The action of Echis carinatus and Naja naja venoms on human neutrophils; an emphasis on NETosis.

BACKGROUND: Neutrophils are the first line defense cells of the innate immunity. As a final defense, they discharge their de-condensed chromatin/DNA fibers, the NETs (Neutrophil Extracellular Traps), by a process called NETosis. Two types of NETosis have been currently described: the suicidal/delayed/classical-type, which is ROS dependent that results in the ejection of nuclear DNA, and the vital/rapid/early-type, which may or may not require ROS but, eject nuclear/mitochondrial DNA or both. Thus, Echis carinatus and Naja naja venoms are comparatively studied for their NET inducing property. METHODS: Formation of NETs, cell viability, ROS, and Ca2+ levels are estimated. An in vivo toxicity study and possible cellular signaling have been addressed using immunoblots and pharmacological inhibitors. RESULTS: E. carinatus and N. naja venoms respectively induce suicidal and vital NETosis. E. carinatus venom induces NETosis by activating NOX and PAD-4 enzymes in a ROS dependent manner via PKC/ERK/JNK signaling axis, while N. naja venom does it by activating PAD-4 enzyme, but independent of ROS requirement and as well as PKC/ERK/JNK activation. CONCLUSION: For the first time our study demonstrates the distinct action of E. carinatus and N. naja venoms on the process of NETosis. NETosis being a newly explored area in snake venom pharmacodynamics, it is important to study its impact on the various pathophysiological properties induced by snake venoms. SIGNIFICANCE: Understanding the varied actions of snake venoms on neutrophils/blood cells and the role of DNase are likely to provide insights for better management of snakebite pathophysiology.

Inhibition of Echis carinatus venom by DNA, a promising therapeutic molecule for snakebite management.

BACKGROUND: E. carinatus bite is a serious threat to South-Asian countries including India, as it causes the highest number of deaths and debilitating sustained tissue necrosis at the bite site. One of our previous studies has demonstrated the strong interaction between DNA and E. carinatus venom. Therefore, in this study, the effect of DNA on E. carinatus venom has been examined. METHODS: Here we show that calf thymus DNA interact strongly with E. carinatus venom and inhibits its enzymatic and pharmacological activities such as proteolytic, hemolytic, hyaluronidase, L-amino acid oxidase, NETosis, hemorrhage, pro-coagulant, and lethality. Further, using immunoblots and immunofluorescence, the study demonstrates the inhibition of proteolytic cleavage of several surface receptors on PMNs, PBMCs, and platelets by the DNA. CONCLUSIONS: This study for the first time explored the efficient inhibition of enzymatic, pharmacological and lethal properties of E. carinatus venom by the naked DNA and demonstrates the possible therapeutic application of it during snakebite management. GENERAL SIGNIFICANCE: This study identifies naked DNA as an effective defense weapon that has got the therapeutic potential to inhibit the detrimental effects of E. carinatus bite.

Melatonin inhibits snake venom and antivenom induced oxidative stress and augments treatment efficacy.

Snakebite is a neglected health hazard. Its patho-physiology has largely been focused on systemic and local toxicities; whereas, venom and antivenom induced oxidative stress has long been ignored. Antivenom therapy although neutralizes venom lethality and saves many lives, remains ineffective against oxidative stress. This prompted us to complement antivenom with an antioxidant molecule melatonin that would protect against oxidative stress and increase the efficacy of the existing snakebite therapy. Here we show that D. russelli and E. carinatus venoms induce strong oxidative stress that persists even after antivenom administration in mice model. Additionally, antivenoms also induce oxidative stress. Polyvalent antivenom induce more oxidative stress than monovalent antivenom. Strikingly, antivenom and melatonin together not only inhibit venom and antivenom induced oxidative stress but also significantly reduce the neutralizing antivenom dose. This study provides a therapeutic potential for enhancing the existing snakebite therapy. The combined treatment of antivenom+melatonin would prevent the upsurge of oxidative stress as well as minimize the antivenom load. Thus the investigation offers immense scope for physicians and toxinologists to reinvestigate, design new strategies and think beyond the conventional mode of antivenom therapy.

NETosis and lack of DNase activity are key factors in Echis carinatus venom-induced tissue destruction.

Indian Echis carinatus bite causes sustained tissue destruction at the bite site. Neutrophils, the major leukocytes in the early defence process, accumulate at the bite site. Here we show that E. carinatus venom induces neutrophil extracellular trap (NET) formation. The NETs block the blood vessels and entrap the venom toxins at the injection site, promoting tissue destruction. The stability of NETs is attributed to the lack of NETs-degrading DNase activity in E. carinatus venom. In a mouse tail model, mice co-injected with venom and DNase 1, and neutropenic mice injected with the venom, do not develop NETs, venom accumulation and tissue destruction at the injected site. Strikingly, venom-induced mice tail tissue destruction is also prevented by the subsequent injection of DNase 1. Thus, our study suggests that DNase 1 treatment may have a therapeutic potential for preventing the tissue destruction caused by snake venom.

Oxidative stress-induced methemoglobinemia is the silent killer during snakebite: a novel and strategic neutralization by melatonin.

Oxidative stress-induced methemoglobinemia remained an untouched area in venom pharmacology till date. This study for the first time explored the potential of animal venoms to oxidize hemoglobin to methemoglobin. In in vitro whole-blood assay, methemoglobin forming ability of venoms varied as Naja naja > Ophiophagus hannah > Echis carinatus > Daboia russellii > Apis mellifera > Mesobuthus tamulus > Hippasa partita. Being highly potential, N. naja venom was further studied to observe methemoglobin formation in RBCs and in combinations with PMNs and PBMCs, where maximum effect was observed in RBCs + PMNs combination. Naja naja venom/externally added methemoglobin-induced methemoglobin formation was in parallel with ROS generation in whole blood/RBCs/RBCs + PMNs/RBCs + PBMCs. In in vivo studies, the lethal dose (1 mg/kg body weight, i.p.) of N. naja venom readily induced methemoglobin formation, ROS generation, expression of inflammatory markers, and hypoxia-inducible factor-3α. Although the mice administered with three effective doses of antivenom recorded zero mortality; the methemoglobin and ROS levels remained high. However, one effective dose of antivenom when administered along with melatonin (1:50; venom/melatonin, w/w), not only offered 100% survival of experimental mice, but also significantly reduced methemoglobin level, and oxidative stress markers including hypoxia-inducible factor-3α. This study provides strong drive that, complementing melatonin would not only reduce the antivenom load, but for sure greatly increase the success rate of antivenom therapy and drastically minimize the global incidence of snakebite deaths. However, further detailed investigations are needed before translating the combined therapy towards the bed side.