Max Planck Institute For Infection Biology

Treatment of autoimmune diseases like lupus has long relied on steroids to knock down the immune system, but more targeted therapies are currently undergoing clinical trials. To make sure these therapies get to the patients who will benefit, work is needed to identify the specific mutations behind each patient's disease. Researchers now report several dozen mutations associated with oversensitive toll-like receptors -- a major cause of autoimmune disease -- and linked two mutations to patients. Lupus is a lifelong, often painful and occasionally lethal autoimmune disease. Few treatments exist today beyond powerful steroids to knock down a patient's immune system -- a therapy that has its own serious risks. The good news is that new and promising treatments are in clinical trials. But the term lupus belies the fact that the disease has a variety of causes, which means that treatments will have to be highly personalized to guarantee that each patient is given the drug that targets the specific genetic mutation responsible for their variety of lupus. Researchers are just now beginning to link specific genetic mutations to subsets of lupus patients, allowing physicians to target therapies to those who will benefit most. In the latest advance, researchers at the University of California, Berkeley, report in a new paper the discovery of two sets of patients with genetic mutations that are nearly identical to mutations that the researchers had earlier pinpointed in mouse and cell lines as linked to autoimmune disease. These two genetic links are among several dozen mutations that the UC Berkeley team recently discovered and linked to lupus, all in one gene that regulates a prime suspect in a subset of lupus patients -- proteins called toll-like receptors (TLR), which enable immune cells to recognize foreign DNA and RNA. According to study leader Gregory Barton, UC Berkeley professor of molecular and cell biology, identifying these mutations could help doctors deliver a personalized treatment to patients with oversensitive TLRs and, in particular, oversensitive TLR7 receptors. "We basically have a map now," said Barton, who is also an investigator in the Howard Hughes Medical Institute. "It's not like everybody that has lupus has a mutation in the gene that causes overactivation of TLRs and TLR7. But there are drugs coming online that very specifically inhibit TLR7. As we sequence more and more people, it will become easier to identify those patients and put them on those drugs. That's a lot better than the current course of therapy for lupus, which is brutal." "This is exciting because the drug will be orally available and is in clinical trials now," said Victoria Rael, a UC Berkeley graduate student who, with fellow graduate student Julian Yano, is a co-first author of the paper. The results of the genetic screens and details of the patients' mutations were published today (May 23) in the Journal of Experimental Medicine. A problem recognizing 'self' Autoimmune diseases, which range from rheumatoid arthritis and Crohn's disease to scleroderma and numerous thyroid conditions, stem from attacks by the immune system on the body's own cells that destroy normal, healthy tissue. Many studies have linked at least two types of autoimmune disease, lupus and psoriasis, to TLRs, which are part of the innate immune system that initially detects foreign invaders, such as viruses and bacteria, and stimulates a first line of attack. Normally, TLRs are delicately tuned to react only to foreign DNA and RNA, but if that tuning is off, they can react to a body's own nucleic acids and proteins associated with nucleic acids, which look much like those of pathogens. What makes this autoimmune reaction so deadly is that the TLRs also activate the body's second-line defense, the more powerful adaptive immune response, mobilizing T and B cells, macrophages, and other cells. These cells then mount a sustained attack that destroys the body's healthy tissue and causes chronic inflammation. The most common form, systemic lupus erythematosus (SLE), for example, is characterized initially by skin rashes -- in particular, a butterfly-shaped rash on the face -- but later by damage to joints, muscles, organs and skin, causing pain and fatigue. It's most commonly seen in females, often starting during the teen years. Lupus, in general, is two to three times more prevalent among women from many ethnic and racial minority groups than among white women. "We think the way the system works is that if nucleic acids find these receptors, most likely they're going to be from a virus," Barton said. "But in some people, the receptor is more responsive, so now levels of self-nucleic acids that otherwise wouldn't stimulate the receptor in a normal person activate the receptor. We think that one of the ways that these mutations are working is that they're making levels of self-nucleic acids that normally wouldn't be stimulatory, stimulatory." Barton and his lab colleagues have been investigating the role of TLRs that are misregulated in lupus, and in particular, one of the main proteins that regulates them: UNC93B1, or UNC for short. Several years ago, a team of postdoctoral fellows and graduate students in his lab screened in cell culture more than 100 genetic mutations in the UNC gene to see which ones overstimulated TLRs and would be good targets for further study. While they published some details in earlier papers, they didn't publish the complete list because there seemed to be little point -- almost no data was available on the genome sequences of lupus patients to compare with the mutations that overstimulated TLRs. But that has changed in recent years, thanks to a plunge in the cost of genome sequencing. That's how the mother of a young girl with severe autoimmune disease found Barton. Her daughter's DNA had been sequenced and showed a mutation in a region of UNC that Barton's team had noted in an earlier paper. Lupus in the family Rael and undergraduate Madeleine Weiss used the same cell culture screening technique to test the novel mutation from the young girl and found that it had an overstimulating effect, similar to the effect of other mutations in that area of the UNC gene. Surprisingly, the patient had the genetic mutation on only one of the two UNC alleles, meaning that she had one normal UNC gene, yet she still suffered severe autoimmune symptoms. Barton and his team also connected with a family of five afflicted with lupus. All had mutations on one UNC allele in another area of the UNC protein that Barton's team had previously identified. That mutation, when screened in cell lines, also produced overactive TLRs. "We were skeptical that just one copy of a gene would be sufficient to cause a disease," Rael said. "It wasn't until we put the patients' mutations into cell lines and saw that they led to very convincing TLR hyper-responsiveness that we realized they had the possibility of being sufficient to be disease-causing." Rael and Yano then repeated the screening work previously performed in the lab and confirmed that 32 distinct mutations in the UNC gene -- about one-third of the mutations tested -- increased the sensitivity of TLR7 to nucleic acids at least twofold. About another five mutants increased TLR7 sensitivity but to a lesser degree. Before these screens, only two mutations in the UNC protein had been linked to increased sensitivity of TLR7 in mice, though three additional human mutations were reported within the last two months. Barton is hopeful that by publishing the complete list of TLR hypersensitivity mutations, doctors can identify other lupus patients who could benefit from the anti-TLR drugs now in clinical trials. One drug, M5049, or Enpatoran, appears to work by latching onto two human receptors, TLR7 and TLR8, and preventing them from binding nucleic acids. Rael, Yano and other members of Barton's lab are investigating further how these unique UNC mutations affect the way a patient manifests the disease. They have recreated these patients' mutations in mice so that they can model human lupus. "With mouse models, you can start thinking about how, even though the mutations are in the same protein, the different mechanisms of TLR regulation break down, which immune cells get activated as a result, and how this can lead to differences in the symptoms patients suffer from," Rael said. The lab also is trying to understand how UNC tunes TLRs, which may be by regulating the number and arrangement of TLRs on immune cells. More TLRs may make a person more sensitive to the small number of self-nucleic acids circulating in the body. "UNC93B1 is important for getting the receptors to the place where they can function, but it also is important for regulating them when they get there," Barton said. "The protein is a very baroque way of trying to make decisions about whether the nucleic acid that you just bound to a TLR is from a virus or from one of your own cells." He hopes that physicians add this gene to the list of lupus-associated genes, "so if they see a mutation like these, even a heterozygous mutation, they will investigate further." Other senior authors of the paper are Bo Liu of the Chinese Academy of Sciences in Shanghai and Olivia Majer of the Max Planck Institute for Infection Biology in Berlin, Germany. The co-authors also include physicians from UC San Francisco, Stanford University, and hospitals in Missouri, North Carolina and Washington. The work was funded in part by the Lupus Research Institute, the Lupus Research Alliance and the National Institutes of Health (R01AI072429).

The transition from the esophagus to the stomach is a delicate region from a medical point of view, often associated with pathological disorders leading to cancer. An international research team has now gained new insights into this region. These pave the way for new prevention and treatment options. The meeting point of the stomach and esophagus, the so-called gastro-esophageal junction, is a region of the human body that is not well-suited to the modern lifestyle. Stress, alcohol, nicotine and severe obesity are often triggers for pathological changes to the mucosal membrane in this area, often resulting in esophageal cancer. An international research team has now gained new insights into the development of the cells, their communication with each other, and their regulation at the junction of the esophagus and stomach. With the help of specially developed mini-organs, so-called organoids, and with techniques that make it possible to track and pro cells, they have been able to follow the development of the gastro-esophageal junction from embryonic to adult stage in detail using animal experiments. New Insights into the Development of the Gastrointestinal Tract Their results reveal the complex communication at the cellular level and the specific pathways that these cells use to communicate. They provide new insights into the development of the gastro-esophageal junction and thus have significant implications for the understanding, prevention and treatment of gastrointestinal diseases. At the same time, they present new starting points for medical research and the development of new therapies. Cindrilla Chumduri is responsible for this study, which has now been published in the journal Nature Communications. Until recently, the infection and cancer biologist was a research group leader at the Department of Microbiology at Julius-Maximilians-Universität Würzburg (JMU); she is now an associate professor at Aarhus University (Denmark). Other participants came from Charité -- Universitätsmedizin and the Max Planck Institute for Infection Biology in Berlin. "This collaboration underlines the importance of different expertise to improve our understanding of the biology of the gastrointestinal tract," says Chumduri. She herself has many years of experience in research with organoids. Among other things, she has used mini-organs she developed to study how cells in the cervix degenerate and turn cancerous -- another region where different types of mucosal cells collide. Where Different Epithelia Meet "The squamous epithelia of the esophagus and the columnar epithelia of the stomach meet at the gastroesophageal junction," explains Dr. Naveen Kumar Nirchal, one of the first authors of the study. The area is known as a "hotspot for the development of metaplasia" -- the replacement of one type of cell by another. Barrett's esophagus, a precursor to esophageal cancer, often develops there, the number of cases of which has increased dramatically in the Western world over the past four decades. "Barrett's esophagus is characterized by the replacement of the resident squamous epithelium of the esophagus by other cell types that are not normally found in this tissue," says the scientist. However, it is still unclear why this region is so susceptible to this process. In order to better understand this transformation, it is therefore first necessary to decipher the normal development process in detail -- from embryo to mature adult. "This is the only way to determine the tissue changes that trigger the progression of the disease, explains Dr. Rajendra Kumar Gurumurthy, another researcher of the study. A Never-Before-Seen Insight into the Development of this Region This has now been achieved: By using a novel approach that combines organoid and mouse models with advanced single-cell transcriptome analyses over time and space, the research team has shed light on the complex developmental process of the gastroesophageal junction. "We were able to provide unprecedented insight into the development of this region from the embryonic stage to adulthood in mice and identify the intricate composition of the cells involved and how they develop," explains Pon Ganish Prakash, another scientist involved in the study. The work shows the sophisticated communication between different cell types within the gastroesophageal junction and the signaling pathways involved. "This understanding opens up new avenues for research into gastrointestinal diseases," says Cindrilla Chumduri. Above all, the precision of the single-cell analysis in their study opens new doors to understanding how pathological processes develop and to developing innovative treatments, the team writes in its study. The work will therefore be a "cornerstone for understanding the development of such diseases" and will significantly influence the approach to the early detection and treatment of diseases in this important part of the digestive system.

Researchers have discovered that Staphylococcus aureus can rapidly evolve within a single person's microbiome. They found that in people with eczema, S. aureus tends to evolve to a variant with a mutation in a specific gene that helps it grow faster on the skin. Human skin is home to millions of microbes. One of these microbes, Staphylococcus aureus, is an opportunistic pathogen that can invade patches of skin affected by eczema, also known as atopic dermatitis. In a new study, researchers at MIT and other institutions have discovered that this microbe can rapidly evolve within a single person's microbiome. They found that in people with eczema, S. aureus tends to evolve to a variant with a mutation in a specific gene that helps it grow faster on the skin. This study marks the first time that scientists have directly observed this kind of rapid evolution in a microbe associated with a complex skin disorder. The findings could also help researchers develop potential treatments that would soothe the symptoms of eczema by targeting variants of S. aureus that have this type of mutation and that tend to make eczema symptoms worse. "This is the first study to show that Staph aureus genotypes are changing on people with atopic dermatitis," says Tami Lieberman, an assistant professor of civil and environmental engineering and a member of MIT's Institute for Medical Engineering and Science. "To my knowledge, this is the most direct evidence of adaptive evolution in the skin microbiome." Lieberman and Maria Teresa García-Romero, a dermatologist and assistant professor at the National Institute of Pediatrics in Mexico, are the senior authors of the study, which appears today in Cell Host and Microbe. Felix Key, a former MIT postdoc who is now a group leader at the Max Planck Institute for Infection Biology, is the lead author of the paper. Bacterial adaptation It is estimated that between 30 and 60 percent of people carry S. aureus in their nostrils, where it is usually harmless. In people with eczema, which affects about 10 million children and 16 million adults in the United States, S. aureus often spreads to eczema patches and infects the skin. "When there's a break in the skin, Staph aureus can find a niche where it can grow and replicate," Lieberman says. "It's thought that the bacteria contribute to the pathology because they secrete toxins and recruit immune cells, and this immune reaction further damages the skin barrier." In this study, the researchers wanted to explore how S. aureus is able to adapt to living on the skin of eczema patients. "These microbes normally live in the nose, and we wanted to know whether when it finds itself on atopic dermatitis skin, does it need to change to live there? And can we learn something about how these bacteria are interacting with atopic dermatitis skin from watching its evolution?" Lieberman says. To answer those questions, the researchers recruited patients aged 5 to 15 who were being treated for moderate to severe eczema. They took samples of the microbes on their skin once a month for three months, and then again at nine months. Samples were taken from the backs of the knees and inside of the elbows (the most common sites affected by eczema), the forearms, which are usually not affected, and the nostrils. S. aureus cells from each sample site were cultured separately to create up to 10 colonies from each sample, and once large colonies formed, the researchers sequenced the cells' genomes. This yielded nearly 1,500 unique colonies, which enabled the researchers to observe the bacterial cells' evolution in much greater detail than has previously been possible. Using this technique, the researchers found that most patients maintained a single lineage of S. aureus -- that is, it was very uncommon for a new strain to come in from the environment or another person and replace the existing S. aureus strain. However, within each lineage, a great deal of mutation and evolution occurred during the nine months of the study. "Despite the stability at the lineage level, we see a lot of dynamics at the whole genome level, where new mutations are constantly arising in these bacteria and then spreading throughout the entire body," Lieberman says. Many of these mutations arose in a gene called capD, which encodes an enzyme necessary for synthesizing the capsular polysaccharide -- a coating that protects S. aureus from recognition by immune cells. In two out of six deeply sampled patients, cells with capD mutations took over the entire S. aureus skin microbiome population, the researchers found. Other patients were colonized with strains initially lacking a functional copy of the capD, for a total of 22 percent of patients lacking capDat the end of the study. In one patient, four different mutations of capD arose independently in different S. aureus samples, before one of those variants became dominant and spread over the entire microbiome. Targeted treatment In tests of bacterial cells growing in a lab dish, the researchers showed that mutations to capD allowed S. aureus to grow faster than S. aureus strains with a normal capD gene. Synthesizing the capsular polysaccharide requires a lot of energy, so when cells don't have to make it, they have more fuel to power their own growth. The researchers also hypothesize that loss of the capsule may allow the microbes to stick to the skin better because proteins that allow them to adhere to the skin are more exposed. The researchers also analyzed nearly 300 publicly available genomes of bacteria isolated from people with and without eczema, and found that people with eczema were much more likely to have S. aureus variants that could not produce the capsular polysaccharide than people without eczema. Eczema is usually treated with moisturizers or topical steroids, and doctors may prescribe antibiotics if it appears that the skin is infected. The researchers hope that their findings could lead to the development of treatments that lessen eczema symptoms by targeting S. aureus variants with mutations in the capsular polysaccharide. "Our findings in this study provide clues as to how Staph aureus is evolving inside hosts and reveal some of the features that might help the bacteria to stay on the skin and generate disease versus being able to be swiped off," García-Romero says. "In the future, S. aureus variants with mutations in the capsular polysaccharide could be a relevant target for potential treatments." Lieberman's lab is now working on developing probiotics that could be used to target the capsule-negative S. aureus strains. Her lab is also studying whether S. aureus strains with capD mutations are more likely to spread to other members of an eczema patient's household. The research was funded by MISTI Global Seed Funds, the National Institutes of Health, the Burroughs Wellcome Fund, the Mexican Government Ministry of Taxes Program for Health Research and Technological Development, and a German Research Foundation fellowship.