The Role of Genetics and Epigenetics in Understanding Systemic Lupus Erythematosus – Causation, Manifestation, and the Search for Therapeutic Targets

An Interview with Amr H. Sawalha, MD, Director, Lupus Center of Excellence at UPMC, and Chief of Rheumatology at UPMC Children’s Hospital of Pittsburgh

Amr H. Sawalha, MD, is professor of Pediatrics, Medicine, and Immunology at the University of Pittsburgh School of Medicine. He holds the Vincent Londino Endowed Chair and is director of the Division of Rheumatology at UPMC Children’s Hospital of Pittsburgh. Dr. Sawalha is director of the Lupus Center of Excellence that spans the clinical and research enterprises of UPMC and the University of Pittsburgh.

Dr. Sawalha’s research program focuses on elucidating genetic and epigenetic contribution to the pathogenesis of systemic autoimmune and inflammatory diseases. His team applies state-of-the-art genomic, epigenomic, and bioinformatics methodologies, as well as subsequent functional studies, using both in vitro and in vivo systems to identify and characterize genetic loci and pathways involved in the pathogenesis of immune-mediated diseases.

Q: Your clinical and research focus is on autoimmunity, with an emphasis on systemic lupus erythematosus, and the role that genetics and epigenetics play in pathophysiology and manifestation of illness. For clinicians unfamiliar with epigenetics and its importance in understanding the nature of lupus as a disease, and possible future treatment developments, can you provide a brief explanation of what epigenetics is and its role in your studies of lupus?

A: Epigenetics refers to mechanisms that regulate the expression of genes. All cells in our bodies have the same DNA makeup (or genes). What makes cells different is the various assortment of genes they express. In a sense, epigenetics determines which genes are going to be silenced in any given cell type or cell state and which genes are going to be active.

There are several epigenetic mechanisms that are involved in “gene regulation.” The key epigenetic mechanism is called DNA methylation, and this is what we primarily study. It is a chemical modification that happens in specific DNA sequences that can be important in gene regulation (for example, gene promoters or enhancers).

When these sequences are methylated genes are silenced, and when methylation is reduced, they are available for the transcription machinery and expression. Because DNA methylation can regulate gene expression, dysregulated DNA methylation can lead to abnormal gene expression levels and therefore, disease. This is what happens in lupus; DNA methylation is abnormal in certain cells and that leads to abnormal cell function and overactivation of some immune cells (for example, T cells) – leading to autoimmunity, as we see in lupus.   

Q: Are there specific epigenetic factors or targets that your lab is particularly interested in understanding that may be at play in influencing gene expression at the individual level and thereby how lupus manifests?

A: As I mentioned earlier, we primarily study DNA methylation. This epigenetic mechanism is dysregulated in immune cells from lupus patients, and we have focused most of our studies on a specific immune cell type called CD4+ T cells. It turns out that a defect in a signaling pathway (called ERK) that regulates the expression of the main enzyme that mediates DNA methylation (DNA methyltransferase 1) is defective in T cells from patients with lupus.

As a result, the expression levels of DNA methyltransferase 1 are reduced, which leads to decreased overall methylation levels in lupus T cells compared to T cells from healthy individuals. This leads to overexpression of key genes in T cells from lupus patients, as DNA methylation puts the “brakes” on gene expression and is defective.

Some of these genes make the T cells overactive, leading to what we call T cell autoreactivity, which means that these T cells are now capable of inducing an exaggerated immune response and attacking self-tissue. This is what happens in lupus. In some of our earlier studies, we were able to demonstrate that if we induce an ERK signaling defect in T cells in animal models, we can reproduce the same DNA methylation defect we see in lupus patients’ T cells and induce the production of autoantibodies and lupus-like disease.

Q: Understanding the role of DNA methylation in lupus has been a focus of research in your lab. For the practicing clinician treating patients with lupus, can you describe the nature of this work and how it may one day help to better understand disease processes and inform the development of treatment or preventive therapies?

A: The work we have been doing in understanding the role of DNA methylation in lupus has provided important insights into the pathogenesis of lupus, but certainly there is much more work that needs to be done. I don’t think anyone now questions that abnormal DNA methylation plays a central role in the pathogenesis of lupus.

Like many complex diseases, we believe both genetic and environmental factors are involved in the disease process in lupus. Epigenetics can mediate the effects of disease-associated environmental factors, and lead to disease in genetically susceptible hosts. For example, oxidative stress (induced by infections, and almost all environmental factors associated with lupus) can result in abnormal T cell DNA methylation similar to what we see in lupus patients.

We have more recently linked oxidative stress (environment) to abnormally high expression levels of a key epigenetic modulator called EZH2, which we showed to be overexpressed and associated with disease activity in lupus patients. Our studies have also demonstrated robust demethylation of interferon-regulated genes in lupus immune cells, which explains hypersensitivity of lupus cells to type I interferons. We also provided evidence for an early epigenetic remodeling in naïve CD4⁺ T cells as the disease becomes more active in lupus patients, providing a clue for early T cell compartmental changes that are associated with lupus flares before we see any gene expression abnormalities. Some of our studies also showed how differences in DNA methylation patterns might explain why lupus is more severe in Black patients for example, and why lupus is more severe in men. Similarly, we have evidence to support specific epigenetic changes that are associated with specific manifestations in lupus, for example, lupus nephritis and skin involvement. These findings might help us one day predict what future manifestations every lupus patient might get, as lupus is a heterogenous disease that can present in many different ways.     

Q: Can you discuss any recent published findings from your lab that have helped to expand the evidence-base for understanding the pathologic features of lupus – understanding that this is a heterogenous disease with varying types of presentation – or findings from your studies that point toward potential targets for which therapeutic development may be?

A: We are excited about our results suggesting EZH2 as a novel therapeutic target in lupus. We also have findings suggesting EZH2 as a therapeutic target for scleroderma, another autoimmune disease, but that’s a different story and involving a different mechanism. Our data showed that naïve CD4⁺ T cells in lupus undergo an epigenetic pro-inflammatory shift implicating specific effector T cell responses as lupus becomes more active. This epigenetic landscape modification occurs before expression changes of corresponding genes, and poises naïve CD4⁺ T cells for specific T helper cell immune responses, and opposes the inhibitory TGF-β signaling.

Our first clue came from bioinformatics analyses that indicated the epigenetic modulator EZH2 might be mediating this epigenetic landscape shift in T cells. We then went on to show that EZH2 is indeed overexpressed in T cells isolated from lupus patients. We did some studies to understand why EZH2 is overexpressed in lupus. It turned out EZH2 is suppressed by two microRNAs that are sensitive to glucose concentration. We showed that the expression of these two microRNAs is low in lupus patients resulting in increased EZH2 expression.

We then linked reduced expression of these two microRNAs to enhanced glycolysis in lupus T cells, increased mTORC1 signaling, and oxidative stress. We then showed that inhibiting EZH2 ameliorated lupus-like disease in animal models. More recently, we demonstrated that EZH2 is overexpressed in other cell types in lupus patients such as B cells. Our unpublished data show that selectively deleting EZH2 in either T cells or B cells can abrogate disease in lupus-prone animal models, and we have a good idea about the mechanisms involved. These data collectively point to EZH2 as a potential therapeutic target for lupus.

Q: Along similar lines, are there emerging epigenetic biomarkers in lupus that either your team or the field at large see of particular interest in pursuing that could aid in earlier identification of disease phenotype and thereby may guide current clinical management options? For example, perhaps there are signatures that may portend accelerated end-organ damage that can be addressed at an early state in the disease course if we know a particular patient has this signature or profile in their genetic or epigenetic characteristics?

A: Our hope is to identify specific epigenetic and genetic signatures that can help us achieve a personalized medicine approach in lupus. We published some proof-of-principle examples of epigenetic changes associated with specific lupus manifestations, and we have reported on genetic associations between lupus-risk genetic polymorphisms with specific “sub-phenotypes” in lupus. However, I don’t think any of this is ready to be applied in a day to day clinical setting yet. We need to do more work.

We do, however, have a very promising epigenetic diagnostic marker for lupus, which I think is ready for development in the clinic. It turns out that hypomethylation of the gene IFI44L has a very high sensitivity and specificity for identifying lupus patients. Our original data were generated with a collaboration with our colleagues in China, and have been since confirmed by multiple groups and in several ethnicities. I don’t know of any single stand-alone test that has a similar high accuracy (sensitivity and specificity) for lupus as IFI44L methylation level. This can be done on whole blood samples, making it very feasible and relatively inexpensive.

Q: Can you discuss any current research projects that you and your team are working on and their specific aims? Have you received any recent grants from the NIH or other funding bodies to help further your studies?

A: Broadly speaking, we are performing studies to identify and characterize the role of key target genes regulated by EZH2 that mediate the pathogenic effect of EZH2 overexpression in different immune cell types in lupus. This will allow us to understand the disease mechanism better, and also identify other more specific targets for therapy in lupus.

The narrower the target, the better likelihood of therapeutic tolerability and effectiveness of any new treatment. In addition, we are working on the interaction between genetic and epigenetic factors in the susceptibility of lupus, and how epigenetic factors and interaction with the microbiome might be involved in the disease. Lupus is part of what we do, and we have active projects in the lab related to other immune-mediated diseases, such as primary antiphospholipid syndrome (stay tuned for the results of a large multi-ancestral genetic study), scleroderma, Behçet’s disease, and Takayasu arteritis. We are very grateful to the funding we receive from the National Institutes of Health and the Department of Defense for our studies.   

Q: For readers who are interested in your work and would like to learn more about your research, can you recommend several research papers or review articles you and your colleagues have recently published?

A: We recently wrote a review on the role of oxidative stress in epigenetics in autoimmunity that readers might find useful and cites many of the key papers I discuss above.

For readers interested in the genetic basis of lupus, I suggest the following comprehensive review: Systemic lupus erythematosus as a genetic disease | Elsevier Enhanced Reader

For a complete and up-to-date list of our publications, please see Dr. Sawalha's publications in PubMed.