RIPK3

A new method of precisely targeting troublesome cells for death using light could unlock new understanding of and treatments for cancer and inflammatory diseases. A new method of precisely targeting troublesome cells for death using light could unlock new understanding of and treatments for cancer and inflammatory diseases, University of Illinois Urbana-Champaign researchers report. Inflammatory cell death, knows as necroptosis, is an important regulatory tool in the body's arsenal against disease. However, in some diseases, the process can go haywire; for example, cancer cells are able to suppress inflammatory signals and thus escape death. "Usually treatments for cancer use pharmacological induction to kill the cells, but those chemicals tend to diffuse throughout the tissues and it's hard to contain to a precise location. You get a lot of unwanted effects," said study leader Kai Zhang, a professor of biochemistry at the U. of I. "We can make the cells responsive to light, and we can focus the light beam to be smaller than one cell. That is how we can use light to very precisely target a cell and turn on its death pathway." The researchers use a method called optogenetics to make the cells respond to light. They borrowed a light-activated gene from plants and inserted it into intestinal cell cultures, attaching it to the gene for RIPK3, a protein that regulates necroptosis. "When activated, RIPK3 undergoes oligomerization -- it forms clusters of protein complexes. Our light-sensitive proteins cluster together when exposed to blue light. So by triggering the light-sensitive proteins to come together, the RIPK3 comes together and oligomerizes, and that's how we mimic the activation pathway," said graduate student Teak-Jung Oh, the first author of the paper published in the Journal of Molecular Biology. However, killing the cell itself is not the only goal. Inducing the inflammatory cell death pathway, rather than outright killing the cell mechanically or chemically, triggers the immune system to respond. The ruptured cells release chemicals called cytokines that irritate nearby cells and attract T cells, white blood cells that play an important role in how the immune system identifies and attacks threats, Zhang said. "Certain cancer cell types create a local immunosuppressive environment, where T cells are either not recruited or, if they do come, they do not recognize it as a threat and do not infiltrate the cancerous area. But by opening up some cancer cells through necroptosis, we hope to modulate this immune suppressive environment and help train the T cells to recognize and attack the cancer," said Zhang, who is a member of the Cancer Center at Illinois. Since the optogenetic system requires light delivery directly to tissues, human clinical applications in tissues deeper than skin are currently limited. However, the Illinois group plans to implement their system in mice next to further study necroptosis and immune response in cancer and other inflammatory diseases. They also will further investigate the in vitro platform's potential for training T cells for immune therapies. "Understanding the cell signaling pathway for necroptosis is especially important because it has been known to be involved with diseases like neurodegenerative disease and inflammatory bowel disease. Knowing how necroptosis affects progression in these diseases is important. And if you don't know the molecular mechanisms, you don't really know what to target to slow the progression," Oh said. The National Institute of General Medical Sciences and National Institute of Mental Health of the National Institutes of Health, the National Science Foundation and the Cancer Center at Illinois supported this work. Zhang also is affiliated with the Beckman Institute for Advanced Science and Technology at Illinois. The National Institutes of Health supported this work through grants R01GM132438 and R01MH124827.

Findings show a newly created drug can prevent runaway inflammation while still allowing the immune system to handle the virus, even when given late into infection. Infection with the influenza virus leads to lung injury through inflammation over-activation that causes collateral damage to cells required for breathing. Such damage can be life-threatening, but scientists have a new preventative treatment. A team from St. Jude Children's Research Hospital, University of Houston, Tufts University School of Medicine and Fox Chase Cancer Center created a drug that can prevent flu-induced lung injury. In a mouse model, the drug achieves a novel balance between shutting down runaway inflammation and allowing the immune system to stop the virus. The findings were published today in Nature. "Our drug significantly increased survival and lowered symptoms of influenza virus infection," said co-corresponding author Paul Thomas, PhD, St. Jude Department of Host-Microbe Interactions. "It dampened dangerous inflammation and even seemed to improve the adaptive response against the virus." In a series of experiments, the drug UH15-38 protected against lethal influenza. Results showed that the drug protected mouse models from similar amounts of influenza that humans experience, even at low doses. Additionally, the team found that a high drug dose could fully protect against an infection with a substantial amount of virus, which would usually be deadly. The models were protected even if they received the dose days after infection, a difficult achievement for an influenza therapeutic. "This drug can also do something we've never seen before," Thomas said. "We're able to start five days after the initial infection and show that we're still providing some benefit." Providers must administer modern antiviral drugs within the first few days of infection to be effective. This study suggests that UH15-38 may fill a currently unmet need, as patients with severe disease have often been infected for several days by the time they get to a doctor. The breakthrough results from understanding how influenza and the immune system interact to cause lung injury. Sending influenza-infected cells down the right path "Infected lung cells create inflammation that alerts the immune system that there's a problem, but too much of it generates runaway inflammation that can cause major problems," Thomas said. "We need to strike a delicate balance between maintaining enough of these processes to get rid of the virus, but not so much that you're getting this runaway inflammation." The collaborating scientists achieved a Goldilocks amount of inflammation using clever chemistry. Their new drug inhibited one part of a major inflammation protein in immune cells: Receptor-Interacting Protein Kinase 3 (RIPK3). RIPK3 controls two cell death pathways in response to infection: apoptosis and necroptosis. Necroptosis is highly inflammatory, but apoptosis is not. Both pathways are used in the antiviral response. UH15-38 was designed to prevent RIPK3 from starting necroptosis while maintaining its pro-apoptotic properties. "Knocking out RIPK3 entirely is not great because then the immune system can't clear the virus," Thomas said. "When we knocked out just necroptosis, the animals did better because they still had apoptosis and could still get rid of infected cells, but it wasn't as inflammatory." Stopping lung inflammation and injury "We also showed that the improved survival was the direct result of the reduction in local inflammation and improved lung cell survival," Thomas said. In a series of prior studies, the Thomas lab found that a specific set of cells in the lung are collateral damage in the runaway inflammatory response. These cells, Type 1 alveolar epithelial cells, handle gas exchange, letting oxygen in and carbon dioxide out. Loss of these cells leads to an inability to breathe. The current study demonstrated that this group of literal breath-taking cells was spared in the presence of the drug. Additionally, inflammation-related immune cells, such as neutrophils, were far less prevalent in the lungs of treated animals. "Often the worst part of influenza illness happens after the virus is controlled when runaway inflammation destroys lung cells," Thomas said. "UH15-38 can dampen that influenza-caused inflammation while leaving viral clearance and the other functions of the immune and tissue responses intact. That makes it a promising candidate to move forward toward the clinic."

Researchers have revealed that common liver diseases are not driven by inflammatory cell death as previously thought, resolving a long-standing controversy in gastroenterology and pointing to a new direction for treatment. WEHI researchers have revealed that common liver diseases are not driven by inflammatory cell death as previously thought, resolving a long-standing controversy in gastroenterology and pointing to a new direction for treatment. The research team studied liver diseases that affect billions worldwide, including non-alcoholic fatty liver disease and hepatitis B, to understand what drives their progression. Their unexpected finding -- that liver cells are unable to undergo an inflammatory form of cell death called 'necroptosis' -- resolves crucial unanswered questions in the field and will help guide the development of new therapeutic interventions. At a glance The findings, published in Gastroenterology, offer clarity on the heavily debated role of necroptosis in the progression of liver pathologies, and provide fundamental insights to guide future pre-clinical and clinical studies in a new direction. The study was led by chief investigator Dr Marcel Doerflinger, former WEHI PhD researcher Dr Simon Preston and principal investigator Professor Marc Pellegrini, in collaboration with researchers from the Peter Doherty Institute for Infection and Immunity and the University of Queensland. Liver damage Liver diseases are a significant and growing global health burden. Non-alcoholic fatty liver disease is the most common liver disease, affecting over 30 per cent of the world's population, while over 296 million people are infected with Hepatitis B globally. To date, researchers have considered necroptosis as critical in the progression of these diseases. However, it was unclear whether this mode of cell death was occurring in liver cells, or in immune cells that had infiltrated the liver in response to infections or diet-related damage. "We sought to address this research gap and define the role and relevance of necroptosis in common liver diseases," said study lead Dr Doerflinger. The researchers used several preclinical genetic models of liver diseases including non-alcoholic fatty liver disease and its advanced form, non-alcoholic steatohepatitis, as well as hepatitis B. The team deleted key genes required for necroptosis from liver cells known as 'hepatocytes' to observe the effects on disease development. They found that deleting these genes had little effect, with disease progression proving comparable to normal hepatocytes. This revealed that necroptosis was not involved in the development of these liver pathologies. "The liver is a vital organ due to its function in the body's metabolism and detoxification," Dr Doerflinger said. "It's unclear why necroptosis is repressed in liver tissue, but we speculate it may be because the liver is constantly bathed in necroptotic signals such as gut microbial products, so limiting necroptosis could potentially protect the liver from excessive inflammation." Molecular mechanisms The research also revealed the molecular mechanisms responsible for the inability of liver cells to undergo necroptosis. After genetically profiling human liver tissue samples, the team discovered that hepatocytes cannot produce a critical protein essential for necroptosis, RIPK3. Production of RIPK3 protein was restricted at the genetic level, where the RIPK3 gene was blocked by a type of epigenetic modification known as 'methylation'. "Methylation acts as a genetic blockade, preventing the body's protein production machinery from binding to the DNA and building RIPK3 protein," said Dr Doerflinger. "As a result, without this essential protein to carry out its necroptotic function, the cell death pathway can't be initiated." Dr Doerflinger said momentum had been growing in the development of inhibitors of RIPK3 for the potential treatment of liver diseases, but their potential clinical applicability had been limited by a lack of fundamental insights. "These findings are a central piece of data that address many unanswered questions in the field that will guide future pre-clinical trials and clinical studies in this direction," he said. The research was supported by the NHMRC and biotechnology start-up Anaxis Pharma Pty Ltd. It was performed in collaboration with Anaxis Pharma and Servier, a global pharmaceutical group. Anaxis is a strategic joint venture established by WEHI and SYNthesis Research Pty Ltd, with a focus on necroptosis as a novel pathway of interest in human disease, developing first-in-class drug candidates for chronic inflammatory diseases including irritable bowel disease, Crohn's disease, liver fibrosis, and reperfusion injury.