A biomedical engineering professor and doctoral student published a paper in the journal PLOS One that reveals new insights into the synergistic relationship of two proteins that are key drivers of immune responses following an infection.
Elebeoba May, associate professor of biomedical engineering and director of the biomedical engineering research program, worked with Ph.D. student Taha Salim to develop a mathematical model to explain the synergistic effects of tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma), two small proteins known as cytokines that signal cells to begin an immune response when an infection is detected inside of the body.
Immunologists have long known that the cytokines TNF-alpha and IFN-gamma are central to the body’s immune response, and although the synergistic relationship between the cytokines was observed in past scientific literature, it had never been quantified dynamically. May and Salim’s paper, “Investigating the Role of TNF-α and IFN-γ Activation on the Dynamics of iNOS Gene Expression in LPS Stimulated Macrophages,” sheds new light on the human immune response that could potentially lead to improved drug delivery and new drugs to modulate the immune response.
During an infection, cells called macrophages are produced by the immune system to identify and destroy invading pathogens. These macrophages, which are the immune system’s first line of defense against an infection, can sense their environment and change along with it.
“We are trying to study how the host immune cells – the macrophages – change and respond along with their environment, and how they produces effector molecules that eliminate bacteria once it’s infected,” said May. “Our goal was to investigate how the different cytokines in this micro-environment affect the immune response.”
With respect to lipopolysaccharide (LPS) induced immune responses – a type of very strong immune response in animals – TNF-alpha acts in synergy with IFN-gamma to produce an enzyme called induced nitric oxide synthase, or iNOS, which helps to regulate the immune response by releasing reactive nitrogen species that can kill the intracellular pathogens. Along with the release of iNOS, macrophages secrete chemokines, another class of pro-inflammatory immunological proteins that help facilitate the migration of other immune cells to the infection site. Because of the cytokines generated in the micro-environment from the initial infection, the incoming cells are able to get activated, or primed, giving them the ability to fight more effectively against foreign invaders.
Using enzyme rate equations and kinetic modeling methods, Salim and May created a mathematical model to capture the intracellular signaling pathways and gene expression systems involved in the release of iNOS, nitric oxide and TNF-alpha from macrophages. Salim combed through past literature on the topic to piece together his model, which went beyond traditional models that isolated each of the cytokines’ pathways.
“Past models looked at individual pathways but didn’t put them together to form the bigger picture, at least with these cytokines,” Salim said. “We had to search the literature for different aspects of our model and piece the different pathways together to show the synergistic relationship between these cytokines. It was like putting together a puzzle.”
In addition to revealing the synergy between TNF-alpha and IFN-gamma, Salim and May’s model quantified the synergistic effect between the cytokines as “exponential, not just additive” to the immune response.
Their model demonstrated that the release of TNF-alpha and INF-gamma leads to new expressions inside of the cell, as the cytokines amplify each other’s impact in the immune response.
The next step for the researchers was to test their model through careful computer modeling experimentation. Salim and May began by looking how much of each cytokine needed to be present in the environment to induce the release of iNOS, and how much iNOS each of the cytokines produced.
TNF-alpha is produced by macrophages when an immune response is activated. Once TNF-alpha is released, the cytokine can activate the same macrophages it was produced from, known as an autocrine effect. IFN-gamma is produced later in the immune response by natural killer (NK) cells. After activation, the NK cells must migrate toward the site of infection, which adds to the time delay to peak expression for IFN-gamma. May and Salim’s model quantified the delay to peak expression for both cytokines and, in doing so, revealed new insights into the synergistic relationship between the two proteins.
In a process called priming, cells in the body are able to prepare themselves for battle against an infection after the release of the cytokines TNF-alpha and IFN-gamma. As with the process of cell activation, cell priming was driven by the synergistic relationship between the two cytokines.
Salim and May found that far less TNF-alpha needs to be present in the environment in order to generate an immune response and release the enzyme iNOS. But despite the speedy initial release of iNOS, the enzyme depletes quickly inside the cell. IFN-gamma is activated later on in the immune response, and much more of the protein must be present in the environment to generate the release of iNOS. However, the immune response driven by IFN-gamma is far greater than the initial response generated by TNF-alpha.
The researchers coined the term “molecular ripple effect” to describe the curious phenomenon. The activation of iNOS and initial release of TNF-alpha signals the beginning of an immune response to all of the surrounding cells, which begin to prime themselves for battling an infection. When IFN-gamma is released later in the immune response, the cytokine essentially activates TNF-alpha once again. Because TNF-alpha can activate the same macrophages they were produced from, the overall immune response is exponentially greater following the release of IFN-gamma.
“Imagine throwing a small pebble in a pond – you get ripples that grow bigger and bigger. But if you throw a boulder into a pond, there will be a large amount of water that’s displaced initially but it all comes back eventually. If the cells in your body activate too strongly during the pro-inflammatory response, you will have an equal and opposite anti-inflammatory response afterwards, and that’s harmful,” Salim said.
In addition to capturing the magnitude and dynamics of this immune response, May noted that one of the key contributions of their paper is quantifying the timing of the response.
“We can use models like this to understand how we can improve drug delivery and design drugs to stimulate a pro-inflammatory response or modulate the immune response in addition to taking an antibiotic.” May said.