Anti-inflammatory drug may prevent death by scorpion
AGÊNCIA FAPESP/DICYT The yellow scorpion (Tityus serrulatus) is a mere 7 cm long and does not look especially threatening, but actually it is the deadliest species of scorpion in South America. Every year, more than 1.2 million people worldwide are victims of its toxic venom. Some 3,000 of them eventually die.
T. serrulatus is found mainly in the southeast region of Brazil. According to data in the scientific literature, most deaths from its sting are due to heart and lung complications that result in respiratory failure.
A study by Brazilian researchers published on February 23 in the journal Nature Communications suggests that such complications could be avoided, or at least minimized, by prompt administration of anti-inflammatory medications found in any pharmacy, such as indomethacin or celecoxib.
“Our experiments were performed with mice, but the results would very probably be replicated in humans because the molecular bases – the mediators involved in the pulmonary inflammatory response – are the same in this case. If this is confirmed, it will be an important tool for the provision of first aid to victims and will certainly reduce mortality,” said Lúcia Helena Faccioli, a researcher at the University of São Paulo’s Ribeirão Preto Pharmaceutical Science School (FCFRP-USP) and one of the authors of the paper.
The study was performed with support from FAPESP during Karina Furlani Zoccal’s postdoctoral research as part of a Thematic Project coordinated by Faccioli at USP.
T. serrulatus’s sting triggers a local inflammatory response that causes sharp pain but does not lead to death, Faccioli explained. In some cases, however, a systemic inflammatory response is also triggered, and this may result in pulmonary edema (accumulation of fluid in the lungs), which impairs breathing.
Until now, scientists have agreed that the severity of envenomation essentially depends on the ratio of a victim’s body mass to the amount of inoculated venom. However, the study performed at FCFRP-USP shows that genetic factors may be involved, in the sense that they may influence the capacity to produce certain inflammatory and anti-inflammatory molecules.
“The mechanisms by which this systemic response is triggered, the mediators involved, were unknown, and our study set out to investigate them,” Faccioli said.
In search of a suspect
Previous research by Faccioli’s group showed through experiments with mice that the venom of T. serrulatus is recognized by Toll-like receptors, a component of the innate immune system. The researchers also found that this induced the production of an inflammatory mediator called interleukin-1 beta (IL-1β), as well as the lipid mediators prostaglandin E2 (PGE2) and leukotriene B4 (LTB4).
“We set out to test the hypothesis that post-envenomation pulmonary edema was induced by the production of IL-1β due to inflammasome activation. This was because data in the literature suggested that inflammasome activation occurs when a substance is recognized by Toll-like receptors,” Faccioli said.
Inflammasomes are complexes of receptors and sensors located in the cytoplasms of defense cells. Inflammasomes are made up of several proteins, including caspase. Inflammasome activation triggers the release of IL-1β, which induces inflammatory processes both directly and indirectly via the production of lipid mediators such as PGE2 and LTB4. These mediators are responsible for attracting other defense cells to the lungs and for pulmonary edema – hence the inflammation. Although inflammation is an essential defense mechanism, it may cause death when excessive.
The first in vitro assays performed with wild-type (not genetically modified (GM)) macrophages from mice confirmed that inflammasome activation resulted from T. serrulatus venom, leading to the production of IL-1β. When the same experiment was performed with GM cells in which some genes that encode inflammasome proteins had been removed, there was no production of IL-1β or lipid mediators.
The next step was to test the hypothesis in vivo. When the researchers analyzed lung tissue samples collected from mice inoculated with venom from T. serrulatus, they observed the formation of edema and an increase in the number of neutrophils (a type of defense cell), both factors that indicate inflammation. In addition, there were large amounts of IL-1β, PGE2 and LTB4.
To verify that IL-1β was the key element in the observed inflammatory response, the group used two GM mouse models: one lacking genes that encode two inflammasome proteins and the other lacking a gene encoding the protein that functions as the cell receptor for IL-1β.
All the animals in both models survived even when inoculated with lethal doses of the venom, confirming that pulmonary edema does not develop to the extent of becoming lethal without the influence of IL-1β.
When the researchers analyzed lung tissue samples collected from GM mice that had survived lethal doses of scorpion venom, they found lower amounts of PGE2 and LTB4 than in wild-type mice.
They then decided to investigate the roles of these two mediators in the inflammatory response. To their surprise, they found that LTB4, hitherto described as a pro-inflammatory molecule, in fact acted to protect tissue against inflammation.
“We performed the experiment with GM animals so as not to produce the enzyme that participates in LTB4 production. We thought they would certainly survive a lethal dose of the venom because they lacked one of the components of the inflammatory response. What we found, however, was that they died much more quickly than wild-type mice and presented with exacerbated lung inflammation involving abundant production of IL-1β and PGE2,” Faccioli said.
Next, the group treated wild-type mice that had received lethal doses of venom with indomethacin, a drug that inhibits the synthesis of prostaglandins, including PGE2. All the animals survived.
In vitro assays showed that PGE2 boosts the production of a molecule called cyclic adenosine monophosphate (cAMP), which in turn leads to an increase in IL-1β and exacerbates inflammation, whereas LTB4 decreases the production of cAMP and hence of IL-1β.
“If we can show that pulmonary edema is also mediated by PGE2 and IL-1β in humans, the public health impact will be significant. Victims can be treated with medications available from any pharmacy while waiting for anti-scorpion serum to arrive,” Faccioli said.
In partnership with the Emergency Unit of the teaching hospital at the University of São Paulo’s Ribeirão Preto School of Medicine (FMRP-USP), the group plans to add all of the mediators involved in the pulmonary inflammatory response to the serum currently administered to patients who are stung by T. serrulatus.
“We’ll also be conducting in vitro studies using venom-stimulated human cells to see whether they display the same molecular pattern as we observed in mouse cells. If the results are similar, we can think about a project in partnership with physicians to treat victims of T. serrulatus with indomethacin,” Faccioli said.