Envenomations by Bothrops and Crotalus Snakes Induce the Release of Mitochondrial Alarmins

et al. (2012) Envenomations by Bothrops and Crotalus Snakes Induce the Release of Mitochondrial Alarmins. PLoS Negl Trop Dis 6(2): e1526. doi:10.1371/journal.pntd.0001526 Envenomations by Bothrops and Crotalus Snakes Induce the Release of Mitochondrial Alarmins Irene Zornetta 0 Paola Caccin 0 Julia n Fernandez 0 Bruno Lomonte 0 Jose Mara Gutierrez 0 Cesare Montecucco 0 Robert A. Harrison, Liverpool School of Tropical Medicine, United Kingdom 0 1 Dipartimento di Scienze Biomediche, Universita` degli Studi di Padova , Padova, Italy, 2 Facultad de Microbiolog a , Instituto Clodomiro Picado, Universidad de Costa Rica , San Jose , Costa Rica Skeletal muscle necrosis is a common manifestation of viperid snakebite envenomations. Venoms from snakes of the genus Bothrops, such as that of B. asper, induce muscle tissue damage at the site of venom injection, provoking severe local pathology which often results in permanent sequelae. In contrast, the venom of the South American rattlesnake Crotalus durissus terrificus, induces a clinical picture of systemic myotoxicity, i.e., rhabdomyolysis, together with neurotoxicity. It is known that molecules released from damaged muscle might act as 'danger' signals. These are known as 'alarmins', and contribute to the inflammatory reaction by activating the innate immune system. Here we show that the venoms of B. asper and C. d. terrificus release the mitochondrial markers mtDNA (from the matrix) and cytochrome c (Cyt c) from the intermembrane space, from ex vivo mouse tibialis anterior muscles. Cyt c was released to a similar extent by the two venoms whereas B. asper venom induced the release of higher amounts of mtDNA, thus reflecting hitherto some differences in their pathological action on muscle mitochondria. At variance, injection of these venoms in mice resulted in a different timecourse of mtDNA release, with B. asper venom inducing an early onset increment in plasma levels and C. d. terrificus venom provoking a delayed release. We suggest that the release of mitochondrial 'alarmins' might contribute to the local and systemic inflammatory events characteristic of snakebite envenomations. - Funding: This work was supported by University of Padua, Progetto Strategico Physiopathology of signalling in neuronal tissues: an in vivo approach to CM, the Fondazione CARIPARO to JF, and the ICGEB-CRP Program (grant COS-08-03) to BL and JMG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Snakebite envenomation is a neglected tropical disease that affects each year hundreds of thousands of individuals in tropical and sub-tropical areas of the world [1][2]. In addition to death, many snake bitten patients develop permanent physical and psychological sequelae which greatly affect their quality of life [3][4][5][6]. In the Americas, species of the family Viperidae are responsible for the vast majority of snakebite envenomations [7][5][8]. In Latin America, most cases are inflicted by species of the genus Bothrops, among which the lance-head vipers B. asper and B. atrox are very important in Central and South America, respectively [7]. In addition, the rattlesnake Crotalus durissus is notorious in South America for inflicting severe envenomations [7][9]. The pathophysiology of envenomations by B. asper (BaV) and C. durissus (CdV) and their predominant toxins has been investigated at experimental and clinical levels [10][11][12][13][9]. These venoms induce strikingly different pathophysiological patterns. BaV, similarly to other Bothrops spp venoms, induce local pathological alterations associated with edema, myonecrosis, dermonecrosis, blistering and hemorrhage [12]. In addition, systemic alterations, i.e. coagulopathies, hemorrhage, acute renal failure and cardiovascular shock, may ensue in moderate and severe cases [11][13]. Such a complex array of local and systemic alterations is mostly induced by the action of metalloproteinases, phospholipases A2 (PLA2) and PLA2 homologues, and serine proteinases, among other components [12][13][14][15]. These envenomations present prominent local inflammatory response, associated with the activation of innate immune mechanisms, which might contribute to the pathogenesis of tissue damage [16]. In contrast to the effects of BaV, the pathophysiological manifestations induced by CdV are characterized by minor local alterations and prominent systemic effects, mostly neurotoxicity, systemic myotoxicity, i.e. rhabdomyolysis, acute renal failure and coagulopathies [9]. Around 60% of CdV is comprised by the dimeric PLA2 complex crotoxin [17], which is composed by a basic PLA2 chain, crotoxin B, and a non-enzymatic acidic subunit, crotoxin A or crotapotin [18]. Cotapotin prevents the binding of crotoxin B subunit to non-specific sites and thus contributes to the high toxicity of this toxin [18]. Crotoxin exerts presynaptic neurotoxicity and systemic myotoxicity, which results in the release of large amounts of myoglobin from damaged muscle fibers, with the consequent impact on the kidney, provoking acute renal failure, which is a common finding in envenomations by this species [19]. Thus, envenomations by BaV and CdV represent different paradigms of tissue damage which greatly differ in the extent of the local inflammatory and pathological responses and in Every year, hundreds of thousands of people in tropical and sub-tropical areas of the world are bitten by poisonous snakes and may develop permanent damages. This is a major tropical disease which is largely neglected by scientific and clinical investigators. Snakes of Bothrops and Crotalus genus are responsible of most cases in Latin America. Here for the first time, we have shown that these venoms cause the release of both mitochondrial DNA and cytochrome c, two well known alarmins. Moreover, the kinetic of these processes are in agreement with the different pathophysiological profiles exhibited by Bothrops and Crotalus envenomations. These elements suggest a correlation between snake evenomations and sterile inflammatory syndrome. Alarmins are reported to have a fundamental role in innate immune response and inflammation; they might contribute to the local and systemic inflammatory events characteristic of these envenomations opening a new prospective in the study of these complex pathologies. the systemic manifestations. On the basis of such different pathophysiological patterns, these venoms constitute valuable experimental tools to assess various aspects of local and systemic muscle damage and inflammation. Snakebite envenomations trigger complex pathogenetic processes that include a range of defense reactions in the bitten organism, whose mechanisms are ill known, but resemble in several aspects muscle trauma [20][21]. It has been long known that following tissue injury such as mechanical (...truncated)