Research seeks new drug for treatment of sepsis-induced immunosuppression

The Center for Research on Inflammatory Diseases, supported by FAPESP, is investigating a novel pharmacological target to combat immunosuppression due to generalized infection.

By Diego Freire

Agência FAPESP – Researchers at the Center for Research on Inflammatory Diseases (CRID), one of the Research, Innovation and Dissemination Centers (RIDCs) supported by FAPESP, may be close to identifying the first pharmacological target for treatment of immunosuppression in patients with sepsis.

They presented their research on the new target at the First International Symposium on Inflammatory Diseases held by CRID in June at the University of São Paulo’s Ribeirão Preto School of Medicine (FMRP-USP) in Brazil.

According to Fernando Queiroz Cunha, a professor at FMRP-USP and coordinator of CRID, the purpose of the symposium was to strengthen translational research in the area of inflammatory diseases.

“CRID’s objectives include the development of translational research,” Cunha told Agência FAPESP. “So we saw the holding of a symposium as an opportunity to bring together scientists who are working on inflammatory diseases, in terms of both basic research and clinical practice, to discuss experimental findings with the aim of prompting new strategies for the treatment of patients, and to collect questions that originate in clinical experience and can be answered in the laboratory, but again with the aim of improving the treatments currently in use.”

The researchers are endeavoring to develop new drugs and diagnostic proposals for inflammatory diseases of infectious, autoimmune or metabolic origin. To this end, they are performing laboratory experiments with genetically modified animals and clinical RNA sequencing assays using samples supplied by patients.

One of their goals is to discover pharmacological targets for the treatment of sepsis, a syndrome characterized by a systemic inflammatory response, especially to bacterial infection. Intense inflammation beginning in the acute phase of the disease may in some cases trigger the production of anti-inflammatory and immunosuppressive mediators, leading to a condition known as sepsis-induced immunosuppression.

Among the key features of this condition is an increase in the number of regulatory T cells (Tregs), a type of lymphocyte responsible for inflammation control. The researchers are attempting to understand the molecular mechanisms associated with this increase during the development of immunosuppression in patients who survive sepsis.

The experimental model used is mouse cecal ligation and puncture (CLP) to induce a bacterial infection, and treatment with antibiotics to simulate what happens in humans.

The researchers then work with the enzyme indoleamine 2,3-dioxygenase (IDO), originally isolated from human placenta. The fetus is a foreign body for the mother’s organism, whose immune system should therefore reject it. Studies have shown that IDO is important for the immune system to “tolerate” the baby’s presence during pregnancy.

Since IDO was discovered, several groups have demonstrated the enzyme’s tolerogenic effect on the immune system. Based on this information, the researchers at CRID hypothesized that IDO might be associated somehow with the growth in Tregs during sepsis-induced immunosuppression.

An increase in protein expression and IDO activity in the mouse spleen after the onset of sepsis was demonstrated by Raphael Gomes Ferreira, a researcher affiliated with FMRP’s pharmacology program, and collaborators. In other experiments, researchers found that IDO is expressed mainly by dendritic cells in the spleen of mice that survive sepsis.

“There are findings in the literature that show IDO to be activated by the metabolization of tryptophan, an essential amino acid, leading to the production of kynurenine, a biologically active molecule variously involved in immune system regulation,” Ferreira said.

Both tryptophan consumption and kynurenine production have also been shown to be associated with the activation of cellular pathways linked to an increase in Tregs. “Once the increase in protein expression and IDO activity in the spleen dendritic cells of sepsis-surviving mice had been demonstrated, the next step was to evaluate whether the inhibition of IDO enzymatic activity was associated with the increase in Tregs observed in animals and patients who survive the disease,” Ferreira said. To test the hypothesis, the researchers treated sepsis-surviving mice with a specific IDO inhibitor. The results showed a decrease in the frequency of Tregs in the spleen of animals treated with the inhibitor, proving the involvement of IDO in the increase in Tregs that occurs after the onset of sepsis.

Finally, to evaluate the role of IDO in the development of sepsis-induced immunosuppression, CLP-surviving mice treated with IDO inhibitor were challenged with B16-F10 melanoma cells, which express the luciferase enzyme.

“Tumor cells in immunosuppressed animals are known to meet less resistance from the immune system, so that they grow more easily,” Ferreira said. “In our experiment, we used mice submitted to sepsis and treated with IDO inhibitor to evaluate whether the enzyme could interfere in the development of sepsis-induced immunosuppression.”

Tumor growth was measured by quantifying the bioluminescence emitted by the cancer cells. For this purpose, the animals were treated with luciferin, the substrate for the luciferase enzyme expressed by cancer cells.

“We injected the substrate for the enzyme into the mice. When it came into contact with the enzyme, the luciferin was broken down and released light, along similar lines to what happens in fireflies. We have a device that quantifies the light emitted by the cancer cells. The larger the number of cancer cells, the more light or bioluminescence is emitted,” Ferreira said.

A pachymeter was also used to measure the volume of tumor cell growth.

Many Tregs were observed in the animals treated with saline solution. Because Tregs suppress the immune system, cancer cells can multiply more easily. The number of Tregs decreased in animals treated with IDO inhibitor, allowing the immune system to perform more efficiently in terms of preventing tumor growth.

For the researchers, the results are a clear sign that the mice became less immunosuppressed after inhibition of IDO enzyme activity. According to Ferreira, the identification of IDO as a participant in the immunosuppression process makes it an important pharmacological target for the treatment of patients with sepsis-induced immunosuppression.

“Treating immunosuppressed patients with IDO inhibitor may perhaps be an interesting approach,” he said. “But if we can discover which mediators produced during sepsis are responsible for promoting the increase in IDO, patients with sepsis can be treated before they develop immunosuppression.”

Because no drugs currently exist for the treatment of patients with sepsis-induced immunosuppression, many patients who survive sepsis die as a result of nosocomial (hospital-acquired) infections, or even simple bacterial infections acquired at home and easily treated in non-immunosuppressed patients.