AIRmin.

Mast cells are considered the primary storage site of histamine in tissues from mammalians. Histamine is stored, together with serotonin, in secretory granules, formed by a highly charged matrix of heparin and protein (28). These cells can be degranulated by a range of agents, including direct action of phospholipases hydrolyzing membrane phospholipids (29), complement fragment 3a (C3a) that induces degranulation stimulated by aggregated IgG, calcium-dependent exocytosis process, and also diverse agents that induce degranulation through Toll-like receptor 2 (TLR-2) activation (30, 31).

Pain is an important signal present during an inflammatory response, and BjV injection induced a significant hyperalgesia in both lines, being more intense in AIRmax mice. Our previous studies have shown the presence of high levels of PGE₂ in the peritoneal cavity of AIRmax mice after BjV i.p. injection (22). Venom components such as phospholipase A₂ (PLA₂) could lead to prostaglandin synthesis (32, 33), and this higher level of PGE₂ associated with mast cell degranulation could partially explain the more intense BjV-induced pain observed in AIRmax in comparison to AIRmin mice. Accordingly, the treatment with CROM partially inhibited BjV-induced hyperalgesia, indicating the contribution of mast cells to the nociceptive response.

The inhibition of BjV-induced pain by CROM could be compared to results where CROM inhibited mast cell degranulation and pain induced by Freund’s complete adjuvant in mice (34). It was already reported, in rats, that the hyperalgesia triggered by BjV is inhibited by meclizine and methysergide, antagonists of histamine (H1) and 5-hydroxytriptamine (5-HT1, 5-HT2, and 5-HT7) receptors, respectively (4). The same authors showed the involvement of mast cells, since CROM inhibited BjV-induced thermal hyperalgesia. The mechanism responsible for BjV-induced mast cell activation was associated with the metalloproteinase component of the venom. Previous treatment with metal chelator abolished the venom effect on mast cells (4). Batroxase, a P-I metalloproteinase isolated from Bothrops atrox venom, induced the edematogenic and hyperalgesic responses involving mast cell degranulation and histamine and leukotrienes as mediators (35). Bernardes et al. (8) analyzed BpirMP, a metalloproteinase isolated from the venom of the Bothrops pirajaí snake, and observed induction of paw edemas and increased nociceptive threshold in rats, which were inhibited by pretreatment with CROM. Our results confirm and extend these observations, since we have used a different animal model and added the interference of genetic components on the evaluated phenotype.

The higher BjV-induced infiltration of neutrophils in AIRmax mice is in accordance with the phenotype used in the selection of these animals. Our group demonstrated previously that in response to an inflammatory stimulus, bone marrow neutrophil production was more intense, concentration of chemotactic factors in inflammatory exudates was higher, and neutrophil resistance to apoptosis was increased in AIRmax mice compared to AIRmin (36). Other authors have described that venom injection induces chemotaxis (37), which can be regulated by mediators released by mast cells, promoting effector functions in Polymorphonuclear leukocytes (PMN) cells (10, 38).

Landucci et al. (39) demonstrated that PLA₂ enzymes were involved in mast cell activation caused by Bothrops jararacussu venom. PLA₂ isolated from the venom of Lachesis muta muta including LmTX-I induces microvascular leakage mediated by mast cell activation when administered via intraperitoneal or intradermal routes (40). The products secreted by mast cells are thus important in the worsening of the injury, and they can be an important therapeutic target (41). On the other hand, some authors have shown that products secreted during the mast cell degranulation can counteract the toxicity of the venoms, especially proteinases, which cleave some of these toxins (42).

Mast cell activation was recently shown to be involved in the early inflammatory response of mesenteric microvasculature to lipopolysaccharide (LPS) inoculation in mice. Treatment with the non-specific mast cell stabilizer CROM prevented mast cell activation toward a pro-inflammatory phenotype, significantly reducing the number of migrated neutrophils (11). Our results suggest that during venom stimulation of AIRmax mice, something similar is occurring, with mast cells contributing to neutrophil migration, since this process is modulated by CROM treatment. We did not observe these effects in AIRmin mice at the time points and doses measured. These mice presented a mild inflammatory response after BjV inoculation in comparison to AIRmax.

In conclusion, our study suggests that mast cells play a role in pain, neutrophil migration, and ROS production triggered by BjV. AIRmax mice are more susceptible than AIRmin mice to BjV-induced inflammatory reactions, which are partially inhibited by pretreatment with CROM, a mast cell stabilizer. The use of these genetically selected mouse strains endowed with extreme divergent inflammatory responses could contribute to a better understanding of the effect that these venoms have in our organism. Clinical reports indicate that predictive factors of worsening of snake poisoning are very variable and difficult to prove; however, it is considered that variations in the genetic constitution of the victim and the venom composition can interfere in the severity and outcome of poisoning (43).

These results point to a possible beneficial effect of drugs that modulate the activation of mast cells in BjV envenomation.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

The raw data supporting the conclusions of this article will be made available by the authors without undue reservation.

The animal study was reviewed and approved by the Ethics Committee on Animal Use of the Butantan Institute.

FK and NS conceived and designed the experiments. FK, MS’A, WC, and AB performed the experiments. NS, FK, MS-F, MS’A, and GP analyzed the data. NS, OR, MF, and OI contributed reagents/materials/analysis tools. NS, FK, and GP wrote the paper. JJ, MF, and OI critically revised the article for important intellectual content. All authors contributed to the article and approved the submitted version.

This work was supported by grants from Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and CAPES. MF and OI have CNPq fellowship.