The research was conducted primarily in the laboratory of Sarkis Mazmanian, Luis B. and Nelly Soux Professor of Microbiology and affiliated faculty member with the Tianqiao and Chrissy Chen Institute for Neuroscience at Caltech. A paper describing the study appears on February 14 in the journal Nature.

Decades of research have shown that the communities of bacteria that inhabit the intestines of animals (the microbiome) influence the immune system and metabolism; studies in the last few years have linked the microbiome to brain function and mood. People with certain neurological conditions have distinctly different gut bacteria communities. Further, studies in mice have shown that manipulating these communities can alter neurodevelopmental and neurodegenerative states, either ameliorating or exacerbating symptoms.

“It’s been really difficult to show causation between something that’s happening in the gut and the brain, rather than just associations between the disease states and the presence or absence of certain microbes,” says Brittany Needham, first author of the new study and a postdoctoral scholar in the Mazmanian lab. “We were interested in trying to understand the molecular messages that are going between the gut and the brain, and how these signals may lead to changes in behavior.”

This study focused on a bacterial metabolite (a by-product of microbes) called 4-ethylphenyl sulfate, or 4EPS. Initially produced by microbes in the intestines, 4EPS is then absorbed into the bloodstream and circulates throughout the body in both humans and mice. In 2013, the Mazmanian lab showed that this particular molecule was present in higher levels in mice with altered neurological development, specifically, a mouse model of autism and schizophrenia. Though other aspects of the altered microbiome differed from the healthy microbiome, 4EPS levels were by far the most different. Additionally, in a screen of human blood samples from 231 individuals, 4EPS levels were about seven times higher in children on the autism spectrum than in neurotypical children.

In this work, the team focused on the effects of 4EPS on mouse models of anxiety. While anxiety disorders in humans are complex, animal models provide a way to study the precise changes in the brain and body that lead to anxious behaviors. “Anxiety” in mice is measured by their willingness to explore or hide in a new space as well as the time spent in a risky environment. Bold mice will explore a new space, sniffing around, but anxious mice will hide, as if facing a predator, instead of exploring.

The study compared two groups of laboratory mice: one group was colonized with a pair of bacteria that were genetically engineered to produce 4EPS; the control group of mice were colonized with bacteria that were identical except lacked the ability to produce 4EPS. Then, the mice were introduced to a new arena, and researchers measured each mouse’s behavior.

The mice with 4EPS spent much less time exploring the area and more time hiding as compared to their non-4EPS counterparts, indicating higher levels of anxiety. Brain scans of the 4EPS mice also showed that some of the brain regions associated with fear and anxiety were more activated in addition to overall changes in brain activity and functional connectivity.

Looking closer at brain cells within these altered regions, the team found that particular cells called oligodendrocytes were altered. These cells are important in part because they produce a protein called myelin, which acts as a protective coating around neurons and nerve fibers called axons, like insulation around an electrical wire. The team found that in the presence of 4EPS, oligodendrocytes are less mature and consequently produce less myelin, leading to thinner insulation around axons.

However, when the 4EPS mice were treated with a drug known to increase myelin production in oligodendrocytes, the drug was able to overpower the negative effects of 4EPS — the mice regained normal myelin production, and the anxious behaviors were reduced.

In a related study appearing simultaneously in the journal Nature Medicine, Needham showed that treating mice with an oral drug to soak up and remove 4EPS from their systems led to reductions in anxious behaviors. This result enabled a small clinical study that also gave humans the drug in an open-label trial (no placebo or control group). Sequestering 4EPS in the human gut led to reduced levels of 4EPS in the blood and urine, and many of the 26 study participants displayed overall decreased levels of anxiety.

“It’s an exciting proof-of-concept finding that a specific microbial metabolite alters the activity of brain cells and complex behaviors in mice, but how this is happening remains unknown,” says Mazmanian. “The basic framework for brain function includes integration of sensory and molecular cues from the periphery and even the environment. What we show here is similar in principle but with the discovery that the neuroactive molecule is of microbial origin. I believe this work has implications for human anxiety or other mood conditions.”

The next steps for the work are to examine the mechanisms through which 4EPS affects oligodendrocytes — which proteins it may be interacting with, whether 4EPS is affecting changes directly in the brain, or if it is affecting another part of the body and those effects are making their way up to the brain. Also, it will be critical to show that the human data have an effect in a well-powered and controlled clinical trial, which is now underway.

In addition to Needham and Mazmanian, Caltech co-authors are former research technician Mark Adame; research technician Joseph Boktor; former postdoctoral scholar Wei-Li Wu (now of National Cheng Kung University in Taiwan); postdoctoral scholar Claire Rabut; EM scientist Mark Ladinsky; lecturer in chemistry Son-Jong Hwang; graduate student Jessica Griffiths; Pamela Bjorkman, David Baltimore Professor of Biology and Bioengineering, Merkin Institute Professor, and executive officer for biology and biological engineering; and Mikhail Shapiro, professor of chemical engineering and Howard Hughes Medical Institute Investigator.

Additional co-authors are Masanori Funabashi of Stanford University and Daiichi Sankyo RD Novare Co.; Zhuo Wang, Yumei Guo, and Daniel Holschneider of USC; Jillian Haney and Daniel Geschwind of UCLA; Qiyun Zhu of UC San Diego and Arizona State University; Rob Knight of UC San Diego; and Michael Fischbach of Stanford University.

Funding was provided by the Center for Environmental Microbial Interactions, the National Science Foundation, the Human Frontier Science Program, the National Institutes of Health, the Ministry of Science and Technology in Taiwan, the Heritage Medical Research Institute, and Lynda and Blaine Fetter. Sarkis Mazmanian is co-founder of Axial Therapeutics, which conducted the clinical trial.

In 2015, the European Network for the Study of Cholangiocarcinoma (ENSCCA) was founded with the aim of setting up a pan-European-wide interdisciplinary cooperative network to address the CCA problem at basic, translational, and clinical levels. Its first initiative was the creation of the Clinical ENSCCA Registry in 2016 to describe in detail the natural course of CCA in Europe, determine potential risk factors, improve the current classification system, investigate early and non-invasive accurate tumor biomarkers for diagnosis and to estimate prognosis, compare the effectiveness of therapies, and select patients for clinical trials.

“CCA is a rare cancer, but its incidence is increasing worldwide, and we need to stop treating it as an orphan disease,” explained lead investigator Dr. Jesus M. Banales, Professor Ikerbasque at Biodonostia, University of Navarra and CIBERehd, Spain. “Although CCA constitutes a major challenge for clinicians, scientists, national health systems, and society, there is a lack of coordinated multidisciplinary pan-European studies. We therefore used the ENSCCA registry to gather vital information.”

In the most comprehensive international observational study to date, investigators evaluated the natural course of histologically proven CCA in more than 2,200 patients who were diagnosed between 2010-2019 and whose details were included in the ENSCCA registry. The data were contributed by 26 referral hospitals in 11 European countries. At diagnosis, 42.2% of patients had local disease, 29.4% had locally advanced disease, and 28.4% had metastatic disease. They compared general and tumor-type specific features at diagnosis, risk factors, biomarker accuracy, as well as the similarities and differences between the three CCA subtypes based on the latest World Health Organization classification according to their anatomical origin (intrahepatic, perihilar, or distal), and a comparison of patient management and outcomes.

The results suggest that the location of CCA tumors along the biliary tree is associated with different risk factors and tumor features. Diagnoses need invasive confirmations by biopsy or cytology, as blood tests for tumor biomarkers show low sensitivity in early stages. Surgical resection remains the only potentially curative therapy, although cure is unlikely with involvement of resection margins or lymph node invasion. Investigators noted that chemotherapy increased patients’ life expectancy compared to patients receiving best supportive care. They also noted that deterioration of the patient’s performance status (ECOG), the presence of metastases, and increased levels of the tumor marker CA19-9 independently affected the outcome.

A comparative analysis of risk factors of the three different locations of CCA tumors revealed some potential lifestyle-related risk factors such as alcohol excess, smoking, overweight/obesity, diabetes, etc. These are highly prevalent in Europe and could predispose at-risk individuals to the development of CCA.

“In conclusion, our study provides a comprehensive analysis of diagnostic, prognostic, and therapeutic aspects of the complex CCA landscape,” commented Dr. Banales. “The results showed that CCA is mostly diagnosed at an advanced stage, a significant proportion of patients fail to receive any cancer-specific therapy, and the prognosis is dismal. Therapeutic options are limited. Accordingly, awareness campaigns and education programs aimed to prevent lifestyle-related risk factors and new techniques for early detection of CCA in high-risk populations are urgently needed in order to decrease cancer-related mortality. Our findings represent valuable knowledge for future comparisons with new targeted therapies and the design of next-generation personalized clinical trials.”

CCA is a highly heterogenous and aggressive malignancy responsible for around 2% of all cancer-related deaths globally each year. It is often diagnosed when it is already advanced, making it difficult to treat successfully. Currently it represents approximately 15% of all primary liver cancers and 3% of all gastrointestinal cancers worldwide.

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A study in the journal Science Immunology now shows how two subsets of one type of immune cell — the CD8 T cell — develop to provide either short-term or long-term immune protection. The study focuses on one factor that guides that developmental bifurcation — interleukin-2, or IL-2.

Understanding details of how naïve immune cells develop into effective infection-fighters or long-lived memory cells is important because it can help us understand which constituents of a complex response are necessary to provide protection against infections or cancers. It can also aid understanding of immune system dysfunction, or it can improve immunotherapy for cancer. Dysfunctions include autoimmune diseases, where the immune system attacks the body’s own cells, and exhaustion, where the immune system loses its ability to fight off continuing infection or the ability to destroy a cancer.

Study senior author Allan Zajac, Ph.D., and co-first authors Shannon M. Kahan, Ph.D., and Rakesh K. Bakshi, Ph.D., all of the University of Alabama at Birmingham Department of Microbiology, used mouse models to follow the traits and fates of the two subsets of CD8 T cells, starting from the peak of their effector phase, eight days after an acute viral infection.

CD8 T cells are a type of white blood cell that has the ability to develop into effector T cells — also known as cytotoxic T cells or killer T cells. These cells can destroy cancer cells or cells infected with viruses or bacteria. IL-2 is a cytokine, one of a large family of small signaling proteins that are released by cells to send messages to other cells, or act as an autocrine signal to the same cell that is making the cytokine. Cells receive the signal at the cell surface, where the cytokine binds to a receptor. The receptor transduces the signal to the cell interior, leading to changes in cell gene expression and phenotype.

Zajac and UAB colleagues were able to isolate and purify CD8 T cells from mice at the effector phase peak. Using fluorescence-activated cell sorting after cell stimulation with a viral antigen, they identified two subsets of this group — a minority of the CD8 T cells that produced IL-2 and a majority that did not produce IL-2.

The two subsets turn out to be strikingly different in the short term, in terms of developmental transitions that lead to fate decisions. However, they then become more alike months later.

The cells that produced IL-2 developed like immune memory cells. They attained stem-like memory traits, resisted exhaustion and preferentially conferred protective immunity upon a secondary viral challenge. Counterintuitively, they did not respond to their own IL-2, which acts inside the cell through a complex called STAT5, or signal transducer and activator of transcription. Even though the IL-2-producing CD8 T cells had normal amounts of IL-2 receptor proteins on the cell surface, the level of phosphorylated-STAT5 inside the cell — which is the activated form of STAT5 — was greatly diminished. The researchers hypothesized several mechanisms acting inside the cell possibly acting to attenuate the ability to receive IL-2-dependent STAT5 signals, but the answer is not yet known.

In contrast to the IL-2-producing cells, the non-IL-2-producing effector cells did respond to IL-2 signals, and they then gained effector traits at the expense of memory formation.

The UAB researchers did RNA sequencing of the cell subsets during the effector phase, nine days after infection, and much later, during the memory phase, about 10 months after infection. The transcriptional profiles of the IL-2-producing and the IL-2-non-producing effector populations were unique and distinguishable from their memory counterparts. However, by the memory time point, the two populations were more related to each other in gene expression. Thus, despite having distinct properties during the effector phase, the IL-2-producing and ?nonproducing CD8 T cells appeared to converge as memory matured to form populations with equal recall abilities to respond to a second viral infection or chronic viral challenge.

“This bifurcation between the ability of IL-2-producing and -nonproducing CD8 T cells to respond to IL-2 couples functional competency with fate decisions, and it implicates IL-2 as a critical differentiation factor,” Zajac said. Zajac notes that the salient findings of this study have not yet been confirmed for humans, and they are restricted to analyzing CD8 T cell responses to a single pathogen.

Co-authors with Zajac, Kahan and Bakshi in the study, “Intrinsic IL-2 production by effector CD8 T cells affects IL-2 signaling and promotes fate decisions, stemness, and protection,” are Jennifer T. Ingram, R. Curtis Hendrickson and Elliot J. Lefkowitz, UAB Department of Microbiology; David K. Crossman, UAB Department of Genetics; Laurie E. Harrington, UAB Department of Cell, Developmental and Integrative Biology; and Casey T. Weaver, UAB Department of Pathology. These UAB departments are all part of the Marnix E. Heersink School of Medicine.

Support came from National Institutes of Health grants AI049360, AI156290 and TR003096; and the American Cancer Society award PF-16-150-01-LIB. Zajac is a professor in the Department of Microbiology.