Regulation Of Metabolism In The Synovium And Blood In Rheumatoid Arthritis

Introduction​

Rheumatoid arthritis is a systemic autoimmune disease that affects the synovial joints, causing severe inflammation. The synovial joint or articular joint is enclosed in a structure called the joint capsule. The joint space, which separates the bones forming the joint, is filled with a lubricating fluid known as the synovial fluid. This fluid is secreted by a thin layer of cells lining the joint, known as the synovial membrane. Rheumatoid arthritis primarily manifests as an inflammation of this synovial membrane. ​

The progression of rheumatoid arthritis can be divided into three phases, summarized in figure 1. An initial pre-articular phase, where genetic and environmental factors cause the formation of autoantibodies, precedes the onset of symptoms. These antibodies may circulate for several years, before an unknown trigger causes an inflammatory attack of synovial tissue in small joints. This second phase, known as the articular phase, is characterised by severe inflammation of the synovial membrane, and activation of the resident synovial fibroblasts and macrophages. The inflammatory reaction may persist, progressing to the third phase of chronic inflammation of the synovial tissue.

At this stage, the synovial fibroblasts multiply and form an invasive, migratory tissue known as the pannus. The pannus invades the underlying bone and cartilage, causing joint destruction and disability. The inflamed synovial membrane becomes hypoxic, leading to angiogenesis and infiltration of several immune cells into the tissue, which secrete cytokines that sustain the inflammation. The stages of rheumatoid arthritis. Each large box indicates the consecutive phases in the progression of the disease in the synovial joint.

In the initial stage, genetic and environmental factors induce the formation of autoreactive antibodies in the circulation. These polyclonal antibodies are produced by circulating autoreactive Bcells, with later involvement of MHC calss II restricted T cells. In the second stage, these antibodies cause an acute inflammatory reaction in the synovial joint. The final stage chronic inflammation causes drastic changes to the synovial membrane architecture, leading to severe erosion of the joint and disability. ​The changes caused in the synovial tissue by the chronic inflammatory process is reflected in the metabolism of the cells constituting the tissue. The activated synovial fibroblasts in the rheumatoid arthritis affected joint are fast dividing cells which secrete pro inflammatory cytokines, whereas under normal conditions they are quiescent cells that secrete synovial fluid components. This altered metabolism in the synovial tissue is of great interest due to the potential for therapeutic targeting. In addition to its effects on joints, RA also causes disruption of systemic metabolism.

This metabolic disruption in RA is manifested as two conditions, metabolic syndrome and rheumatoid cachexia. The presence of metabolic disruption in RA patients has several consequences. It affects the treatment efficacy and increases the likelihood of life-threatening comorbidities like cardivascular disease. Hence a detailed understanding of the metabolic disruptions present in rheumatoid arthritis is necessary for creating effective treatment strategies. ​

Literature survey

Synovial metabolism

The synovial membrane in a normal joint consists mainly of synovial fibroblasts, along with resident macrophages. The metabolism of normal synovial fibroblasts have not been studied in detail. However, based on a study in quiescent dermal fibroblasts, it is thought that synovial fibroblasts depend primarily on glycolysis for energy metabolism. Pyruvate from glycolysis drives the tricarboxylic acid (TCA) cycle. The pentose phosphate pathway is used to generate NADPH, which is required to maintain redox homeostasis in the cell. In rheumatoid arthritis affected synovial tissue, hypoxic conditions cause a greater reliance on anerobic glycolysis for energy production. The TCA cycle is downregulated and pyruvate is converted to lactic acid. Cellular metabolism must adapt to changes in the microenvironment of the cell. The adaptations of the metabolic pathways in response to the microenvironment is regulated by several signalling pathways.

These pathways are summarised in figure 2. 5' AMP activated kinase (AMPK) is a sensor of the energy state of the cell. It is activated by low ATP conditions, and though several downstreams proteins, causes increase in mitochondrial biogenesis and oxidative phosphorylation. The mTOR pathway acts downstream of signalling from growth factors, cytokines etc. It drives biosynthetic processes like fatty acid synthesis and protein synthesis, activates glycolysis while inhibiting oxidative phosphorylation. The mTOR and AMPK pathways act as master regulators of cellular metabolism. Hence, the metabolic changes present in the RA affected synovium may be regulated by proteins in these pathways.

However, dysregulation of these pathways in the diseased synovium has not yet been demonstrated. Genes activated by the mTOR and AMPK signalling pathways are shown in the blue boxes. The processes they regulate are shown in the red and green boxes. Inhibition is indicated by the red line ending in a square and activation by the green arrow. The regulator of mTOR are shown in the purple boxes. Systemic metabolismSeveral studies have found associations between RA and obesity or metabolic syndrome. Metabolic syndrome is characterised by increased amount of visceral adipose tissue, high blood pressure, high blood sugar levels and dyslipidemia. It also creates a proinflammatory state in the body and is a risk factor for cardiovascular disease and type 2 diabetes. Adipose tissue performs a variety of functions in the body in addition to fat storage. It secretes a variety of proinflammatory cytokines including TNF-α and IL-6. It also secretes adipose tissue specific cytokines called adipokines. Adipokines function mainly to control metabolism by regulating energy expenditure. Their levels vary with the amount of white adipose tissue present in the body. Adipokines are known to play a role in inflammation.

For example, leptin is a proinflammatory adipokine, that is known to be upregulated by TNF-α and IL-1β. Its proinflammatory effects include increased differentiation of proinflammatory Th1 cells, increased secretion of TNF-α, IL6 and IL12. Similarly other adipokines affect the immune system in various ways that have not been completely characterised. Levels of the adipokines leptin, adiponectin and visfatin are seen to be increased in RA synovial fluid and in blood. Thus the increased amount of adipose tissue present in RA patients with metabolic syndrome influences the inflammatory state already present in the body due to RA. The other metabolic condition associated with RA is rheumatoid cachexia. It involves high disease activity and functional impairment. At high levels of inflammation that is present in treatment resistant RA, there is severe loss of muscle mass, while the visceral fat mass is maintained or increased.

The wasting in this condition is thought to be caused by systemic inflammation and high levels of proinflammatory cytokines like TNF. Wasting is also present in other chronic illnesses like cancer, chronic infection etc. However the metabolic disruption in rheumatoid cachexia is different from other wasting conditions in that the visceral fat mass is maintained or increased, so that body weight and BMI are not necessarily reduced. Rheumatoid cachexia is associated with greater cardiovascular mortality even though cholesterol and triglyceride levels are low. The central adiposity coupled with low cholesterol and triglyceride levels indicate a profoundly altered lipid metabolism in rheumatoid cachexia. These conditions illustrate the close interaction between the inflammatory state of the body and control of metabolism (figure 3). Cytokines and adipokines influence both the immune system and metabolism. Their varying levels in RA affect body composition and the amount and distribution of body fat, while the amount of visceral fat mass influences the inflammatory state, including the levels of adipokines and cytokines. Body composition, lipid levels and disease activity in various RA states. Pre-RA, a state which manifests little or no disease activity, progresses to early RA with the onset of inflammation in the joints.

This state may progress in one of the three directions shown in the figure: obese RA, characterised by the presence of the markers of metabolic syndrome and moderate disease activity; well-controlled RA which responds to treatment; and treatment resistant RA, marked by rheumatoid cachexia and high disease activity. In pre RA and early RA, there is increase in fat mass and dyslipidemia characterised by increased triglycerides(TG), total cholesterol(TC), LDL cholesterol and decreased HDL cholesterol. Dyslipidemia and changes in visceral fat mass are seen in all the three advanced states. Although the connection between RA and metabolic disruption is well studied clinically, the underlying molecular pathways are not well explored. ​ Gaps Identified in understanding the influence of inflammation on the regulation of metabolism in RA​ Metabolic changes in the synovial tissue of rheumatoid arthritis affected joints have been characterised.

However, the regulation of these pathways by signalling pathways, in response to the altered environment of the joint has not been examined in detail. We hope to characterise these signalling pathways that regulate the changes in metabolism. Similarly, the signalling underlying the changes in systemic metabolism is largely unexplored. It is known that the blood metabolome and transcriptome reflect organism wide processes. To our knowledge, no studies have examined the state of genes related to regulation of metabolism in the blood transcriptome of rheumatoid arthritis. Hence, although it is well known that the immune system and metabolism are intimately linked, the underlying network that connects the two aspects in rheumatoid arthritis has not been elucidated, in the synovial tissue or at the level of the organism.

Objectives

1. Analysis of genes associated with the regulation of metabolism in the synovium of rheumatoid arthritis affected joints.
2. Analysis of the blood transcriptome of rheumatoid arthritis: How is the expression of genes associated with the regulation of metabolism affected?
3. Modelling specific aspects of regulation of metabolism that are identified as modified in rheumatoid arthritis.
4. Analysis of the effect of rheumatoid arthritis drugs on the genes associated with the regulation of metabolism​

Methodology​

For Objective 1 and 2

Gene expression analysis of datasets from public databases: Coexpression expression analysis of RA and healthy synovial tissue:

• Consensus and differential coexpression analysis of the samples using WGCNA and DiffCoexp packages in R
• Annotation of coexpression modules
• Identification of modules relevant to regulation of metabolism
• Identification of transcription factors regulating coexpression using transcription factor database TRRUST.
• Generation of gene regulation networks using cytoscape.
• Identification of cytokines and adipokines upstream of the identified transcription factors, relevant in RA using literature.

Gene expression analysis of datasets from public databases: Differential expression analysis of RA vs healthy:

• Differential expression analysis using limma and standard t test in R.
• Identification of differentially expressed genes relevant to regulation of metabolism.
• Construction of protein protein interaction network of differentially expressed genes using String database and literature.
• Analysis of the network using Cytoscape.
• Identification of differentially expressed genes in the gene regulation networks generated in the previous step, using cytoscape.

For Objective 3:

1. Dynamic model building (depends on the result of the previous steps).
2. Identify metabolites that are affected in rheumatoid arthritis from literature.
3. Create a network model of the regulation of the specific metabolite using the results from objective

Create an ordinary differential equation model for the metabolite. ​

For Objective 4

1. Gene expression analysis of Pre and post treatment blood samples in rheumatoid arthritis.
2. Differential expression analysis to identify genes altered by treatment using limma and t test in R.
3. Identification of the treatment altered genes in the protein protein and gene regulation networks created in objective 2.
4. Identification of known drug targets for RA and insulin resistance using the database DrugBank.
5. Predict the effects of treatment on metabolism based on these networks. ​

Expected outcome​

Using the methodology described here, gene regulatory networks describing the altered control of metabolism in the synovial membrane and blood of RA affected individuals can be created. It is expected that these networks will reveal the role of cytokines and adipokines in RA related metabolic disruption at the level of the synovial and systemic metabolism. By identifying metabolites that are affected by the important genes in the regulatory network, we hope to establish the mechanism behind the alterations of these metabolites that are seen in RA. The effect of inflammatory mediators on the specific RA altered metabolites can be examined using the model. Additionally, the effect of RA drugs on the control of metabolism can also be explored.

​Importance of research

Due to the close relation between cellular metabolism and inflammation, metabolic pathways and their regulators are promising drug targets in the treatment of RA. Understanding the mechanisms that regulate metabolism in the synovial tissue may allow us to predict the effects of drugs in the tissue. It is well known that RA patients are at higher risk for cardiovascular disease than the general population. The components of metabolic syndrome are also individual risk factors for CVD.

In addition, RA patients with metabolic syndrome are at a greater risk of developing CVD. However, due to several factors including the influence of inflammatory mediators on lipid metabolism, the indicators of cardiovascular risk in the general population such as cholesterol and triglyceride levels, cannot be directly applied to RA patients. By elucidating the network responsible for systemic metabolic disruption in RA, we hope to clarify the effects of the inflammatory mediators on CVD risk factors in RA patients.