The study presents a new therapeutic target for t

image: The study also proved that a drug originally indicated to treat alcohol dependence can inhibit the action of the molecule and prevent complications
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Credit: CRID-USP

A new strategy for preventing complications associated with sepsis is presented by researchers affiliated with the University of São Paulo (USP) and collaborators in an article published in the review Blood.

The proposal is to inhibit the action of a protein called gasdermin D, which the authors show is possible with disulfiram, a drug already approved for human use and originally indicated to fight addiction to alcohol. alcohol.

The study was conducted at the Inflammatory Disease Research Center (CRID), one of the Centers for Research, Innovation and Dissemination (INCD) funded by FAPESP. CRID is hosted by the Faculty of Medicine Ribeirão Preto of the University of São Paulo (FMRP-USP) in Brazil.

“We know the drug is safe because it has been used since the 1950s, and we are proposing that it be repurposed to treat sepsis. We have seen it work in animal trials in the lab. Now we need of a clinical trial to assess its effectiveness in septic patients,” said Camila Meirelles Silva, postdoctoral fellow at CRID and first author of the article.

Popularly known as “generalized infection”, sepsis is actually systemic inflammation, frequently triggered by a bacterial infection that has entered the bloodstream. When the immune system fights off the pathogen, it ends up damaging the whole body. Patients with severe sepsis develop lesions that impair the functioning of vital organs, especially the lungs.

In previous research, the CRID group has shown that an immune mechanism known as neutrophil extracellular trapping (NET) is directly involved in tissue damage in septic patients. As their name suggests, NETs are a defense strategy used primarily by neutrophils, a type of white blood cell that phagocytose (kill) bacteria, fungi, and viruses. In extreme situations, these immune cells themselves can die and the material contained in their nuclei is ejected to the outside environment in the form of an extracellular network toxic both to pathogens and to the cells of the organism.

“In our latest study, we investigated the mechanism that allows the release of NET, in which gasdermin D is directly involved,” Silva said. “We show that when we prevented neutrophils from releasing these mesh-like traps by inhibiting gasdermin D, we were able to reduce the level of tissue damage and improve patient outcome.”

Methodology

Some of the experiments involved white blood cells isolated from 24 septic patients hospitalized 24 hours previously (12 men and 12 women). Confocal microscopy analysis showed that NETs were released in vitro by a large proportion of the cells. The tests also showed a large amount of active gasdermin D in the patients’ neutrophils. This had already been observed in neutrophils isolated from septic mice.

“Gasdermin D is a pore-forming protein,” Silva said. “Previous research has shown that when this molecule is activated in cells, it forms pores in the nuclear membrane that allow genetic material to pass into the cytosol. Further pores are then opened by gasdermin D in the plasma membrane, allowing all of this content to be released into the extracellular medium. We have proven that in sepsis the protein also acts in this way.

In vivo experiments were performed with mice subjected to a procedure designed to induce sepsis. Some normally expressed gasdermin D. The other group consisted of mice that had been genetically modified so that they did not produce the protein in any cell in their body (gasdermin D knockout mice).

“When we induced sepsis, we found that the knockout mice produced a smaller amount of NETs, ​​developed less organ damage [with no pulmonary edema, unlike the rest], and survived more,” Silva said.

In vitro assays involving cells from knockout mice confirmed that neutrophils did not release NET even in the presence of LPS, a molecule in the membranes of pathogenic bacteria usually interpreted as a threat by the immune system.

Inhibition assays

Data from the scientific literature have shown that disulfiram binds to gasdermin D to prevent the protein from forming cell membrane pores. Based on this evidence, the CRID scientists decided to test the effect of the drug in the context of sepsis, for which there are no specific drugs.

They found from in vitro tests that after treatment with disulfiram, human and mouse neutrophils stopped releasing NETs when stimulated with LPS.

In another experiment, neutrophils isolated from septic patients were incubated with disulfiram. The treatment inhibited the release of NETs.

Finally, septic mice were treated with disulfiram and their progress was compared to that of untreated mice. Treated animals had less tissue damage (including less pulmonary edema), less NETs in the blood and a better prognosis: 60% survived, compared to 20% in the untreated group.

The results have aroused the interest of the scientific community and motivated a remark, also published in Blood, by researchers Maksim Klimiankou and Julia Skokowa of Tübingen University Hospital in Germany.

“Neutrophils display multiple defenses against pathogens, including phagocytosis, production of reactive oxygen species, secretion of bactericidal enzymes, and formation of NETs. Neutrophils pay a high price for all of these defensive actions , dying in the process of digestion, neutralization and destruction of invaders. However, not everything is perfect in this line of defense: it is almost impossible to regulate the degree of activation of neutrophils. Once activated, they frequently exhibit hyperresponsive phenotypes, causing deleterious effects both at the site of inflammation and systemically, and playing a critical role in triggering the process of multi-organ dysfunction and the lethality of sepsis. deleterious effects of neutrophils during sepsis while preserving other functions of these cells could represent a therapy ie precious,” the German researchers write in the commentary.

The research results were also subject to podcast produced by the staff of Blood.

Next steps

According to Fernando de Queiroz Cunha, a professor at FMRP-USP and principal investigator of CRID, the production of disulfiram was stopped in Brazil in 2019, which prevented the group from carrying out a clinical trial.

“It was such a cheap drug that the company decided to take it off the market, and psychiatrists who treat alcohol-dependent patients now have to import it,” Cunha said. “As an alternative, we are in talks with a Brazilian pharmaceutical company to develop a slightly modified molecule that could be patented and marketed domestically.”

Cunha also revealed CRID’s intention to test whether disulfiram can be used to prevent organ damage in patients with COVID-19. A previous study by the group showed that NETs participate in the rampant inflammation triggered by SARS-CoV-2 (More than: agencia.fapesp.br/33523/).

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About the São Paulo Research Foundation (FAPESP)

The São Paulo Research Foundation (FAPESP) is a public institution whose mission is to support scientific research in all areas of knowledge by granting scholarships, fellowships and grants to researchers linked to educational institutions University and Research from the State of São Paulo, Brazil. FAPESP is aware that the best research can only be done by working with the best researchers at the international level. Therefore, it has established partnerships with funding agencies, higher education institutions, private companies and research organizations in other countries known for the quality of their research and encourages scientists funded by its grants to develop further their international collaboration. You can find out more about FAPESP at www.fapesp.br/en and visit the FAPESP press agency at www.agencia.fapesp.br/en to keep abreast of the latest scientific advances that FAPESP helps to achieve through its many programs, prizes and research centers. You can also subscribe to the FAPESP press agency on http://agencia.fapesp.br/subscribe.


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