PNAS study reveals why organs fail following massive trauma

Massive trauma, say from a sabertooth tiger attack, meant immediate death for the primitive human. Modern man is more likely to survive severe injury caused by a car crash, gunshot or fall thanks to high-tech emergency medicine.

Unfortunately, the body does not know what to do when it survives an injury that would have been fatal until recently in human evolution. Nearly one third of the time, mechanisms in place to protect people from disease misfire seven to ten days after severe injury, causing multiple organ failure. That is one reason why trauma is the leading cause of death for Americans aged 44 and younger.

A nationwide team of researchers is working urgently on the problem of post-trauma immune system and organ failure, and has discovered several new biochemical pathways that play a central role, according to a study published today in the Proceedings of the National Academy of Sciences (PNAS). Inventing new techniques along the way, the team is changing emergency room guidelines, building the foundation for earlier diagnosis of post-trauma organ failure and making possible the design of drugs to reverse it.

Trauma has become a federal research priority because the survival rate has not improved in 10 years. Excitement is growing, however, because new evidence suggests that major diseases involving the immune system share some of the same mechanisms and may be cured by manipulating the same molecules. A protein called programmed cell death 1 (PD1), for example, suggested by the PNAS paper as playing a role in post-trauma organ failure, is also involved in the immune system breakdown leading to AIDS, according to an article in the August edition of Nature.

“Our study proves for the first time that it is possible to identify the genetic and protein changes in specific immune cells that play a significant role in determining whether or not trauma is fatal,” said Carol L. Miller-Graziano, Ph.D., professor of Surgery and of Microbiology & Immunology at the University of Rochester Medical Center, and an author of the PNAS study. “Beyond trauma, we believe that the techniques established here can provide insights into many disease that involve human immune system failure.”

No Respite from Toxins

The human immune system is actually two systems developed at different points in human evolution. White blood cells of the more primitive innate system, like neutrophils and macrophages, form the first line of defense against disease. They swarm to the injury to engulf and destroy bacteria with toxic molecules and biochemicals. Because these same molecules and chemicals are also toxic to nearby human cells, the innate response is normally shut down quickly by the onset of the more time-consuming, precise and thorough adaptive immune system.

This second system pumps out an endless variety of immune cells on the hope that one or more will be the right shape to link up with, and become activated by, any invader encountered. When one of those immune cells, called lymphocytes, recognizes a bacterium or virus presented by the innate system, that lymphocyte expands into army of clones specifically designed to attack the invader at hand. Unfortunately, lymphocytes too are capable of mistakenly destroying human cells. Thus, they have developed quality control mechanisms where they can choose to stop expanding into an army (anergy) or to commit suicide (apoptosis).

The PNAS study offers new evidence that an overwhelming wave of biochemical signals created by the innate response after massive trauma can mistakenly trigger anergy and apoptosis, especially in T cell lymphocytes, shutting down the adaptive response before it can begin. Without the normal transition, the innate system continues to pump out toxic chemicals (cytokines) and molecules (free radicals) that tear at organs until they fail.

The goal of the current study was to identify the few genes and proteins that play a central role in T-cell mediated immune and organ failure. Given that severe trauma effects as many as 20 percent of the 20,000 or so human genes, finding the genes and proteins involved in T cell anergy and apoptosis amid this storm has been a challenge.

Researchers took blood samples from 22 healthy subjects, and from 18 consenting patients who had experienced severe trauma, and were in the midst of organ failure. The team then used unique, clinically applicable techniques to separate out each immune cell type in the patients' blood into pure samples for study. Only when researchers compared pure samples of the same immune cell types (e.g. T cells to T cells) did meaningful patterns emerge. The authors achieved a statistically significant picture of the differences between traumatized and healthy patients for the first time, and despite skeptics that said it could not be done.

With pure samples in hand, researchers used microarray technology to generate a long list of genes that change when a person undergoes massive trauma. They then ran that list through a database of genetic information to see which of the genes changed by trauma were listed in the literature as having a role in T cell function. What emerged was a map of the likely functions of hundreds of genes and proteins related to T cell post-trauma dysfunction, and their likely partners in signaling pathways. The final step was to demonstrate that the proteins associated with those genes have an actual role in organ failure via real-world biochemical tests.

Results showed that the expression of nearly 5,700 genes related to T cell function is changed in cases of massive trauma, as are 2800 genes related to the function of macrophages, partnering immune cells necessary to T cell activation. Trauma had the most profound effect on just 338 of the genes, an at least two-fold change in their expression, the process by which the information they store is converted into proteins that carry out bodily functions. Post-traumatic genetic changes had two major effects on T cells: a marked increase in regulatory protein pathways that diminished their function, and a decrease in signals that turn them on, researchers said.

Whether T cells continue to multiply depends on the action of sensitive receptor proteins on their surfaces. Some of the receptors, once activated by signaling molecules, cause the T cell to multiply, while others stop the process or cause the cell to self-destruct. Researchers in the current study identified as many as 20 new receptors on the surfaces of T cells or signaling molecules within T cells, that increase their activity in the case of massive trauma to either cause anergy or apoptosis. Each pathway represents a target for the design of new drugs to reverse T cell shutdown.

The research for the current paper was conducted under the auspices of a Large-Scale Collaborative Award from the National Institute of General Medical Sciences (NIGMS), a division of the National Institutes of Health. Also called “Glue Grants,” the awards bring together large teams of researchers to handle extremely complex problems. While the PNAS study patient sample was small, techniques based on it are now being used to study 150 patients per year throughout the Glue grant infrastructure. The glue grant is an open access endeavor as well. The results are posted publicly and hundreds of researchers have already accessed the PNAS study results and applied them to their own projects.

The grant is organized into seven research cores, with the PNAS study written within the Protein Analyses and Cell Biology (PACB) Core working closely with the Genomics Core. PACB include analytical sites, including the one led by Miller-Graziano. She oversaw the separation of white blood cells into pure samples for the PNAS study, and continues to determine cell surface protein expression for the larger Glue grant team.

Along with Miller-Graziano, the University of Rochester Medical Center team is led by Paul E. Bankey, M.D., Ph.D., director of Trauma and Critical Care in the Department of Surgery, and includes Asit De, Ph.D., assistant professor of surgery, and Krzysztof Laudanski, M.D., a post-doctoral fellow. Fifteen authors from eight universities and one pharmaceutical company collaborated on the PNAS article, including the Stanford Human Genome Center.

The trauma glue grant was launched in 2001 with an original $30 million award over five years, and has just been renewed with an additional $30 million in funding. The University of Rochester Medical Center team will receive about $3.9 million for its role between the two periods of the grant.

“These findings strikingly reveal that novel interactions among signaling pathways can be deconstructed in hospitalized patient populations,” Bankey said. “For the first time, we can uncover changes in cells that tell us which patients are recovering, and which are getting worse, in almost any disease that involves the immune system.”