PARMITA MISHRA
Immunofluorescence image of mTECs, with Aire genes shown in red. At the University of São Paulo (USP) in Brazil, CRISPR/Cas9 was used to block Aire in murine mTECs and to study the effect of loss of this gene’s function. CRISPR/Cas9 opens up new questions and answers about epigenetic regulation in autoimmune diseases, and therefore a clearer understanding of immune response.
The Role of Epigenetics in Autoimmune Diseases
Autoimmune diseases (ADs) are a group of complex diseases originating from a loss of immunological tolerance to self-antigens (“self tolerance”). This manifests in the demonstration of “autoimmune phenomena” such as autoantibodies and/or autoreactive lymphocytes. While the mechanisms of autoimmune disease are varied and well-understood, the cause for the initial loss in self-tolerance is undiscovered. It is traditionally believed that this cause is the subset of three factors: genetic predisposition (raising susceptibility), environmental change (triggering an immune response), and the immune reaction (causing a specific AD).1
Epigenetics refers to “stable and heritable changes in gene expression that do not involve alterations in DNA sequence.”2 Recent findings suggest epigenetic mechanisms could be the link between genetic and environmental factors in AD, causing specific long-term phenotypic consequences arising from “environmental influence on slowly evolving genomic DNA”.3 If specific environmental factors are localized, huge leaps would be observed in disease prevention. In this paper, I would like to discuss current findings in the field of epigenetics in the context of three common autoimmune diseases.
I. Epigenetics and Epigenetic Mechanisms
Epigenetic changes are potentially heritable changes to genetic function which are affected by environmental triggers. As previously mentioned, these changes are not chemical changes made to the DNA itself, and are often considered reversible. Since the 1940s, this concept has been a very important step forward from a purely “naturalist” theory - one relying solely on hereditary variation to explain how the <30,000 genes in a cell translate to the wider phenotypic variations in human beings.4
Epigenetic marks - gene expression features that are not directly linked to genetic code - are affected by epigenetic mechanisms. In this regard, epigenetics are key to controlling gene expression, both during initial cell genesis, and as a response to environmental stimulus. The robustness of epigenetic changes causes even monozygotic twins to be identified based on their epigenetic history. This robustness is also the cause of long-term phenotypic changes which may lead to, and explain, complex disease such as cancers and ADs. Programming of the epigenome begins as early as the fetal stage in the uterus, where the maternal environment during gestation affects both the somatic and gamete cells of the fetus, causing a possible link between an offspring and its grandmother’s environment.5 Paternal epigenetic contribution has also recently been discovered to be transmitted through the germline.6 Experiences which could alter the epigenome include nutritional, developmental, hormonal, social, toxin and stress experiences. Three of the most widely-accepted epigenetic mechanisms today include DNA methylation, histone modifications, and transmission through microRNA.
(i) DNA Methylation
DNA methylation occurs due to the addition of a methyl group from S-adenosylmethionine (SAM) cosubstrate to the cytosine ring in CpG pair clusters called CpG islands (although non CpG-sites are also being studied for methylation patterns7). This converts cytosines to 5-methylcytosine, and this part of the DNA has reduced accessibility to gene transcription. When embryonic stem cell methylosomes (methylation profiles) were compared to those of fetal fibroblasts, the gene areas where DNA interacts with proteins showed remarkably lower methylation, and general cell type differences, proving the importance of methylation in reduced gene expression.8
DNA methylation is considered one of the most traditional epigenetic mechanisms for disease causation and susceptibility. A breakthrough study in nutritional epigenetics was the study of Agouti pregnant rodents. In the study, rodents were fed methyl-group-rich foods, and found that their offspring had a different coat color compared to the control group. Additionally, these rodents developed statistically-significant frequencies of adult-onset diabetes, obesity, and tumorigenesis.9 Alongside maternal transmission, paternal transmission was also observed, originally in the murine AxinFu allele.10 This shows a long-term phenotypic change causing disease onset years after the environmental stimulus, and is a key reason why methylation is studied rigorously in complex diseases such as cancers and ADs.
(ii) Histone modifications
Histones are conserved proteins of the kinds: H2A, H2B, H3 and H4 (core histones), as well as H1 and H5 (linker histones). The nucleosome (subunit of chromatin, the substance making a chromosome) consists of DNA wrapped around two copies of the four core histones (octamer). Linkers bind to heterochromatin DNA to “seal off” the nucleosome.11
Some modifications to the ten millions of cellular histones include: acetylation, methylation, ubiquitination, phosphorylation, SUMOylation, and ADP ribosylation. These modifications occur during cell genesis and the cell development cycle. The key processes of these are methylation and acetylation. Histone acetylation (increasing transcription) is countered by deacetylation of terminal lysine (decreasing transcription). Lysine/arginine methylation (using histone methyltransferases/HMTs) in some specific sites (H3K4me2, H3K36me2/3 and H3K79me2) results in increased transcription, methylation at some other sites (H3K9me2/3, H3K27me3, and H4K20me2/3) inhibits transcription, and demethylation (using demethylating enzymes lysine-specific/histone demethylase) reverses this kind of methylation.12
These changes differ in their effect from DNA methylation, in that DNA methylation acts in a black-or-white way in terms of allowing or disallowing expression, while histone modification may be balanced between expression and disallowed expression on a spectrum (for instance, by balancing acetylation and deacetylation). The two mechanisms may also act concurrently: deacetylation combined with methylated cytosine may cause especially powerful transcription inaccessibility, while DNA demethylation alongside acetylation may cause accessibility.13 Some cations such as polyamine spermine may cause further loosening of nucleosomes, facilitating evermore transcription.14
(iii) MicroRNA transmission
Epigenetic markers, in DNA methylation and histone modifications, often directly alter gene expression in disease onset. Certain microRNAs (miRNAs) may themselves be involved in chromatin regulatory machinery, or may be controlled through the former two mechanisms. About 20 base pairs long, miRNAs are small, non-coding molecules which bind to specific sequences in the 3' untranslated region of mRNAs, and inhibit protein synthesis. While there is still a lot of uncertainty about the exact biogenesis of miRNAs and their link to complex diseases, a few miRNAs have been identified for their direct link to complex diseases.15
II. The Relationship Between Epigenetics And Autoimmunity
Autoimmune diseases have varied epidemiology, etiopathology, and physiological manifestations. However, they have the same immunological aberrations: autoreactive immune responses (presence of autoantibodies and/or autoreactive lymphocytes.) In the normal adaptive immune system, immune cells are regulated to obliterate autoimmune reactions and maintain self tolerance. A failure to maintain this environment causes dysfunctional immunity, leading to a variety of complex autoimmune etiologies, based on the specific autoreactivity produced.
ADs have a hereditary aspect to their susceptibility. Polymorphisms in over 200 bona fide loci are considered contributors to various ADs.16 17 Additionally, maintenance of normal immune response inherently requires epigenetic regulation. B cell self tolerance operates through several mechanisms including some complex epigenetic regulations.18 T cell central self tolerance takes place in the thymus through negative selection; any remaining self-reactive T cells are either suppressed by regulator cells (Tregs) or by destruction using anergy induction (“cell-intrinsic programs that lead to a state of functional unresponsiveness”), which is controlled by histone mechanisms in T cells.1920 Thus, it follows that considering genetic and epigenetic modifications is integral to understanding autoimmune pathology. A key statistical clue of epigenetics in autoimmunity is the monozygotic twin studies.
Twin studies: Comparison between monozygotic twins help eliminate genetic differences between samples - genetic differences are relatively insignificant. With such similar genomes, concordance rates in autoimmune disorders were less than 100% in common ADs. These rates were even lower for siblings. Epigenetic differences were established to also increase with age, and were much higher for twins that had spent their lives further apart and developed an AD,
supporting “epigenetic drift” playing a role in divergence of twin phenotypes.21
Table: Concordance rates from twin studies in ADs22, 23
Environmental factors of ADs such as “drugs, ultraviolet exposure, infection, smoking, silica, hormones, and nutrition” are also linked to epigenetic changes. Concurrent to these findings, epigenetic interplay in ADs has become widely accepted in the scientific community.24
There are a multitude of theories regarding specific AD causation. This can be studied through the lens of three common ADs: systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and ulcerative colitis (UC). A lot of the mechanisms in each section may be common amidst the three ADs, as well as some other ADs, due to baseline commonalities in pathology. These would be noted wherever appropriate.
(i) Systemic lupus erythematosus
SLE (or lupus) is a systemic AD which targets multiple organs, including joints, kidneys, nerve cells (central nervous system or CNS), and the skin. Cell apoptosis, triggered by environmental factors such as UV radiation, smoking, and drug intake, causes nuclear antigens to be presented; “susceptible” immune systems form autoantibodies such as anti-double stranded DNA and anti-Smith antibodies, causing local inflammatory reaction (rashes, ulcers, blood cell dysregulation). There may be a hypersensitivity cascade, causing additional body cells to be targeted, increasing symptoms of SLE.25
The factors underlying susceptibility have been associated with several epigenetic mechanisms, most common of which is DNA hypomethylation. There is hypomethylation in over ten promoter regions as well as the rRNA (ribosomal RNA) gene promoter.26 Since this hypomethylation is global, it causes structural changes in the chromatin, causing further overexpression, hyper-autoreactivity, and chronic inflammation in lupus patients.
Hypomethylation and demethylation is noted in several peripheral blood mononuclear cells, but especially in CD4+ T cells, helper cells which suppress or regulate immune response.27 Global histone H3 and H4 hypoacetylation has also been observed in these cells.28 DNA hypomethylation also occurs in cell activation - B cells, where CD5 proteins function to mitigate activating signals to self cells, are thus dysregulated, promoting autoimmune response.29
Drug-induced SLE: Some interesting breakthroughs in the epigenetics of SLE have concerned drug-induced lupus symptoms, caused by drugs procainamide and hydralazine. In fact, the first evidence of the drug-autoimmunity relationship was reported in 1950 in patients treated with hydralazine. Intake of these drugs causes marked DNA hypomethylation. However, studies in mice also showed a reversal in these effects once the drug stimulus was removed.30 This is a clear case for epigenetic effects on SLE symptoms: a stimulus causing a reversible epigenetic mechanism. It is believed around 10% of SLE cases are drug-induced.31
Epigenetics acting as a biomarker: MiRNA have been found to be modified in T cells. Research on B cells for miRNA is quite limited and non-conclusive. Research findings in the last decade have shown an increase in abnormal miRNA function to be associated with the progression of SLE, making it a strong biomarker for diagnosis and prognosis, or target for epigenetic therapy.32
X-Inactivation and Other Theories For Sex Discrepancy: When discussing a disease like SLE, it is also important to remark on the sex-linked susceptibility of the disease. The sex ratio between females and males is ~10:1 in SLE, 3:1 in RA and 2:1 in another common AD, multiple sclerosis.33 Any potential model for AD causation and mechanism must explain this discrepancy. Several physiological explanations such as sex hormones, fetal microchimerism, and sex-related environmental factors have been proposed but have not garnered enough evidence for a strong conclusion.
A possible explanation is offered by the X-Inactivation hypothesis. The inactive X chromosome (Xi or the Barr body) “is a major epigenetic feature in female cells; maintenance of the Xi’s inactive state is essential to avoid loss of X-linked dosage compensation that could result in overexpression of genes.”34 Overexpression of genes may directly cause susceptibility to hyper-autoreactivity. Demethylation in the Xi chromosome was observed in SLE in studies in the CD40LG molecule, contributing to overexpression and potentially the “striking female predilection” of SLE.35
Similarly, the X-linked inhibitor of apoptosis protein (XIAP) was identified as a direct target of a miR-34a microRNA, which is linked to RA. Additionally, females saw a skewed X-chromosome inactivation pattern, and the promoter CD40L in X-chromosome gene was demethylated in CD4+ T cells in females with RA history (and not males).36
(ii) Rheumatoid arthritis (RA)
RA consists of an excess in production of inflammatory messengers. Pathology of RA includes detection of cytokines and chemokines beyond a threshold value in the synovial fluid (found in cavities of synovial joints), hyperplasia of synovium (increased cell density causing synovial thickening/cell resistance to apoptosis) and presence of inflammatory cells in the synovium. The rigidity of hyperplasia causes it to present a “tumor-like phenotype”, eventually causing production of enzymes that degrade the cartilage.37
Global hypomethylation is observed in synovial cells, indicating overexpression and causation for the extreme inflammatory response (e.g. hypomethylation in IL-6 promoter genes, correlation between RA synovial fibroblast hypomethylation and Line-1 overexpression/altered gene expression38). Interestingly, there is also evidence for hypermethylation in RA, for instance, in CpG islands in the death receptor 3 (DR-3) protein promoter. DR-3 causes apoptosis, and hypermethylation reduces this function, therefore increasing apoptosis resistance and increasing cell density further.39
RA is an important disease to study when analyzing the link between epigenetics and ADs. This is because some of the greatest technological advancements in epigenetics have traditionally occurred in RA: in fact, RA was one of the first complex diseases to assertively be associated with DNA methylation using EWAS (epigenome-wide association study).40 RA also has a wide range of research on epigenetic therapy: on identified epigenetic targets in RA which are/could be used therapeutically.
Epigenetic Therapy in RA: Eraser proteins (histone deacetylases) are very well-studied in RA. Histone deacetylase inhibitors have had strong positive effects in RA mouse studies.41 Accordingly, “isoform-specific” HDAC inhibitors have recently been developed (isoforms are proteins functionally similar to each other).42 There have been surprising epigenetic discoveries in these inhibitors - they were shown to deactivate and disengage proteins within the nucleus responsible for reading, and therefore block transcription.43
(iii) Ulcerative colitis (UC)
Ulcerative colitis is one of the principal types of inflammatory bowel disease (IBD), an AD common in Western countries, with an increasing prevalence in Asia, Eastern Europe and Latin America. A search for the genetic basis of UC began in this century. Concordance rates were found to be roughly 10%, relatively low for ADs, opening up the potential for higher environmental dependence. Additionally, there are many environmental factors already identified for UC, and natural history “suggests a major role for epigenetic events.”42
There is direct evidence of DNA hypomethylation in UC: in patients of UC, rectal T lymphocytes showed lower levels of 5-methylcytosine than control donors.15 UC is uniquely relevant when studying epigenetics of ADs. Although there is not as much specific epigenetic research for UC as in SLE and RA, its occurrence shows interplay of a multitude of environmental stimuli. Therefore, UC is a good model for exploring the factors affecting the epigenetics of AD.
Socioeconomic factors and ethnicity: As early as 2004, familial IBD literature found an influence of ethnicity and cigarette smoking.44 Further studies also cited exposure history of appendectomy.
Biological exposure: Enteric infections, such as salmonella and campylobacter, may be linked with UC and IBD onset.
Gut microbiome: The epidemiological link between gut microbiome and UC was observed by association between childhood exposure to antibiotics and the development of IBD in later childhood.45 Recently, the enteric microbiota has been accepted as an etiologic factor in IBD occurrence.46 It has also been found that “bacteria mediated epigenetic effects on the mucosal immune system in early life have been shown to affect the development of immunologically mediated disease in adulthood.”47 Therefore, epigenetics may affect UC, and potentially other ADs, not just through the traditional epigenetic pathways, but also through the microbiota.
III. Environmental Triggers And The Autoimmune Response
We have discussed some of the specific environmental triggers in the epigenetics of ADs, including hydralazine-induced epigenetic responses in systemic ADs like SLE, and specific gut microbiome and biological exposures in UC. Due to shared pathologies, various ADs may also share certain environmental triggers. This can potentially explain the regional gradients and upward trends in ADs we see today.
Autoimmune diseases affect around 5% of the world population, with a large share of incidence in developed countries.48 There is a net increase of AD in every country per year, with there being up to a 7.2% increase for certain ADs in North American and European countries.49 Genetic backgrounds may help explain certain regional differences, but environmental exposure may hold an equivalent importance for time trends. Environmental triggers currently linked to ADs include: drugs, pollutants, viruses/other pathogens, sex hormones, heavy metals, and stress. There are multiple hypotheses which link these triggers to ADs in common literature. These hypotheses are all compatible with the epigenetic model of ADs.
Hapten hypothesis states chemical substances react with “self” cells to form novel antigenic molecules.50 Tetramethylpentadecane (TMPD), a chemical that induces AD response, has been known to induce chromatin changes and changes in gene expression. Trichloroethylene (TCE), known to induce AD symptoms in mice, influences DNA methylation patterns.51
Radiation and sex hormones, although less studied, are theorized to impact epigenetic marks, either by “regulating transcriptional machinery or controlling epigenetic enzymes” (DNA methyltransferases and histone deacetylase, etc)48. In America, higher prevalence for SLE exists in women of African-American descent. It has been hypothesized that this is due to systemic issues such as segregation, drawing parallels with stress biology. 52 Stress hormones, “through its corresponding receptors”, are theorized to act similarly to sex hormones to facilitate a stress-AD epigenetic relationship.48
Epstein-Barr virus (EBV) has been linked with SLE, RA, Kawasaki disease, autoimmune hepatitis, and multiple sclerosis, some of the most common and complicated ADs. For instance, in SLE patients, there are serum autoantibodies that recognize pathogenic epitopes such as the anti-SM autoantibody. These also react against the Epstein-Barr virus antigen EBNA-1. Studies have shown the EBV DNA that enters B lymphocytes at the time of infection causes a chain of reactions that causes viral genetic code to regulate transcription and associate to epigenetic machinery of the adaptive immune system.53 Such a chain of reactions may help explain why microbial infection of SLE-susceptible individuals can contribute to SLE disease activation.31
It is difficult to establish direct links between specific environmental factors and complex diseases; it is nearly impossible to enlist either all the gene sites or all the environmental triggers responsible for the shaping of a disease trajectory. However, these examples shed light on some ways in which environmental triggers relate to ADs in the light of epigenetics.
Conclusion
Due to technological development, epigenetics - a concept first theorized 80 years ago - has grown to provide new insights into complex diseases. Epigenomic analyses have allowed for greater assertion and understanding of causal relationships between epigenetic mechanisms and autoimmune diseases. Despite these improvements, there are certain hurdles that need to be considered. We do not have enough information on specific repetitive elements in autoimmunity. Additionally, even with the progression of EWAS, we do not have any mechanism to prove that epigenetic changes such as hypomethylation were the cause, rather than the effect, of autoimmune disease onset.
Regardless, the current scientific consensus on epigenetics and autoimmunity opens the possibility of future epigenetic therapy - the reversibility of epigenetic modifications could act as a catalyst for curing autoimmune diseases. In fact, several epigenetic drugs are used already in the treatment of pre-leukemias. Research on drug treatment of epigenetic alterations in ADs is an important step forward in this prospect. Public health policy and the research on evolutionary medicine should also be rooted in epigenetic therapies and the epigenetic basis of autoimmune disease for faster implementation.
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