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Special Report on Inflammatory Diseases/Autoimmune Research: The undiscovered country
Tires crunch against loose gravel as the car pulls to a stop, the highway a distant memory. Dust eddies around the door as it swings open, and you crawl out from behind the wheel.
Silently, your feet tread toward a metal railing, and as the horizon presents itself, you find yourself staring into a canyon that stretches for miles in all directions.
Rain-washed gullies, pillars of stone, loose boulders and multicolored strata of eons past assault the eye, the brain struggling to take it all in. For all intents and purposes, you are witness to an undiscovered country, or at least an unpopulated one.
There are no signs of order or infrastructure. No roads to crisscross the rocky channels. No homes dot the highlands. And even the rivers, streams and wadis seem chaotic and illogical.
A quick scan of your tour pamphlet, however, tells you the vista that spreads below you is filled with explorers. Down in the deepest shadows and narrowest crevices, men and women are making intriguing discoveries.
But the time for organization and planning is not yet ripe. There is still work to be done until we can begin to make sense of it all, to see the patterns and turn them into a plan to move forward.
Welcome to the landscape of autoimmune disease and the valley of epigenomics.
Complications on complexity
Despite the best efforts of genomics initiatives to link all human disease to individual or collections of genetic markers, autoimmune diseases remain somewhat aloof from simple characterization, and efforts to treat the diseases are often complicated by a limited understanding of their underlying pathology. In many cases, the best for which clinicians and patients can hope is the amelioration of symptoms (see the sidebar below, Autoimmune also-rans).
Although the dozens of autoimmune conditions can be quite distinct from each other, they are all linked by the erroneous attack of a patient’s immune system against self-antigens, triggering organ-focused (e.g., type-1 diabetes) or widespread (e.g., systemic lupus erythematosus, or SLE) tissue damage. Collectively, they represent a medical burden for upward of five percent of the global population.
As hinted above, autoimmune conditions often display discordance in studies of monozygotic twins, indicating that non-genetic factors play a significant role in the susceptibility to and trigger of disease. This idea is supported by genome-wide association studies (GWAS), which have identified very few genetic changes that correlate with disease.
“Complexity is probably the understatement here,” says Gregory Dennis, vice president of global product development and therapeutic area head for immunology, rheumatology and immuno-inflammation at PPD. “We think about multiple mechanisms, multiple factors that play a role in their development.”
He points to not only nutritional factors, but also environmental factors that influence whether a person will experience any of these conditions and how severe the condition will be.
“Within each of the disease categories, there is a tremendous amount of heterogeneity, as well as variability in the activity of the disease over time,” he continues. “And all of those things together make the approach to understanding them extremely complex.”
This intricate dance of internal and external factors in the development and progression of autoimmune disease has led several research groups to consider the biological space between the genetic and external worlds: the realm of epigenomics.
Briefly, epigenomics includes processes that influence gene expression and protein translation via three often interrelated mechanisms:
In fact, the evidence for epigenomic influences in autoimmune disease goes back decades.
Clinicians noted the development of diseases like SLE in patients receiving treatment for other conditions. Two such drugs are hydralazine and procainamide, which are known to inhibit DNA methylation and induce self-reactivity. Likewise, it is not unusual to read case studies of patients being treated for one autoimmune disorder (e.g., rheumatoid arthritis, or RA) developing a second autoimmune condition (e.g., uveitis) potentially as a result.
DNA methylation and chromatin condensation—which are critical processes in X chromosome inactivation—may also help to explain some of the gender bias seen in many autoimmune diseases, where women are more susceptible to disease. In the cases of RA and multiple sclerosis (MS), women are two to three times as likely to develop disease, while in SLE and Sjögren’s syndrome, the ratio is 9-to-1.
Several research groups have demonstrated the significant role of epigenetic modifications in the differentiation and maturation of cell types, including those of the immune system. Such is the case for Eric Verdin and colleagues at the University of California, San Francisco’s Gladstone Institute, who are examining the activity of two families of HDACs in a mouse model of MS.
In April, Verdin’s group described the results of a study of Sirtuin 1 (SIRT 1) in the regulation of a transcription factor (RORγt) involved in the maturation of T-helper cells, specifically Th17.
As noted in the Journal of Experimental Medicine, when the researchers either deleted the SIRT 1 gene or used pharmacologic SIRT 1 inhibitors, Th17 differentiation was suppressed. When the compound was given to mice with experimental autoimmune encephalomyelitis, disease progression was strongly blocked.
“In light of the significant effect the treatment had on inflammation, the implications of these results will likely extend beyond multiple sclerosis to other types of autoimmune disorders,” Verdin said in announcing the findings. “We are particularly interested in testing this in type-1 diabetes given the similar pathways involved, and we are already seeing very promising results in preliminary experiments.”
In a recent review in Immunology and Cell Biology, Bhawna Gupta of India’s KIIT University and David Hawkins of University of Washington offered their perspectives on the role of epigenetics in cell differentiation.
“Epigenetic modifications contributing to the proper acquisition of cell fates of the immune system are well illustrated,” they suggested. “miRNA and histone deacetylase (HDAC)-mediated regulation of CD8+ T-killer cells and dendritic cells, and broader epigenetic regulation of T-helper cell lineage establishments, further document the role of epigenetic mechanisms in manipulating immune systems and a concomitant progression towards autoimmunity.”
“Though in most cases of autoimmune diseases the precise epigenetic mechanisms involved remain to be resolved,” they added, “improving our understanding of the role that epigenetic modifications play in the development of autoimmunity is likely to increase the prospects for controlling or preventing autoimmune diseases.”
A first step in that understanding comes in simply cataloguing the epigenomic changes that correlate with disease.
In 2013, Devin Absher and colleagues at the HudsonAlpha Institute for Biotechnology and the University of Alabama at Birmingham conducted what was then described as the most comprehensive analysis of SLE epigenetics, examining the genome-wide methylation patterns of more than 460,000 CpGs covering more than 95 percent of the genes in CD4+ T cells, CD19+ B-cells and CD14+ monocytes. The analyses were conducted on cells from both SLE patients and control subjects (see sidebar below, How’d they do that?).
Publishing their findings in PLoS Genetics, the researchers identified hundreds of CpGs in all of the cell types that showed significantly different methylation patterns between SLE and control samples, and that almost all of these changes involved hypomethylation of SLE DNA. They also noted that the majority of these CpG pattern alterations clustered around 60 genes, and most of these genes were involved in interferon signaling.
This result was not particularly surprising, as interferon-regulated genes tend to be overexpressed in SLE patients. What was interesting, however, was that whereas gene overexpression tended to occur during SLE flares, there was no significant difference in the methylation patterns between patients experiencing flares or in a quiescent phase.
“The persistence of the hypomethylation in patients during quiescence is important,” the authors wrote, “as it may help explain the chronic nature of the disease and the potential for recurrent flares in SLE patients.”
They also raised the point that as patients experiencing flares would typically increase their medication levels, the lack of a change in methylation patterns suggested that the treatments had little or no effect on disease epigenetics.
“Nonetheless, studies that examine the epigenetic impact of anti-inflammatories, as well as the epigenetic states that modulate their efficacy, may have an impact on the clinical management of SLE,” they added.
Gupta and Hawkins caution over-interpretation of results like these, however, by raising the adage of correlation and causation.
“Another confounding factor regarding causation is distinguishing causative epi-mutation from epigenetic changes induced from the cell being in a diseased state,” they added to their discussion.
In 2013, Xiaoxia Zuo and colleagues at Hunan, China’s Central South University reviewed the microRNA landscape of scleroderma, a disease characterized by vascular dysfunction and the build-up of extracellular matrix (ECM), leading to fibrosis and failure of affected organs such as lung, kidney and heart.
As they wrote in Experimental and Molecular Medicine, microarray and real-time PCR analysis had already pointed to upward of 40 microRNAs that were linked to fibrosis in various diseases and organ systems. And many of these microRNAs were regulated by transforming growth factor-ß, already considered an important molecule in scleroderma pathogenesis.
Perhaps the best-characterized of the collection is miR-29, which is known to directly repress expression of several collagens and therefore is a direct regulator of ECM synthesis. Studies from several labs, including their own, demonstrated that miR-29 levels were reduced in skin samples from systemic sclerosis (SSc) patients, and that supplementation of miR-29 led to decreased collagen expression in SSc fibroblasts.
Similarly, a handful of other microRNAs showed altered expression levels in SSc samples, not only of their own expression but that of downstream genes involved in various aspects of fibrosis pathology. This led the researchers to speculate on the use of the microRNAs as potential biomarkers for the disease, offering not just diagnostic advantages but possibly also indications of therapeutic targets.
Similar studies have identified dozens, if not hundreds, of microRNAs involved in self-reactive immune pathways and the pathogenesis of other autoimmune conditions including SLE, type-1 diabetes, RA and MS. But from the perspective of biomarkers, this very wealth of information may provide its own challenges.
Bringing order to chaos
“There are multiple factors—including nutrition and environmental factors—that have the ability to influence epigenetics by impacting the expression of genes,” PPD’s Dennis reiterates, suggesting this adds to the variability of the diseases and their responses to therapy. “And that further complicates our ability to identify biomarkers that have sufficient sensitivity and specificity to be meaningful to us.”
The conversation hearkens back to a biomarker discussion with Igor Jurisica of Princess Margaret Cancer Centre (formerly Princess Margaret Hospital), who suggested that a single molecular marker may not be sufficient. Rather, panels of a few to several biomarkers may be necessary to produce meaningful results, and not just biomarkers of one molecular type.
“When we started, we were looking for a better algorithm for identifying these prognostic and predictive signatures,” he said, explaining his group’s efforts to use bioinformatics approaches to decipher biomolecular data (see Signs of Intelligent Biomarkers, February 2015 DDNews). “But then we realized that in order to solve that problem, we had to have a much better handle on all these networks: which microRNAs control what genes, what proteins interact with what other proteins, what are the important signalling cascades and so on.”
Moving away from diagnostic and prognostic markers and into the realm of therapeutics, the sequence specificity of microRNAs make them attractive targets for clinical intervention, at least in theory. And as indicated above in the scleroderma discussion, some work has been done on the regulation of microRNA levels to alter cellular pathology. But much of this work is still in its early stages and despite the advances in antisense and RNAi technologies, will likely be years or decades from reaching a clinical phase.
Of more immediate interest, however, are the enzymes involved in DNA methylation and histone modification.
“Reversibility of epigenetic modifications makes enzymes such as [DNA methyltransferases (DNMTs)], HDACs and histone methyltransferases (HMTs) attractive drug targets,” offered Gupta and Hawkins. “Small-molecule inhibitors of histone modifiers and DNA methyltransferases are becoming increasingly available.”
With their lengthy history in cancer research (more on that below), HDAC inhibitors (HDACis) have garnered a lot of attention in the treatment of autoimmune disorders. As described above in the Verdin study, HDACis have demonstrated protective properties in EAE, the rodent model of MS, and also in murine models of SLE, RA and inflammatory bowel disease.
HDACs represent a large family of enzymes, however, with each isoform involved in its own specific pathways. Efforts to inhibit the entire family of proteins and particularly under conditions of chronic treatment with pan-HDACis come at the cost of significant adverse events ranging from diarrhea and vomiting to thrombocytopenia and cardiac issues.
“For this reason, a more targeted approach is warranted if HDAC inhibitors are to be used in the treatment of autoimmune disease,” argued Nicole Regna and Christopher Reilly of Edward Via College of Osteopathic Medicine in a recent review.
“The identification of aberrant HDAC specific isoforms to each autoimmune disease may be important in reducing toxicity,” they concluded in Clinical & Cellular Immunology. “Isoform-selective HDAC inhibition has the potential to correct aberrant immune regulation by altering the function of components of the inflammatory cascades without the deleterious side effects associated with traditional pan-HDAC inhibitors.”
As with any therapeutic approach, however, it is likely that a combination of therapies will be required to not only quell the underlying disease, but also to reverse the damage. And this may be particularly true for many autoimmune conditions, which may not demonstrate any significant symptoms until well into disease progression.
With this in mind, Maja Jagodic and colleagues at the Karolinska Institutet and elsewhere looked at the role of combination therapy in EAE.
“MS therapies act via immunosuppressive or immunomodulatory mechanisms and are effective only in the relapsing-remitting stage,” they wrote. “The permanent neuronal loss that starts early and characterizes the progressive stage of MS remains untreatable.”
Although Verdin and others showed HDACis offer protection in EAE, they do nothing to promote remyelination of the nerve fibers. Thus, therapeutic protocols with HDACis require chronic daily treatment, which unfortunately also brings a build-up of side effects.
According to Jagodic, however, thyroid hormone treatment correlates with increased expression of markers for oligodendrocyte precursor cells (OPCs) and enhanced remyelination. Thus, the researchers decided to investigate if the combination of HDACi valproic acid and thyroid hormone would not only slow EAE progression but also reverse demyelination.
As they described in Neurobiology of Disease, a brief but acute treatment plan not only induced significant improvements in clinical symptoms such as locomotor activities, but also showed significant improvements in nerve myelination, particularly in the brain.
“Our results suggest that, as we observed in embryonic neural stem cells [(NSCs)] and OPCs, the HDACi and thyroid hormone can overturn a repressive transcriptional checkpoint present in NSCs/OPCs in the EAE brain that prevents their differentiation and myelination,” the authors concluded.
“This advocates future efforts to develop novel treatments that would combine immunomodulatory and remyelinating properties of HDACis and thyroid hormone to treat MS.”
But as is often the case in drug development, what works in animal models does not always work in the clinical setting.
As University of Michigan’s Theresa Mau and Raymond Yung recently noted in a Frontiers in Genetics review, the growing catalogue of preclinical success has yet to significantly move past the exploratory stage.
“Although much is now known about the epigenetic mechanisms of lupus and epigenetics-based therapies have entered into clinical practice,” they wrote, “it is disappointing that much of the basic research in the field has not resulted in clinical trials in lupus or other autoimmune diseases.”
A quick survey of ClinicalTrials.gov shows just how true this statement is.
“I’m not surprised at all that you found few investigations of microRNAs or other genetic interventions in the autoimmune diseases,” offers Dennis, who suggests that despite the breadth of research at the preclinical level, the concept of applying epigenetic interventions at the clinical level is simply too novel at this stage and is nowhere near as mature as it is with something like cancer.
Looking to cancer’s lead
“There seems to be a little uncertainty in terms of the direction of the research of these particular autoimmune conditions,” Dennis says. “A lot of what we get in terms of directing our research comes from the area of cancer.”
Only in about the last three to five years, he suggests, have researchers started to consider the role of impacting the immune system on those other diseases. (For more on immunotherapeutic approaches to cancer, see Body, heal thyself, June 2015 DDNews.)
“When we identify agents or mechanisms that are particularly effective and impact the immune system in the treatment of cancers, we’ll very often explore their utilization in the treatment of autoimmune diseases,” Dennis continues. “And that’s where we’re going to be able to leapfrog or advance the field in autoimmunity: by paying attention to what’s happening in cancer, taking that into consideration and then move forward in utilization of that knowledge in the conduct of autoimmune disease clinical trials.”
Thus, it appears that much of what happens in the still largely undiscovered country of autoimmune disease will take its cues from the wildly populated and infrastructure-laden land of cancer the next valley over.
Most advances in the understanding and treatment of autoimmune disease have occurred in what could be termed the Big Six conditions: rheumatoid arthritis, psoriasis, ulcerative colitis, Crohn’s disease, ankylosing spondylitis and psoriatic arthritis. To a large extent, suggests Gregory Dennis of PPD, this focus is the result of patient population sizes.
“While we have not had a specific sequence of autoimmune diseases that we explore,” he says, “I think our approach has always been to explore those that have the greatest numbers. That’s why you see rheumatoid arthritis being studied first.”
But whereas the Big Six account for the majority of patients, there are dozens of other autoimmune conditions that impact thousands of people across the United States. The following are just a few:
Addison’s disease: Insufficient production of adrenal hormones (e.g., sex hormones, glucocorticoids) leads to chronic vomiting and diarrhea, extreme weakness and fatigue, weight loss and mouth lesions. Hormone replacement therapy addresses the symptoms but not the disease.
Graves’ disease: Hyperactivity of the thyroid gland can lead to anxiety and moodiness, vision trouble, fatigue, weakness and sleeplessness and rapid or irregular heartbeat. Antithyroid medications may be given, although surgery or radiotherapy is also used in conjunction with hormone supplementation.
Hashimoto’s thyroiditis: In many ways the opposite of Graves’ disease, chronic inflammation of the thyroid leads to hypothyroidism and symptoms of fatigue, hair loss, intolerance to cold and constipation. Hormone replacement therapy may be given to treat the symptoms.
Multiple sclerosis: Destruction of the protective myelin sheath around nerve cells leads to progressive neurological failure. This can lead to muscle spasms and difficulty moving, loss of coordination, weakness and tremors, vision issues and focus issues.
Sjögren syndrome: Autoimmune attack of the tear and salivary glands leads to dry eyes and mouth, but can also produce symptoms of fatigue, fever and joint swelling. Symptomatic relief is often in the form of topical lubricants, but may also include disease-modifying drugs commonly used for other conditions such as rheumatoid arthritis (e.g., anti-TNFs).
Systemic lupus erythematosus: Broader destruction of tissues of the skin, joints and organs, SLE’s symptoms can be far-reaching and highly variable but include fatigue and general discomfort, skin rash and mouth sores and fever. Organ involvement can lead to more severe symptoms. Milder forms of the disease are treated with NSAIDs and steroids, but more severe SLE may require immunosuppressants.
Type 1 diabetes: Autoimmune destruction of pancreatic beta cells leads to hyperglycemia and symptoms of numbness and fatigue, vision difficulties and in more severe cases, rapid breathing and gastrointestinal issues. Insulin treatment and lifestyle changes are often prescribed both to control blood sugar and limit the risks of complications such as diabetic nerve and blood vessel damage.
(More information about autoimmune conditions can be found at the American Autoimmune Related Diseases Association at www.aarda.org)
How’d they do that?
To better understand the role of DNA methylation in SLE, Devin Absher and colleagues at the HudsonAlpha Institute for Biotechnology and the University of Alabama at Birmingham undertook genome-wide DNA methylation analysis across three immune cell types—T cells, B cells and monocytes—from healthy controls and SLE patients. They described their efforts in PLoS Genetics.
Collecting peripheral blood samples from each patient, the researchers used immunomagnetic beads to isolate CD4+ T cells, CD19+ B cells and CD14+ monocytes, and flow cytometry when looking at cell subpopulations within samples. They then extracted DNA from these cells types and examined the CpG methylation patterns by treating the DNA with sodium bisulfite prior to amplification, hybridization and imaging.
The results suggested not only significant differences between the healthy and SLE blood samples but also that almost all of the pattern differences involved hypomethylation of DNA from SLE patients.
Applying gene ontology (GO) and pathway analysis, the researchers tried to identify whether there were overlapping patterns between the cell types and if the hypomethylated stretches clustered around any specific genes. They determined that the most prominent hypomethylation patterns tended to occur near genes involved in interferon signalling, consistent with clinical data.
The analysis also showed that these hypomethylation patterns remained steady whether the patient was experiencing an SLE flare or was in the quiescent phase, quite in contrast with measured interferon responses in patients, which tend to spike during flares. The researchers suggested that this continual hypomethylation may explain the chronic nature of SLE.
“Future studies of DNA methylation in early progenitor populations from SLE patients will be needed to establish the responsible cells, and to define the events that might induce this epigenetic state in progenitor cells,” the authors wrote.