Burroughs Wellcome International Ltd.

Chlamydia, the leading cause of sexually transmitted bacterial infections, evades detection and elimination inside human cells by use of a cloaking device. But researchers have grasped the hem of that invisibility cloak and now hope they can pull it apart. This evolutionary arms race between the immune system and the pathogen has been going on for millions of years and its parts are very finely tuned. Chlamydia, the leading cause of sexually transmitted bacterial infections, evades detection and elimination inside human cells by use of a cloaking device. But Duke University researchers have grasped the hem of that invisibility cloak and now hope they can pull it apart. To enter the cell and peacefully reproduce, many pathogenic bacteria, including Chlamydia, cloak themselves in a piece of the cell's membrane, forming an intracellular free-floating bubble called a vacuole or, in the case of Chlamydia, an inclusion. Chlaymydia's cloak appears to be especially effective at evading the cell's built-in immunity, allowing the infection to last for months. A Duke team led by graduate student Stephen Walsh and Jörn Coers, PhD, an associate professor of molecular genetics and microbiology in the Duke School of Medicine, wanted to know how the cloaking worked. "We knew there was the potential to kill Chlamydia, but when we did experiments with the human-adapted form, Chlamydia trachomatis, it was very good at growing in human cell cultures," Coers said. Even after the scientists used an immune stimulant to alert the cell's defense systems of the presence of Chlamydia, nothing happened. "We said, there's the pathogen. Our defense system should see it. Why does it not see it?" They ran their experiments again using a mouse-adapted version of the Chlamydia bacteria in human cells to see how the cell's immune system responded to a non-human pathogen. "Humans, don't get mouse Chlamydia because it evolved with mice and human Chlamydia evolved with humans," Coers said. "So there's this really fine-tuned adaptation that the pathogen has undergone." The mouse version of the bacterial inclusion was readily identified and labeled for destruction in human cells. "Chlamydiatrachomatis is so good at evading our human responses," Coers said. "It still causes an inflammatory disease, but it's a very slow disease." This evolutionary arms race between the immune system and the pathogen has been going on for millions of years. "Mouse and human adapted Chlamydiahave a common ancestor," Coers said. "However, this common ancestor may go back as far as when humans and rodents basically split from each other. This is a long time for the bacteria to really fine-tune their interactions with their host species." Working with Duke MGM colleagues Raphael Valdivia and Robert Bastidas, the researchers ran a large genetic screen of Chlamydia that identified a protein, GarD (gamma resistance determinant), that appeared to be blocking the host cell's ability to mark a Chlamydia inclusion for destruction by the immune system. Mutating their GarD genes left the bacteria vulnerable. "GarD is the stealth factor," Coers said. Specifically, GarD interferes with the ability of a giant signaling protein called RNF213 or mysterin to sense small bits of bacterial molecules poking out of the shell of the inclusion. "RNF213 is basically the eyes of the immune system," Coers said. Having blinded mysterin in this fashion, the signal for immune flagging and destruction is never started. The inside of a cell is swarming with these little bubbles of membrane-covered vacuoles; most are friends, but some, like the Chlamydiainclusion, are foes. "There's so many different types of membranes and vacuoles that live inside a cell," Coers said. "How is the immune system able to find the rare vacuole that contains a pathogen? In the case of Chlamydia, we really don't have the answer to that question. But whatever it is, we believe this enzyme (mysterin) is seeing it." Unfortunately, that's all the further this story goes for now, Coers said. This is a great new insight into a pernicious infection, but several steps away from a therapy. Researchers still need to figure out how mysterin sees those bacterial molecules in the first place and how GarD blinds mysterin. "If you could find a mechanism to deactivate GarD, then you can turn human Chlamydiainto mouse Chlamydia," Coers said. "That would allow us to harness the powers of our own immune system to clear infection." New Chlamydiainfections occur in 200,000 Americans per year and are often asymptomatic for months or even years while being transmissible through sexual contact. With time, an untreated infection can lead to pelvic inflammatory disease, ectopic pregnancy and female infertility. The US Centers for Disease Control recommends that young women be tested forChlamydia annually. This research was supported by grants from the National Institutes of Health (A030801, AI150106, AI140019,U19-AI084044) and the Burroughs Wellcome Fund.

The new immune networks have implications for developing immunotherapies and understanding autoimmune diseases SAN FRANCISCO, July 11, 2022 /PRNewswire/ -- Using new technologies to study thousands of genes simultaneously within immune cells, researchers at Gladstone Institutes, UC San Francisco (UCSF), and Stanford School of Medicine have created the most detailed map yet of how complex networks of genes function together. The new insights into how these genes relate to each other shed light on both the basic drivers of immune cell function and on immune diseases. Alex Marson (center) and his colleagues created the most detailed map yet of how complex networks of genes function together. Photo: Michael Short/Gladstone Institutes "These results help us flesh out a systematic network map that can serve as an instruction manual for how human immune cells function and how we can engineer them for our benefit," says Alex Marson, MD, PhD, director of the Gladstone-UCSF Institute of Genomic Immunology and co-senior author of the new study, published in Nature Genetics. The study, conducted in collaboration with Jonathan Pritchard, PhD, professor of genetics and of biology at Stanford School of Medicine, is also critical to better understand how variations in a person's genes are connected to their risk of autoimmune disease. Immune Insights from CRISPR Researchers know that when the immune system's T cells—white blood cells that can fight infections and cancer—become activated, levels of thousands of proteins within the cells change. They also know that many of the proteins are interconnected such that changes in the level of one protein can cause changes in the level of another. Scientists represent these connections among proteins and genes as networks that look somewhat like a subway map. Mapping these networks is important because they can help explain why mutations in two different immune genes might lead to the same disease, or how a drug might have an impact on many immune proteins at once. In the past, scientists have mapped out a part of these networks by removing the gene for each protein, one at a time, and studying the impact on other genes and proteins, as well as on immune cells' overall function. But this kind of "downstream" approach only reveals half the picture. "We really wanted to look at what is controlling key immune genes," says Jacob Freimer, PhD, a postdoctoral fellow in both the Marson and Pritchard labs, and first author of the new paper. "This kind of upstream approach hadn't been done before in primary human cells." This upstream approach would be like mapping out the subway routes by first identifying main hubs and then figuring out the routes to those key stations, rather than by painstakingly reconstructing the whole network from disparate satellite stations. Freimer and his collaborators turned to the CRISPR-Cas9 gene editing system, which allowed them to disrupt thousands of genes at once. They concentrated on genes that make a type of proteins known as transcription factors. Transcription factors are the switches that turn other genes on or off and can control many genes at once. The scientists then studied the impact of disrupting these transcription factors on three immune genes known to play an important role in T cell function: IL2RA, IL-2, and CTLA4. These three genes were hubs that anchored the upstream mapping efforts. "This let us go through over a thousand transcription factors and see which ones have an impact on these immune genes," says Freimer. An Interconnected Network The researchers suspected that they would find connections between the genes regulating IL2RA, IL-2, and CTLA, but they were surprised by the extent of the connectivity they discovered. Among 117 regulators found to control levels of at least one of the three genes, 39 controlled two of the three, and 10 regulators simultaneously altered levels of all three genes. To help fill out the immune gene map even more, the team next took a more traditional downstream approach, removing 24 of the pinpointed regulators from T cells to show the full list of genes they regulate—other than IL2RA, IL-2, and CTLA4. The researchers showed that many of the regulators controlled each other. The transcription factor IRF4, for instance, altered the activity of 9 other regulators and was itself regulated by 15 other regulators; all 24 controlled levels of IL2RA. In other cases, regulators were themselves regulated by IL2RA, in so-called "feedback loops." As in a dense subway network, each hub was connected to many others, and the connections ran both ways. "There were cases where a transcription factor was regulating IL2RA, but then IIL2RA itself also controlled that same transcription factor," says Freimer. "It appears that these kinds of feedback loops and regulatory networks are much more interconnected than we previously realized." Back to Patients Among the full list of genes controlled by the regulators studied, the research team found a high number of genes already linked to immune diseases, including multiple sclerosis, lupus, and rheumatoid arthritis. The new map helped reveal how genetic changes associated with these diseases can appear in different genes but—because of regulatory connections between genes—end up having the same net effect on cells. It also points to key groups of genes that might be targeted by drugs to treat immune diseases. The study suggests there's a central network of important genes, and when this network is perturbed, it can increase a person's disease risk. "When we understand the ways in which these networks and pathways are connected, it starts to help us understand key collections of genes that need to function properly to prevent diseases of the immune system," says Marson. About the Research Project The paper "Systematic discovery and perturbation of regulatory genes in human T cells reveals the architecture of immune networks" was published in the journal Nature Genetics on July 11, 2022. Other authors are Christian Garrido of Gladstone; Oren Shaked and Jessica Cortez of UCSF; and Sahin Naqvi, Nasa Sinnott-Armstrong, Arwa Kathiria, Amy Chen, and William Greenleaf of Stanford School of Medicine. The work and the authors were supported by National Institutes of Health (R01HG008140, RM1-HG007735, T32AI125222, and 5F32GM135996-500 02); the Burroughs Wellcome Fund; the Chan Zuckerberg Biohub; the Innovative Genomics Institute; the American Endowment Foundation; the Cancer Research Institute; the Jordan Family; Barbara Bakar; the Parker Institute for Cancer Immunotherapy; a Helen Hay Whitney Fellowship; a Stanford Graduate Fellowship; and a Stanford Center for Computational, Evolutionary, and Human Genomics Fellowship. About Gladstone Institutes To ensure our work does the greatest good, Gladstone Institutes focuses on conditions with profound medical, economic, and social impact—unsolved diseases. Gladstone is an independent, nonprofit life science research organization that uses visionary science and technology to overcome disease. It has an academic affiliation with the University of California, San Francisco. About UCSF The University of California, San Francisco (UCSF) is exclusively focused on the health sciences and is dedicated to promoting health worldwide through advanced biomedical research, graduate-level education in the life sciences and health professions, and excellence in patient care. UCSF Health, which serves as UCSF's primary academic medical center, includes top-ranked specialty hospitals and other clinical programs, and has affiliations throughout the Bay Area. Learn more at ucsf.edu, or see our Fact Sheet. Sources Gladstone Institutes: Julie Langelier | [email protected] | 415.734.2019 UCSF: Robin Marks | [email protected] | 628.399.0370 SOURCE University of California, San Francisco; Gladstone Institutes

The fossil record tells us about ancient life through the preserved remains of body parts like bones, teeth and turtle shells. But how to study the history of soft tissues and organs, which can decay quickly, leaving little evidence behind? In a new study, scientists use gene expression patterns, called transcriptomics, to investigate the ancient origins of one organ: the placenta, which is vital to pregnancy. The fossil record tells us about ancient life through the preserved remains of body parts like bones, teeth and turtle shells. But how to study the history of soft tissues and organs, which can decay quickly, leaving little evidence behind? In a new study, scientists use gene expression patterns, called transcriptomics, to investigate the ancient origins of one organ: the placenta, which is vital to pregnancy. "In some mammals, like humans, the placenta is really invasive, so it invades all the way through the wall of the uterus, into the maternal tissue. In other mammals, the placenta just touches the wall of the uterus. And then there's everything in between," says senior author Vincent J. Lynch, PhD, associate professor of biological sciences in the University at Buffalo College of Arts and Sciences. "So what kind of placentas were early placentas?" he says. "We use gene expression patterns to reconstruct the evolution of the placenta and predict what the placenta of the last common ancestor of eutherian mammals looked like. Our data tells us that this placenta was invasive, and that non-invasive placentas evolved multiple times among mammals. This addresses a 150-year-old mystery: People have been debating what kind of placenta the first one was since then." As Lynch explains, all living mammals other than marsupials and egg-laying monotremes are eutherians, which have long pregnancies in which the developing fetus evokes a strong physiological response in the mother. The research was published on June 30 in eLife. Lynch led the study with first author Katelyn Mika, PhD, University of Chicago postdoctoral scholar in human genetics and in organismal biology and anatomy. Camilla M. Whittington, PhD, and Bronwyn M. McAllan, PhD, both at the University of Sydney, are also co-authors. "Our ability to ask how the placenta might have functioned at different points during its evolution by using the gene expression profiles of currently existing animals to reconstruct the ancestors is a really cool approach and provides us more information on how changing gene expression can contribute to the evolution of a new trait," Mika says. To conduct the analysis, the team compared the genes active in the uterus of various mammals during pregnancy. After finding that these gene expression profiles correlated with the degree of placental invasiveness, the scientists used their data to predict what ancestral mammalian placentas looked like. The study included about 20 species, such as the egg-laying platypus, pouch-bearing marsupials, and a range of eutherian mammals that give birth to live young. The small subset is one limitation of the analysis: The authors write in eLife that research on a larger number of species is needed to help determine the strength of the findings. Nevertheless, the study makes important contributions in understanding how pregnancy evolved, Lynch says. The results could also benefit modern medicine. "Knowing which genes are active among different species during pregnancy tells us about how evolution works," he says. "But it also tells us about what makes a healthy pregnancy, and how things might go wrong. We're finding the genes that establish the right kind of environment for healthy human pregnancies. If those genes are not expressed in the right way, that might give rise to problems." This study was supported by grants from the March of Dimes and the Burroughs Wellcome Fund Preterm Birth Initiative.