Will the Real Immune Issue Please Step Forward?

Patrick O. McGowan, PhD, is an assistant professor in the Department of Biological Sciences, University of Toronto at Scarborough. He starts off his paper by listing the array of immune problems found in ME/CFS – some of which seem almost at odds with each other.  There’s the proclivity towards Th2 (anti-inflammatory) dominance, but pro-inflammatory findings are also found. There’s natural killer cell dysfunction found in the innate immune response but there’s also the cytotoxic T-cell findings in the adaptive immune response.

McGowan asks if all of these are happening at once or are different studies finding different issues in different study populations. The question of what’s really going on is big one in a field that generally gets very small studies which use varying methodological approaches in a disorder with a vague definition.

(Webinar on Epigenetics and ME/CFS – Dr. McGowan will talk about his study in a webinar tomorrow, Thursday, August 21, 2014, 2-3:00pm Eastern (1pm Central/Noon Mountain/11am Pacific) time.  Space is limited –  RSVP to Reserve Your Slot – Click HERE)

The “Second Genetics”

Epigenetics refers to the changes in sites in our DNA that are susceptible to change over time which can modify the expression of our genes.  Our genetic heritage, it turns out, only sets the tableau for how our genes express themselves. Just because you’re borne with a genetic  makeup that suggests, say, your immune system will immediately jump on and take out any pathogen that dares disturb you, that doesn’t mean it’s going to stay that way.

Epigenetics has provided an entirely new slant on genetics and gene expression

Things happen as you age that will change how your genes respond to events. Epigenetics isn’t so much an ‘if’ as it is a ‘how much’.  These processes occur in our body all the time. The older we get the less we are our mother’s and father’s sons and daughters and the more our genetic makeup takes on an identity of its own.  Twin studies indicating identical twins are epigenetically indistinguishable at first but diverge widely over time indicate how malleable our patterns of gene expression are.

How this change in gene expression over time occurs and the implications it has for health and disease is what epigenetics is all about.  In a short time it’s become an enormous field.

There’s no question epigenetic modifications play a role in  disease – the only question is how much of a role.  A couple of years ago the National Institutes of Health was interested enough in the possible effects of epigenetic changes on aging, heart disease, and mental illness to devote 190 million dollars to its study. Some researchers think epigenetics will end up having a greater impact on disease than genetics.

Epigenetic modifications of immune genes have been implicated in autoimmune and other inflammatory diseases as well as neurodegenerative diseases. Researchers are now arguing which factor – genes, the environment, or epigenetics – plays the biggest role in autoimmunity.  A recent review paper described probable epigenetic effects on neuroinflammation.

An identical twin study containing one twin with lupus and one twin without lupus, for instance, found altered methylation rates in the sick twin. Increased rates of hypomethylated immune genes suggested epigenetic changes had increased immune activation in the afflicted twin.

Epigenetic Processes

Over fifteen types of epigenetic processes have been identified. Damage to our DNA from toxic chemicals such as benzene and styrene can also, through increased oxidative stress, cause epigenetic changes. This, of course, suggests that the increased rates of oxidative stress present in ME/CFS may cause epigenetic changes.

DNA Methylation

Methylation plays a major role in epigenetics

DNA methylation which involves the addition of methyl groups at CpG sites that convert cytosine to 5-methylcytosine may be the most common way epigenetic changes occur.  CpG sites can either be hypomethylated (exhibit reduced methylation) or hypermethylated (exhibit increased methylation).  Hypomethylation results in increased gene expression, while hypermethlation results in reduced gene expression.

The miRNA Slant

Micro RNA (miRNA) will not be talked about in McGowan’s study, but they are entangled in the epigenetic question. These small bits of RNA down-regulate many biological functions by decaying mRNA.  As many as 60% of proteins in our body are believed to be regulated by miRNAs. One miRNA (miR-146a) that has been proposed as a ‘master regulator of immunity’ impacts both the innate immune and T-cell responses.

A study suggests that one miRNA may play a significant role in the natural killer cell problems in ME/CFS.  Because miRNAs themselves appear to be strongly regulated by epigenetic processes, they’re something to keep an eye on in disorders characterized by high rates of epigenetic alterations.

The Study

Small Patient Set: Big Data Set

This study looked at the epigenetics of females with post-infectious onset, post-exertional malaise, and who met certain other criteria on symptom scores and who had not been taking immune-altering medications.

Even a relatively small epigenetic study (12 patients and 12 controls) generates an enormous amount of data.  This study of the DNA ‘methylome’ of peripheral blood lymphocytes started out with an array covering 480,000 DNA sites subject to methylation (CpG sites). After microarray normalization 327,409 probes remained.  Applying ‘probe selection criteria’ dropped the number of different methylation sites found  to 1,192 sites.  That corresponded to 826 genes. But then there was the ‘region analysis’, ‘gene pathway enrichment’, and much more.  It’s complex  stuff – far too complex for me.

Let’s get to the results.

Results

McGowan found significant differences in DNA methylation in the immune cells of healthy controls and the ME/CFS patients in almost 1200 CpG sites in over 800 genes. Genes involved in metabolic regulation, kinase activities, cellular processes and the immune response were highlighted. Significant numbers of both hypermethylated and hypomethylated CpG sites were found.

McGowan’s results suggested immune regulation was ‘off’

High rates of epigenetically altered immune regulating genes suggested ME/CFS patient’s immune systems are skewed to a Th2 immune response. Altered methylation rates in the genes involved in regulating the adaptive immune response (T and B cells) could help explain the inflammation present in ME/CFS.

The authors highlighted a few genes that had been epigenetically altered. One hypermethylated gene (BCL10) in the ME/CFS patients activates a transcription factor called NF-kB that’s responsible for triggering a wide inflammatory response. Maes has proposed that the NF-kB inflammatory pathway is enhanced in both ME/CFS and depression. Another epigenetically altered gene in ME/CFS is associated with EBV infection of B-cells.

It was interesting that no genes expressly associated with natural killer cells were highlighted although many genes associated with T-cells were.

The rubber meets the road in epigenetics when epigenetic modifications are shown to actually result in changes in gene expression. The study did not assess gene expression, but the authors noted that the epigenetic changes they found fit with prior ME/CFS gene expression results.

Different Epigenetic Patterns in Fibromyalgia

They also noted that a similar analysis of whole blood (not PBMCs) in Fibromyalgia suggested a very different pattern of epigenetic modifications existed in ME/CFS and FM. Methylation appears to be primarily affecting structural nervous system development and neuronal genes in FM, and immune and metabolic genes in ME/CFS (with the proviso that immune cells were examined in ME/CFS).

Epigenetics has the potential to alter gene expression in so many ways that it simply has to be addressed. This study suggested that epigenetically modified immune dysregulation could play a role in ME/CFS.

How to go from 800 plus methylated immune genes to identifying a gene or set of genes that can provide a basis for treatment is another question.

 A Look at Epigenetic Effects on Rheumatic Disorders

“It is clear that aberrant epigenetic regulatory mechanisms play important roles in the development and pathogenesis of rheumatic disease.” – Gray, 2014

(Much of the information in this section is taken from a review paper  – Perspectives on epigenetic-based immune intervention for rheumatic diseases  Steven G Gray*, Arthritis Research & Therapy 2013, 15:207)

Epigenetics may play a particularly strong role in autoimmune disorders

Epigenetic studies in other disorders are beginning to do that.  Lupus studies, for example, have identified epigenetic modifications that alter the expression of CD4 T-cells and contribute to the development of autoreactivity and increased antibody production in that disease.

The fact that epigenetic modifications of the histone strands in our DNA create prime targets for autoantibodies suggests that epigenetic modifications may have special relevance to autoimmune disorders. Researchers have identified damaging epigenetic processes in Sjogren’s Syndrome that were reversed using Rituximab.

One epigenetic targeting drug (vorinostat) that is able to damp down pro-inflammatory cytokines and regulate T-cells may be effective in a range of autoimmune disorders.  It is currently being used to treat lymphoma.

Given the broad pattern of hypomethylation found in ME/CFS and other disorders, it’s possible that drugs able to target factors that increase methylation rates across the board could be helpful. Lupus researchers appear to have uncovered a hypomethylated CpG site that regulates DNA methyltransferase –  a prime factor in the methylation  process.

McGowan noted that a DNA methylating drug that alters the methylation of immune pathways (including HLA Class I pathways) affected in ME/CFS has been produced.  That drug, now apparently being tested in lung cancer cell lines, could be applicable to ME/CFS at some point.

Dietary Control of Methylation

A large number of naturally-occurring compounds that can regulate epigenetics suggest that dietary modification or supplementation may be helpful. Cucurmin, for instance, is able to regulate epigenetic processes believed active in some rheumatic and autoimmune disorders.

Cucurmin may be helpful in reducing harmful epigenetic effects

Two placebo-controlled studies, one with a product called Meriva (Thorne Research, Inc.,Dover, ID, USA), containing a formulation of curcumin complexed with phosphatidylcholine, was found to have significant benefit on rheumatoid arthritis patients, including decreased expression of pro-inflammatory cytokines IL-6 and IL-8. A cucurmin analog appears to be able to induce the expression of the major miRNA immune modulator (miR- 146a).

Delphinidum appears to be able to reduce activity in the NK-kB inflammatory pathway that the authors highlighted in their results. Resveratrol is a natural compound that appears to be able to modulate inflammation by regulating pro-inflammatory cytokines.

Sulforaphane’s(SFN) ability to reduce immune responsiveness is currently being assessed in clinical trials using ‘broccoli smoothies’. SFN’s ability to reduce the Th2 response, if proven, could come in handy in a disease like ME/CFS that appears to be characterized by an increased Th2 response. Injecting SNF into mice with arthritis reduced synovial problems and bone destruction as well as pro-inflammatory cytokine production.

The Future

McGowan outlined several steps that are needed to validate the effects epigenetics is having in ME/CFS.  Longitudinal studies following ME/CFS patients over time which associate epigenetic alterations with specific symptoms is important.  Researchers need to be able to show that epigenetic changes show up in changes in symptom expression.

Noting that immune abnormalities can be unmasked by challenges such as exercise, McGowan et. al. proposed that both epigenetic modifications  and gene expression be analyzed before and after exercise. Researchers also need to be able to show that the epigenetic modifications are actually altering the expression of the genes they’re associated with.  A combined epigenetics/gene expression study is a necessity at some point.

Getting the next studies done will clearly require substantial financial input, and that probably means NIH funding. Gathering the data needed to get the big bucks from the NIH is, of course, what these pilot studies are all about.

Webinar on Epigenetics and ME/CFS

Dr. McGowan will talk about his  study in a webinar tomorrow, Thursday, August 21, 2014, 2-3:00pm Eastern (1pm Central/Noon Mountain/11am Pacific)   Space is limited – RSVP to Reserve Your Slot – Click HERE)