Elsevier

Molecular Immunology

Volume 116, December 2019, Pages 45-55
Molecular Immunology

Contribution of endothelial cell and macrophage activation in the alterations induced by the venom of Micrurus tener tener in C57BL/6 mice

https://doi.org/10.1016/j.molimm.2019.09.009Get rights and content

Highlights

  • M. t. tener whole venom can induce pro-inflammatory and hemostatic effects in mice.

  • Pro-coagulant and pro-inflammatory properties in LSEC were seen after activation with venom.

  • Pro-inflammatory cytokines release in macrophages were detected at 24 h.

  • Both cell activation and the whole venom contribute to the immune response and the hemostactic disorder seen in vivo.

Abstract

An acute inflammatory response, cellular infiltrates, anemia, hemorrhage and endogenous fibrinolysis activation were previously described in C57BL/6 mice injected with M. tener tener venom (Mtt). As the endothelium and innate immunity may participate in these disturbances and due to our poor understanding of the alterations produced by these venoms when the neurotoxic component is not predominant, we evaluated the effects in an in vitro model. At 24 h, the release of pro-inflammatory mediators was detected in peritoneal macrophages. At different times, the release of pro-inflammatory (TNF-α, IL-6, NO and E-Selectin), pro-coagulant (vWF and TF) and pro-fibrinolytic (uPA) mediators were seen in liver sinusoidal endothelial cells (LSECs). These results suggest that Mtt venom activates macrophages and endothelium, thus inducing the release of mediators, such as TNF-α, that orchestrate the acute inflammatory response and the later infiltration of mononuclear cells into liver in C57BL/6 mice. In addition, endothelium activation promotes TF expression, which may in turn modulate the inflammatory and hemostatic response. These findings suggest crosstalk between inflammation and hemostasis in the alterations observed in Micrurus envenomation, where the neurotoxic manifestations do not predominate.

Introduction

Animal venoms, including snake venoms, have been widely studied, firstly to elucidate the mechanisms of action of their toxic compounds, but also due to their enormous potential as diagnostic tools and therapeutic agents (White, 2005). Venoms are deadly cocktails, which comprise a unique mixture of peptides and proteins that can disturb homeostasis and the activity of proteins, enzymes, receptors and ion channels that might alter the central and peripheral nervous systems, as well as the cardiovascular, muscular, hemostatic and immune systems (Calvete et al., 2009). For this reason, snake envenomation is a major public health problem in rural areas of tropical and subtropical countries in Africa, Asia, Oceania and the Americas (WHO, 2007).

Snake venom components may induce a systemic inflammatory response, specifically pro-inflammatory cytokine release (Petricevich, 2004; Salazar et al., 2018). In addition, active hemostatic compounds in snake venom have been identified and isolated that can decrease the coagulability of blood, induce bleeding and associated secondary effects (Sajevic et al., 2011; White, 2005). Although it is evident that components of snake venom may affect the hemostatic and inflammatory systems separately, it is not clear whether the interaction between these systems may contribute to the disturbances seen in victims.

The acute inflammatory response begins with the activation of resident immune cells including macrophages in the injured tissue, which release pro-inflammatory mediators such as IL-1 and TNF-α that in turn trigger the inflammatory response in the neighboring endothelial cells of post-capillary venules (Barton, 2008; Medzhitov, 2008). Likewise, in pathological conditions, a pro-inflammatory state combined with pro-thrombotic events has been demonstrated. These may produce multi-organ failure as a consequence of fibrin deposition in the microvasculature (Petäjä, 2011). Pro-inflammatory cytokines produce hemostatic alterations including endothelial activation, platelet activation and aggregation, thrombin generation, and suppression of the fibrinolytic system (Levi et al., 2002), but the hemostatic system may also reciprocally affect the immune system and promote inflammatory activity (Margetic, 2012; O’Brien, 2012). Hence, the study of the mechanism involved in these alterations has led to the evaluation of the crosstalk between hemostasis and inflammation, where the innate immune response plays a central role.

Micrurus envenomation is characterized by neurotoxic symptoms and may lead to death from muscle paralysis and respiratory arrest. Some reports have shown that these venoms may also contain hemorrhagic, hemostatic, hemolytic and edematogenic activities (Barros et al., 1994; Casais-e-Silva and Teixeira, 2017; Salazar et al., 2011, 2018; Tambourgi et al., 1994; Vivas et al., 2016). In the United States, only two species are responsible for all coral snake toxicity, Micrurus fulvius fulvius (Eastern coral snake) and Micrurus tener tener (Texas coral snake), and most of the information available rely on the Eastern coral snakebites. Few studies have described the clinical manifestations of patients bitten by Micrurus tener tener (Mtt), which represents 2.3% of the snakebites in Texas, which include local symptoms including pain, edema, erythema, and long-lasting paresthesia, and, in severe cases, skeletal muscle weakness and cranial nerve dysfunction (McAninch et al., 2019; Morgan et al., 2007). These effects can be attributable to the most abundant protein families shared by all species from the elapids of the New world, which are phospholipases A2 (PLA2) and three-finger toxins (3FTxs), although, members of other protein families, that are common but not omnipresent, including metalloproteinases, L-amino acid oxidases, Kunitz-type serine protease inhibitors, serine proteinases, and C-type lectin-like proteins can also modulate these effects (Alape-Girón et al., 1996; Bénard-Valle et al., 2014 Corrêa-Netto et al., 2011; Lomonte et al., 2016a).

In a previous article, we reported results showing that Mtt venom induced a pro-inflammatory response in addition to hematological and hemostatic disturbances in C57BL/6 mice that were injected intraperitoneally (i.p.) with a sub-lethal dose of this venom (Salazar et al., 2018). As the endothelium and innate immunity may participate in the disturbances seen in C57BL/6, we evaluated the inflammatory response and hemostatic alterations induced by Mtt crude venom in an in vitro model. This was done by employing cells from the same mouse strain; specifically, liver sinusoidal endothelial cell (LSEC) line and peritoneal macrophages, to extend our understanding of other mechanisms of action, besides neurotoxicity, that may be involved in the acute manifestations of Micrurus snake bites.

Section snippets

Reagents

Cadmium granules, potassium nitrate, bovine thrombin, sodium chloride, calcium chloride, lipopolysaccharide (LPS, from Escherichia coli serotype O26:B6), trichloroacetic acid, trizma base, sulphorhodamine B, 3′diaminobenzidine, HEPES, and other reagents were purchased from Sigma Aldrich, USA. Avidin peroxidase and streptavidin peroxidase, anti-VCAM-1, anti-ICAM-1, anti-E-Selectin antibodies were obtained from Santa Cruz Biotechnology, USA. 3,3′,5,5′-tetramethylbenzidine (TMB) was purchased from

Effect of Mtt venom on LSEC and peritoneal macrophages

The SRB assay showed that the pool of Mtt venom used in this study induced a LC50 for LSEC of 15 and 10 μg/mL at 24 and 48 h, respectively (Fig. 1A). Meanwhile, for peritoneal macrophages, the LC50 values were 57 and 27 μg/mL at the same times (Fig. 1B). After 24 h, the venom produced in LSEC an increase in cytoplasmic granules along with a decrease in cell-cell contact and cell density at concentrations higher than 3.8 μg/mL (Fig. 1C and E). Meanwhile, all the concentrations tested produced

Discussion

Coral snake envenomation are relatively uncommon, however, these venoms are very potent and may produce neurotoxicity characterized by muscle paralysis and respiratory failure (Sánchez et al., 2008). The biological characterization of Mtt venom identified SVMP and PLA2, as well as fibrino(geno)lytic, fibronectinolytic, gelatinolytic and hyaluronidase activities (Salazar et al., 2011). In addition, previous studies have shown that Mtt venom triggers an acute systemic inflammatory response within

Declaration of Competing Interest

The authors declare that they have no conflicts of interest.

Acknowledgements

Financial support from the Science and Technology Foundation (FONACIT, Grant PG-2005000400), the Instituto Venezolano de Investigaciones Científicas, Venezuela, and the grant from the NIH/ORIP, Viper Resource Grant # P40OD01960-15 (National Natural Toxins Research Center (NNTRC), Texas A&M University-Kingsville, Dr. E.E. Sánchez) are gratefully acknowledged.

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