ScientistSpotlight

Mady Hornig: The battle against

Introduction Chronic Fatigue Syndrome, known medically as myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), is a debilitating disease that by some estimates affects as many as 4.5 million individuals in the US alone. Symptoms, which include extreme fatigue, difficulty concentrating, headaches, and muscle pain, typically begin in the aftermath of a viral-type infection and often last for years. There are no lab tests for ME/CFS. The disorder remains a mysterious and sometimes controversial illness. It is not yet part of any medical school curriculum; physicians as well as the general public have long disagreed about whether it qualifies as a biological disease or a psychological disorder. As a consequence, people with ME/CFS often feel stigmatized.

A sharper understanding of ME/CFS has emerged. Researchers at the Center for Infection and Immunity (CII) at ’s Mailman School of Public Health recently published research findings that identify distinct immune changes in patients with ME/CFS. Showing distinct phases in the trajectory of the disease, this work provides perhaps the strongest support yet that chronic fatigue syndrome is a biological illness, not a psychological disorder. Mady Hornig, MD, Director of Translational Research at CII and an associate professor of Epidemiology at Columbia’s Mailman School, is the lead author of the research, published in Science Advances.

Affymetrix: Why is ME/CFS getting more attention these days?

Hornig: Renewed interest in studying ME/CFS began heating up around 2010 with a suggestion that one or more might be implicated in the disease. Even though we eventually ruled out the two suspected viruses, the momentum had begun and new initiatives were funded. One of the key groups is the Chronic Fatigue Initiative (CFI), formed in 2010 by the Hutchins Family Foundation.

“Technological advances have enabled us to discover many more immune molecules and their signaling pathways and to understand how essential these are to brain development and brain function. Improvements in the sensitivity and reliability of multiplexed immunoassays such as the ProcartaPlex® Immunoassay from Affymetrix.”

The CFI-funded work has several prongs, including recruitment of the cohort, establishment of a unique sample biobank, and creation of a database that links clinical data to biological samples in the biobank. These components of the CFI form a powerful resource for both ongoing and future research. CII, directed by W. Ian Lipkin, MD, was a logical fit with the goals of the CFI given our longstanding focus

Mady Hornig, MA, MD, is Director of Translational Research

in the Center for Infection and Associate Professor of Epidemiology at the Mailman School of Public Health at Columbia University. A physician-scientist, she is internationally recognized for her animal model work and clinical research in human cohorts on the role of microbial, metabolomic, immune, and toxicologic factors in brain disorders, including , , attention- deficit/hyperactivity disorder, obsessive-compulsive disorder, mood disorders, and myalgic encephalomyelitis/chronic fatigue syndrome. She was the lead author of a paper published in Science Advances in February 2015, describing distinct sets of immune biomarkers in blood across the disease trajectory of ME/ CFS, firmly supporting the concept that the disorder has a biological basis, as well as of a second paper defining immune signatures in the cerebrospinal fluid of ME/CFS patients that was published in Molecular Psychiatry in March 2015.

1 “Through immune profiling of blood samples from patients diagnosed with ME/CFS, we have detected distinct changes in these immune molecules, identifying patterns in the immune system that have important relationships with different phases of the disease.”

on pathogen discovery and immune and infectious connections to brain diseases. I have been working in this area for well over 20 years, studying disorders such as depression, bipolar disorder, and schizophrenia, as well as several brain disorders that first manifest in childhood and in which immune disturbances appear to play a role, including autism, attention-deficit/hyperactivity disorder (ADHD), and PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal infection), an acute autoimmune disorder that follows strep throat in some vulnerable children, manifesting as acute obsessive-compulsive disorder (OCD)-like features, , and eating problems.

Technological advances have enabled us to discover many more immune molecules and their signaling pathways and to understand how essential these are to brain development and brain function. Improvements in the sensitivity and reliability of multiplexed immunoassays such as the ProcartaPlex® Immunoassay from Affymetrix are accelerating our ability to discover new biological markers of disease based on samples that are easily accessible, such as peripheral blood. A few decades ago it would have taken more blood than could be drawn at one sitting to produce some of the information we can now acquire using even just a few drops of the blood collected into a standard blood tube. These new capabilities at the lab bench herald exciting new directions for research into brain disorders like ME/CFS, OCD, ADHD, depression, mild cognitive impairment, Alzheimer’s, and even autism.

Affymetrix: Can you tell us more about the relationship between ME/CFS and the immune system?

Hornig: Brain-immune interactions are at the heart of the disease processes we study. Disturbances among the many microbes that inhabit our intestinal tracts can disrupt the balance of the so-called metabolome. The metabolome comprises the bacterial and host metabolites present in blood and other biological samples, including amino acids, neurotransmitters, short chain fatty acids, and a host of other inflammatory and neuroactive molecules. Distortions in an individual’s metabolome can skew their immune molecules, some of which interact with brain receptors, including those located in key regions of the brain’s stress-response circuitry like the hypothalamic- pituitary-adrenal axis. Through immune profiling of blood samples from patients diagnosed with ME/CFS, we have detected distinct changes in these immune molecules, identifying patterns in the immune system that have important relationships with different phases of the disease. Different mechanisms appear to be active in different stages of the illness; where patients lie along their disease trajectory may help predict which treatments will be most effective for them.

The study we published in February 2015 used immunoassay testing methods to determine the levels of 51 immune biomarkers in blood plasma samples collected through two multicenter studies. These two studies represented a total of 298 ME/CFS patients and 348 healthy controls. In patients who had the disease three years or less, we found specific patterns that were not present in controls or in patients who had had the disease for more than three years. Patients at three years or less had increased amounts of many different cytokines; notably, even small increases in interferon gamma, an immune molecule linked to the fatigue that follows infection with viruses like Epstein-Barr , the cause of infectious mononucleosis, were more common in the early phases of illness. These discoveries are closely linked to the improved sensitivity of our immunoassays; even small increases in immune molecules such as interferon gamma could be detected.

A month after our first study came out, we published a second paper in the journal Molecular Psychiatry that reported our discovery of another unique pattern of immune molecules among people with ME/CFS, this time in their cerebrospinal fluid. The group of ME/CFS patients we examined in this study were largely further along in the course of their illness; not unexpectedly, the immune patterns we found in cerebrospinal fluid for these patients were quite similar to those we found in the plasma of long-duration ME/CFS patients. This discovery provides insights into the basis for cognitive dysfunction in ME/CFS—frequently described by patients as “brain fog”—as well as offering new hope for improvements in diagnosis and treatment. Our finding that the immune changes observed in the peripheral blood are also present in the central nervous system (as represented by cerebrospinal fluid) supports the concept that this illness is a systemic one, and may help to explain the presence of symptoms in both peripheral parts of the body and the brain.

Affymetrix: What do you make of your findings that there are different stages of the disease?

Hornig: ME/CFS patients often report first getting sick with illnesses that are time-limited, like infectious mononucleosis, with swollen glands in the head and neck, sore throat, and fever; however, unlike most patients with infectious mononucleosis, they never fully recover. Our research suggests that in these patients, the immune response to the initial infection-like illness may get stuck in “high gear,” with cytokine levels remaining very high for around three years. After the three-year mark, the levels of most of the cytokines we studied begin to drop, with the immune system beginning to show evidence of exhaustion. Rapid detection of these immune changes may both facilitate early diagnosis and open up unique opportunities for initiation of treatments that can dampen down the levels of cytokines that we found to be elevated in early-phase disease.

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Affymetrix: How has technology from Affymetrix contributed to your work?

Hornig: We’ve been working with eBioscience® unit of Affymetrix for about 10 years. For these recent studies, in which we used ProcartaPlex Immunoassay to analyze both blood and cerebrospinal fluid samples, we were impressed with the reliability of the assay as well as the diversity of the analytes we could include—specifically, 51 immune molecules. The early adoption of the magnetic bead format by Affymetrix helped immensely. We also value the very real connection that we have with respect to product development. We have developed customized products with their team; they have partnered with us to ensure that the solutions available to us match our research needs. This active engagement has helped us not only to troubleshoot technical issues, but has also accelerated our ability to establish a firm foundation for ongoing research into ME/CFS and other immune-mediated brain disorders.

Affymetrix: How did you decide which analytes to look for?

Hornig: We chose target molecules to represent the varied types of immune responses that occur in a wide range of infections as well as in disorders involving autoimmunity. We focused on infections and autoimmune conditions hypothesized to contribute to brain disorders as well as the kind of physical problems observed in the ME/CFS population. In earlier studies, we used ProcartaPlex Immunoassay and similar panels to distinguish between viral and bacterial signatures in individuals with respiratory tract infections. Because we are interested in many potential infectious triggers of and immune contributors to brain disorders, we sought broad representation of these immune molecules. The panel also includes immune molecules that play a critical role in neuroimmune communication, and that have been implicated in the pathogenesis of the diverse set of brain disorders that we study.

The panel is very flexible, making it possible to compare results across a wide range of illnesses. The ability of companies like Affymetrix to produce each individual assay type in large, quality-controlled lots has been critical. Much of our research is based in very large, longitudinal, population-based cohort studies; thus, to maintain inter-assay comparability on top of the usual quality control requirements, we must adhere to stringent criteria. Within a large project, assays may span years from beginning to end. Inter-lot comparisons are also facilitated by consistency in the monoclonal antibody pairs used to capture and detect immune molecules and other analytes. This provides key support for even longer-term studies that seek to examine how diseases change over time or to compare patterns found within the disease of interest with patterns present in specific comparator diseases.

“Our findings suggest that objective diagnostic tests for ME/CFS may be possible. We also believe that the patterns of immune disturbances we uncovered may spur the discovery of new, effective treatments that can be tailored to different stages of the disease.”

Affymetrix: What’s the next step in your work on ME/CFS?

Hornig: Our findings suggest that objective diagnostic tests for ME/CFS may be possible. We also believe that the patterns of immune disturbances we uncovered may spur the discovery of new, effective treatments that can be tailored to different stages of the disease. Additionally, they offer great comfort to ME/CFS patients who for years have been told that the disease is only “in their heads.” I get e-mails daily from people around the world asking how they can help our efforts or enroll in our studies. The new lines of research emerging from our group and other research groups around the world hold great promise for changing mainstream conceptions about this disabling disease and accelerating investigation into its biological underpinnings.

As part of the next step in our research, we have launched a longitudinal immune-profiling study in which we will collect multiple blood and stool samples over the course of a year. The framework is one in which we will query, multidimensionally, the gut microbiome- metabolome-immune-brain axis, in conjunction with extensive clinical characterization of patients by our esteemed medical partners. We anticipate that ME/CFS is a heterogeneous disorder with multiple triggers; thus, patients are likely to have variable clinical phenotypes. Their microbiota, metabolomes, and immune profiles are likely to differ across stages of the disease and in accordance with both the severity of illness and the presence of different disease features. We are also planning a parallel microbiome analysis of samples from the oral cavity, frequently the site in which the symptoms of ME/CFS first appear.

We have several other studies we hope to initiate, including an epigenetics study to look at the genes turned on and off after exposure to infection or in conjunction with altered immune states. By identifying the genes that are turned on and off at different phases of the illness, we may better understand the contribution of these genes to the immune and metabolomic signatures of these patients, enabling us to develop new hypotheses as to how these relate to the impaired physical and cognitive function of ME/CFS.

On a broader scale, we are gathering funding to establish an ME/CFS Center of Excellence, intended to engage a global consortium of clinician-scientists and other researchers and to fuel innovative research. We hope to incorporate core components focused on clinical

3 assessment, treatment trials, pathogen/microbiome discovery; studies employing /epigenetics, metabolomics, proteomics, and toxicology; as well as a critical medical education and training initiative. With the ME/CFS Center of Excellence, we intend to identify key therapeutic targets and to accelerate the process of matching patients to the safest, most effective treatment avenues. We even consider that it may be within the realm of possibility to prevent the development of ME/CFS in the first place. For example, if certain genetic variants or gut or oral microflora are associated with the development of ME/CFS after infectious mononucleosis, we might be able to stave off longer-term, post-infectious fatigue and clinical impairment by identifying vulnerable individuals when their viral infection first begins and rapidly implementing targeted, preventive measures. We are also dedicated to moving ME/CFS away from its current status as an orphan illness within the medical community; broad programs of intensive education for established physicians as well as for upcoming generations of doctors are critically needed. Our other goal is to create a strong educational program for patients, family, and the community that will combat the extensive misinformation that exists about the disorder and those afflicted by it.

Beyond ME/CFS, I believe that there will be great utility, as yet untapped, in applying immune profiling to achieve a better understanding of a whole host of brain disorders that may be initiated or exacerbated by environmental factors (infectious, dietary, xenobiotic/toxic, extreme psychosocial stressors) that then exert their influences on the innate or adaptive arms of the immune system and/or oxidative or nitrosative stress response machinery. These approaches may ultimately help us to understand the pathogenesis of disease among subsets of patients with depression (which is now thought to have an inflammatory subtype), bipolar disorder, schizophrenia, OCD, ADHD, mild cognitive deficit, Alzheimer’s disease, and autism.

“There will be great utility, as yet untapped, in applying immune profiling to achieve a better understanding of a whole host of brain disorders that may be initiated or exacerbated by environmental factors (infectious, dietary, xenobiotic/toxic, extreme psychosocial stressors).”

Reference: Hornig, M., et al., Distinct plasma immune signatures in ME/CFS are present early in the course of illness. Science Advances 1(1): e1400121 (2015).

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