A study by the INCLIVA Health Research Institute, the University Clinical Hospital of Valencia, and the University of Valencia (UV) has analyzed the role of extracellular histones in cell damage processes associated with sepsis, a serious illness that can lead to multiple organ failure.

Histones are proteins normally found within the cell nucleus, where they help organize DNA. However, during sepsis, they can be released outside the cell and circulate in the bloodstream, where they have been linked to damage in various tissues.

The study demonstrates that these extracellular histones are able to enter the cells lining blood vessels (endothelial cells) and that this process contributes to triggering cell death.

“The goal was to discover whether extracellular histones (molecules released during sepsis that significantly contribute to multi-organ damage) are able to enter the cells lining blood vessels and whether this entry significantly contributes to the damage that leads to their death,” explains Dr. Carlos Romá Mateo, coordinator of the study and member of the INCLIVA Research Group on Cellular and Organic Pathophysiology of Oxidative Stress, led by Dr. Federico Pallardó Calatayud.

A serious illness with high mortality

Sepsis is a serious condition caused by an abnormal immune response to an infection. It occurs when the immune system fails to eliminate the pathogen and the infection spreads throughout the body via the bloodstream. In these cases, the immune response itself also generates collateral damage to tissues. If the situation worsens, sepsis can progress to septic shock, a critical phase in which multi-organ failure occurs, significantly increasing the risk of death. Sepsis is estimated to cause approximately 11 million deaths annually worldwide, including in high-resource countries, as it often occurs in hospital settings. Among the major challenges to improving its clinical management are early diagnosis, rapid identification of the responsible microorganisms, and the development of biomarkers and targeted therapies that reduce the damage caused by molecules released during the immune response, such as circulating histones. These histones are the subject of this study and are initially released by the infected patient's own immune system cells.

“Previous studies by our group had already shown that released histones, upon contact with blood vessel cells (endothelial cells), triggered various cell death processes, which were more direct the higher the histone concentration. Other work by the group demonstrated that measuring histones in the blood of sepsis patients can contribute to improved diagnosis and prognosis, making understanding the mechanisms involved increasingly crucial for managing this complex disease,” adds Romá Mateo.

How histones enter cells
The results of this research were recently published in the journal Frontiers in Immunology.

For the study, an experimental model consisting of human endothelial cells was used. These cells were exposed to different concentrations of extracellular histones, in varying amounts and for different durations, and the internalization of these histones was tracked using fluorescent labeling.

Using fluorescent labeling techniques, researchers were able to track the path of these proteins and confirm that they penetrated cells and contributed to cell death. Furthermore, they observed that once inside, the proteins followed an intracellular pathway that directed them to cellular mechanisms related to autophagy, a process by which the cell degrades damaged or unnecessary structures.

The use of different specific inhibitors allowed researchers to identify the precise molecular pathway that histones followed to enter cells. When this pathway was experimentally blocked, the cells were protected from cell death. This result suggests that interfering with this pathway could become a therapeutic strategy to reduce the toxicity of extracellular histones in patients with sepsis.

Confirmation in Three-Dimensional Models

To corroborate the results, the researchers conducted additional experiments in a three-dimensional multicellular model: so-called vascular organoids, cellular structures that more realistically reproduce the environment of blood vessels, as they include different cell types and a three-dimensional structure similar to that of tissues. This part of the work was carried out during a research stay by Concepción Garcés in the laboratory of Dr. Josef Penninger in Austria, as part of her doctoral thesis.

The scientific article was led by Dr. Carlos Romá Mateo, from the Department of Physiology at the Faculty of Medicine and Dentistry of the University of Valencia. The first co-author is Patricia Rodríguez Tascón, a predoctoral researcher in the same INCLIVA research group.

The work was carried out in collaboration with researchers from Canada, Germany, and Austria. The University of Valencia researchers participating in the study are affiliated with INCLIVA and the Biomedical Research Networking Center for Rare Diseases (CIBERER) of the Carlos III Health Institute.

The infrastructure of the Central Research Unit in Medicine (UCIM-UV-INCLIVA), specifically the Cytometry and Confocal Microscopy services, was crucial to the development of the work. Guadalupe Herrera and Antonio Ibáñez, the technicians responsible for these services, are also co-authors of the article.

The work was primarily funded by the Spanish Ministry of Science and Innovation (PID2020-119127RA-I00) and the University of Valencia (UV-INV-AE24-3662073).

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