An overview of the immune modulating effects of enzymatic toxins from snake venoms

Abstract

Snake venoms are complex mixtures of organic and inorganic compounds, including proteins belonging to the protease (serine and metalloproteinases), oxidase (L-amino acid oxidases), and phospholipase (especially phospholipases A₂) enzyme classes. These toxins account for the serious deleterious effects of snake envenomations, such as tissue necrosis, neurotoxicity, and hemorrhage. In addition to their toxic effects, snake venom toxins have served as important tools for investigating the mechanisms underlying envenomation and discovering new pharmacologically active compounds with immunotherapeutic potential. In this sense, the present review discusses the new findings and therapeutic perspectives in the immune modulating potential of enzymatic toxins from snake venoms belonging to the classes metalloproteinase, serine protease, L-amino acid oxidase, and phospholipase A₂.

Introduction

The immune system is composed of cells (leukocytes and platelets) and soluble factors (complement system proteins, antibodies, acute-phase proteins, and arachidonic acid-derived lipid mediators) that work together to keep the host homeostasis by killing pathogenic microorganisms, clearing antigens, and maintaining the self-tolerance. As immune system imbalances can result in inflammatory and autoimmune diseases, hypersensitivity, cancer, and immunodeficiency, this system must be tightly regulated [1]. Modulation of the immune system may help to control exacerbation of the immune response in autoimmune and inflammatory diseases, as well as to activate the immune system components in antitumor responses and immunodeficiencies.

Recently, several medical and scientific studies have been searching for new treatment strategies, known as immunotherapy, which consists in the use of substances able to modulate the immune cells to battle the progression of different disorders, such as cancer and autoimmune diseases [2]. Considering the biodiversity of snake venoms, which are complex mixtures of organic and inorganic compounds, including enzymatic and non-enzymatic proteins, peptides, and non-protein molecules [3], one would expect that they could be potential sources of new immune modulating agents. Thus, the comprehension of the mechanisms underlying the immune modulating action of snake venom toxins may help to design new synthetic compounds or to engineer novel nanoconjugated molecules to pharmacologically manipulate the immune response. An example is the use of nanotechnology to build nano-engineered drug carrier systems with advantages in targeted delivery and controlled release [4,5]. Some recent studies have shown the therapeutic potential of the nanoparticle-sustained delivery of snake venom toxins in cancer treatments by using silica or gold nanoparticle conjugates as a strategy to enhance the cytotoxicity of venom components on targeted tumor cells and to reduce their toxicity towards normal cells [[5], [6], [7], [8], [9], [10]].

The present review focuses on the four main classes of enzymatic proteins that exist in snake venoms and whose immune modulating potential has already been reported in the literature, i.e. metalloproteinases, serine proteases, L-amino acid oxidases (LAAOs), and phospholipases A₂ (PLA₂s). Although the immune modulating properties of these classes of toxins are still debatable, their ability to regulate hemostasis, renin-angiotensin, and repair systems are well-known [11]. As the hemostasis system is closely associated with the inflammatory response, enzymatic toxins from snake venoms that modulate the hemostasis and repair systems probably interfere in the immune response; however, this relationship has not been properly explored yet.

Serine proteases and metalloproteinases are proteolytic enzymes. Serine proteases are one of the most abundant toxin classes in Viperidae venoms. During envenomation, the proteolytic action of serine proteases mainly affects the hemostasis processes by interfering in different coagulation checkpoints and deregulating the fibrinolytic and kallikrein–kinin systems. Most snake venom serine proteases exert thrombin-like effects [[12], [13], [14]], but the role that they play in the immune response is controversial. Snake venom metalloproteinases (SVMPs) belong to a subclass of zinc-dependent enzymes that are divided into the classes P-I, P-II, and P-III, according to their structural domains [15,16]. Due to their proteolytic effects, SVMPs play major roles in the hemorrhagic events triggered by snake venoms [17,18]. SVMPs also induce apoptosis, activate prothrombin and factor X, and inhibit platelet aggregation [[19], [20], [21]]. This enzyme class is associated with the inflammatory response in envenomations, which culminates in edema formation, neutrophil recruitment, activation of the complement system, and release of cytokines like interleukin 1β, 6, and 10 (IL-1β, IL-6, and IL-10, respectively), and tumor necrosis factor α (TNF-α) [15,[22], [23], [24]].

LAAOs are flavoproteins that catalyze the stereospecific oxidative deamination of L-amino acids, producing H₂O₂ and ammonia [[25], [26], [27]]. This enzyme class occurs in a variety of sources [28], and those isolated from snake venoms have attracted the scientific interest in the last decades due to their antitumor, antimicrobial, and proinflammatory effects [[29], [30], [31], [32]]. PLA₂s constitute a protein superfamily with enzymatic activity that specifically catalyzes the hydrolysis of sn-2 ester bonds of phospholipid substrates in the presence of calcium [33]. Snake venom PLA₂s display a variety of pharmacological and toxic effects, such as edema, myotoxicity, neurotoxicity, hemolysis, hypotension, and anticoagulation [[33], [34], [35]].

Several snake venom toxins, especially those belonging to the four aforementioned enzyme classes, exert their immune modulating action by interfering in the inflammatory process and immune cell functions, and stimulating leukocytes to release cytokines and eicosanoids that are key mediators of inflammation. When an antigen or pathogen invades or disrupts the integrity of a natural barrier such as the skin, mucosa or respiratory tract, resident immune cells recognize danger signals and trigger the immune response. Pathogen- and/or damage-associated molecular patterns usually represent such danger signals, which bind to pattern-recognition receptors and elicit the immune response − this is an innate mechanism of immunosurveillance [36]. During envenomation, snake venom toxins − peptides, enzymes, and non-enzymatic proteins – can directly bind to pattern-recognition receptors or cause tissue damage and release damage-associated molecular patterns that are recognized by local cells and elicit the inflammatory response [37]. Other venom components can exert the opposite effect and could be used to downmodulate the effects of inflammation and its associated pain [38,39].

Activation of innate immune cells by venoms and their toxins can trigger the production of proinflammatory cytokines, such as IL-6, TNF-α and IL-1β, chemokines, and lipid mediators, which in turn contribute to additional leukocyte influx and activation. Lipid mediators are eicosanoids derived from the arachidonic acid metabolism, such as prostaglandins, leukotrienes, and thromboxanes. These mediators, in combination with cytokines and chemokines, trigger various inflammatory events, including edema, pain, chemotaxis, cytokine release, and leukocyte activation [[40], [41], [42], [43]]. Targeting and inhibiting these inflammatory mediators may help to design therapeutic schemes to alleviate the symptoms of envenomations and optimize anti-inflammatory therapeutic approaches. Furthermore, identification of the components that account for the immune modulating action of snake venoms could generate a platform to develop new drugs to modulate the immune response.

Several studies carried out in the past decades have supported the immune modulating action of snake venoms. Naja naja atra snake venom selectively increases interferon-γ and IL-4 secretion and suppresses IL-17 production, which in turn improves the innate and humoral immune responses and inhibits the action of CD4 Th17 and CD8 T cells [44]. Bothrops atrox [30], Bothrops erythromelas [45,46], Bothrops jararaca [47], Bungarus caeruleus [48], and Crotalus durissus cascavella [49] snake venoms modulate the immune response by increasing secretion of proinflammatory cytokines, and by regulating the release of antibodies and the function of leukocytes. Bothrops jararaca and Bothrops jararacussu snake venoms upregulate the neutrophil functions and chemotaxis [50,51]. On the other hand, Naja kaouthia and Thailand cobra venom suppress the chronic inflammatory response in animal models of arthritis [52,53], while Crotalus durissus terrificus venom exerts anti-inflammatory action by inhibiting the spreading and phagocytic activity of mice macrophages and neutrophils [54]. Additionally, several Bothrops snake venoms are able to modulate the complement system [23,29,47,55].

Hence, crude snake venoms and their isolated toxins may up- or downregulate the innate and adaptive immune system response, depending on the cell context and snake species. In this sense, the present review reports an overview of the immune modulating potential of four classes of enzymatic toxins from snake venoms: metalloproteinases, serine proteases, LAAOs, and PLA₂s. Table 1 summarizes some of their effects on the immune system that will be discussed in the following sections of this review.