1. Introduction
Crotamine is a basic polypeptide toxin found in the venom of the rattlesnake
Crotalus durissus terrificus and a member of the α-myotoxin family. Its three-dimensional structure αβ1β2β3 is similar to that of other human proteins intrinsically related to antimicrobial activity, such as β-defensins. Furthermore, positively charged regions distributed throughout the structure and a small area of negative charge optimize electrostatic interactions between crotamine and diverse cell membranes [
1,
2,
3,
4,
5].
This toxin displays different cellular and molecular targets as well as several activities, including neurotoxicity and myotoxicity. Its myotoxic potential is related to the electrophysiological changes in sodium and potassium channels, changes in mitochondrial calcium homeostasis and degeneration of myofibrils, with consequent structural damage to muscle fibers [
6,
7,
8,
9]. Moreover, studies have shown that the mechanism of action of crotamine is not restricted to the muscle tissue, involving other tissues, mainly liver and kidneys or involving other cells such as fibroblasts, neural and embryonic stem cells [
2,
10].
In addition to its toxic effect, crotamine has been shown to potentiate insulin release [
11] and to have a strong antimicrobial activity [
9,
12,
13,
14,
15]. Other properties, still poorly understood, include analgesic and hemolytic activities, as well as stimulation of the immune system by interfering with the activity of mast cells, macrophages, lymphocytes and monocytes. [
12,
16,
17,
18].
Crotamine also has cell-penetrating ability and nuclear specificity, acting through independent mechanisms of energy expenditure from interaction with extracellular matrix proteoglycans [
10,
19]. Therefore, crotamine has been studied as a nucleolar targeting peptide (NrTP) for biomolecules and antitumor agents on different tumoral strains [
3,
17,
20,
21]. The cytotoxic effects of crotamine have been demonstrated in vivo and in vitro using tumor cell lines, allowing the study of the mechanisms by which the molecule can alter cellular homeostasis by inducing damage to cytoplasmic organelles such as lysosomes and mitochondria [
19,
22].
Due to its pharmacological potential, crotamine is considered a promising molecule for clinical use in different biomedical fields [
2,
3,
23]. However, data on its local and systemic safety in biological models are scarce [
16,
24,
25]. Thus, to better understand the in vivo proinflammatory activity of crotamine, we assessed the effects of this toxin on different immunological parameters.
3. Discussion
Snake venoms contain a diversity of biologically active polypeptides with remarkable applications in biotechnology due to their ability to cause physiological and biochemical changes on different biological systems [
26]. In addition to their use in cancer cells, cardiovascular system and in neurochemistry, advances have been made on the composition and toxicity of poisons for the development of antivenoms [
27,
28,
29,
30,
31]. Thus, a number of toxins and peptides isolated to date has been found to interact with ion channels, enzymes and components of the cell membrane [
26,
29,
32], resulting in activities such as analgesic [
26], antimicrobial [
33,
34,
35], antihypertensive [
32], antiviral [
36], antiparasitic [
37,
38] and antitumor [
39,
40,
41,
42,
43].
The pharmacological properties of crotamine on different biological models include its analgesic potential [
16], insulin release potential [
11], memory persistence enhancement without psychomotor alterations [
44] and antimicrobial and antiparasitic actions in several species [
5,
12,
13,
14,
15,
23]. Moreover, due to its cell-penetrating capacity and site-specific interactions, crotamine has been studied as a drug-mediating peptide and as a model for the discovery of new antitumor molecules [
10,
17,
19,
21,
22,
23]. However, data on crotamine safety for local and systemic application in biological models are scarce [
24,
25].
In this study, the intradermal (
id) route of administration was evaluated, considering the ability of crotamine to penetrate cells and also considering the presence of many lymphatic structures in the dermis, which contribute to the drainage and diffusion of molecules [
45]. We demonstrated that a single
id injection of crotamine, at all studied concentrations and time-points, induced acute changes in immunological parameters and promoted oxidative stress but did not present a dose- or time-dependent response.
Although assessed separately, the metabolic and immune pathways are interdependent, since hormones, cytokines, transcription factors and signaling proteins act in both pathways to maintain the body’s homeostasis. The innate and the adaptive immune systems have numerous components with protective functions [
46]. The inflammatory response, on the other hand, is initiated by inflammatory cytokines such as IL-1, TNF-α and IL-6 released from an affected tissue inducing hepatic synthesis of acute phase proteins, such as CRP [
47].
In this study, crotamine increased CRP levels on the third day after intradermal injection (
Figure 1). CRP is an acute inflammation signaling protein, mainly produced by the liver but also by adipocytes and arterial tissue and regulated by cytokines IL-6, TNF-α and IL-1 [
48]. CRP levels are increased in response to active infections and acute inflammatory processes [
48]. Thus, our results suggest that crotamine stimulates the production of CRP either in response to the local aggression or via TNF-α signaling, whose levels were also shown to be high (
Figure 2A).
The elevated TNF-α level on the first day of analysis after intradermal injection of crotamine indicates its potential effect on an initial inflammatory response and the increase of other immunological markers involved in the process. TNF-α is mainly responsible for the recruitment and activation of neutrophils and monocytes in the injury site. In low concentrations, it acts on endothelial cells promoting vasodilation and stimulating the secretion of other chemotactic cytokines and fibroblasts [
49,
50,
51].
In contrast, our results showed that crotamine might exert two opposite effects on the synthesis and signaling of cytokines in inflammatory and immunological processes. Animals injected with lower crotamine doses (200 and 400 μg) showed significantly increased levels of IL-10 when compared to histamine control group (
Figure 2B). IL-10 has an anti-inflammatory action by regulating the activity and production of pro-inflammatory cytokines by macrophages, monocytes, mast cells and dendritic cells, thus limiting the inflammatory and immunological response [
46,
49].
Levels of proinflammatory cytokines, such as IL-6, were correlated with the myotoxic and edematogenic effects of crotamine isoforms isolated from
C. d. cumumensis in the study by Ponce-Soto et al. [
52]. Studies on the proinflammatory effects of crotamine demonstrated that this peptide increased the phagocytic activity in macrophages associated with NO, TNF-α and IL-1β, which are strictly related to inflammatory responses [
18].
The cell response to stimuli depends on a complex signaling process. The stimuli are transmitted from the extracellular medium to the intracellular medium through an orderly sequence of reactions, some of them dependent on oxidation reactions, generally referred to as redox-sensitive signaling [
53]. Thus, along with the analysis of cytokines and signaling molecules that mediate an inflammatory response, oxidative stress biomarkers can provide information on the relationship between oxidative damage, macromolecules (DNA, lipids and proteins) and various inflammatory and immunological processes [
54].
Herein, crotamine was shown to induce systemic oxidative stress, which was evidenced by the higher serum levels of MDA-TBARS, when compared with the histamine control group (
Figure 4B). However, this effect diminished with increasing doses of crotamine, suggesting a positive correlation between crotamine-induced pro-oxidant profile and the high serum levels of MPO (
Figure 3D), since increased levels of MPO activity represent an important marker of inflammation and oxidative stress [
51]. On the other hand, our results suggest that the highest concentration of crotamine (800 μg) stimulated the anti-oxidant system in order to reduce the oxidative damage, as indicated by the high serum levels of -SH groups (
Figure 4B). Consequently, TNF-α and IL-10 levels appear to peak at medium dose and then decrease with a higher concentration of crotamine (
Figure 2), suggesting an anti-inflammatory activity of this toxin.
Regarding to the NAG activity, an opposite trend was observed, as presented in
Figure 3C. Upregulation of NAG was observed on the first day of analysis demonstrating that anti-inflammatory activity of crotamine was not persistent. Additionally, an expressive increase of NO levels was recorded along the seven days of analysis, at all crotamine concentrations tested (
Figure 3A,B). NO is a potentially toxic agent, whose toxicity could be particularly denoted in stress oxidative conditions. Thus, our results suggest that NO may play a key role in the activity of macrophages [
55,
56,
57].
Using an intra-hippocampal application route, Gonçalves et al. [
25] observed that crotamine altered the oxidative parameters in the serum of animals after 21 days. These results suggest that crotamine has the ability to induce an imbalance in the systemic redox system for a long time, regardless of the route of administration. Previous studies on
Crotalus durissus terrificus venom and its main component, crotoxin, have shown that NO is closely related to antinociceptive effect [
58], modulation of macrocytic activity [
41,
59,
60] and the myotoxic effect of the venom [
61].
Defensins and cathelicidins are the best characterized antimicrobial peptides which act as effectors of the innate immune response [
62]. In addition, several antimicrobial peptides appear to initiate the process of tissue repair, mainly by inducing an angiogenic response at the site of injury [
63]. Crotamine has a heterogeneous cytotoxic profile on different microorganisms, as well as structural and/or genetic similarities with antimicrobial peptides such as β-defensins [
64].
Morphometric analysis of immunohistochemistry data presented in
Figure 5A,B showed an upregulation of VEGF 24 h after the intradermal injection of crotamine. It was also observed that between the 3rd and 7th day of analysis there was a downregulation of the number of newly formed blood vessels. These values, however, were still greater than or equal to those determined for the histamine-treated control group.
To date, little is known about the immunomodulatory and immunogenic properties of venom components. Cell-penetrating peptides (CPPs) have attracted considerable attention as a new class of ligands for delivery of specific therapeutic and diagnostic agents mainly due to several advantages compared to conventional antibodies, such as easier synthesis, smaller sizes, lower immunogenicity and cytotoxicity, besides offering simpler and improved conjugation to nanocarriers. Immunogenicity is the main cause of failure of biological products in clinical trials and therefore it is imperative that drug development studies include an immunogenicity risk assessment, leading to a clinical strategy [
64,
65,
66,
67,
68].