Transcriptome Analysis of Orbital Adipose Tissue in Active Thyroid Eye Disease Using Next Generation RNA Sequencing Technology

1. INTRODUCTION

Thyroid Eye Disease (TED) is caused by a systemic autoimmune attack on the orbit and other target tissues, including the thyroid, skin, and pretibial soft tissues [1]. Circulating lymphocytes and humoral agents infiltrate the orbital soft tissues and induce orbital fibroblasts to cause the characteristic pathological changes of TED, such as orbital adipose tissue expansion, muscle fibrosis, and deposition of glycosaminoglycans within the extraocular muscles [2, 3]. Various molecular factors have been implicated in TED pathogenesis, including insulin like growth factor-1 and interleukins [4], although most studies have relied on cultured cell lines from TED patients [5].

TED follows a stereotypical disease course (“Rundle’s curve”) consisting of an “active phase,” characterized by inflammation and dynamically worsening orbitopathy, followed by a “quiescent phase” of disease stability. About 3-5% of patients develop severe TED associated with vision loss and compressive optic neuropathy [6]. Treatments for active TED include systemic corticosteroids, orbital radiation, biological immunomodulatory agents, and induction of euthyroid status, in some cases by thyroidectomy. Previous studies have used microarray technology to study differential gene expression in orbital fat in TED and have identified Wnt signaling genes, adipocyte-related immediate early genes, and IGF-1 signaling genes as being potentially implicated in pathogenesis [7-10]. Other in vitro studies on orbital adipose-derived stem cells harvested from patients with TED used RNA Seq and found downregulation of early neural crest markers and ectopic expression of HOX genes [11]. This study aimed to characterize the RNA transcriptome in the orbital adipose tissue of patients with severe, active TED compared to that of matched, healthy controls. We utilized Next Generation Sequencing (NGS) to identify differential gene expression patterns and potential therapeutic targets for translational research and prospective clinical trials.

2. METHODS

2.1. Study Design

This prospective case-control study was approved by the University of California, San Diego Institutional Review Board, and was performed in accordance with the Declaration of Helsinki. Study cases all had severely affected phenotypes of TED in the active phase of the disease when orbital decompression surgery was performed Fig. (1). All cases had actively worsening signs and symptoms, and two had compressive optic neuropathy while the third had a Thyroid Stimulating Immunoglobulin (TSI) level > 500. Clinical Activity Score (CAS) was determined by a board-certified ophthalmologist and oculoplastic surgeon based on history and examination findings¹. Two cases had CAS scores of 7 and one had a CAS score of 8 on a scale with a maximum score of 10. Controls had dermatochalasis and no history of thyroid abnormalities or TED.

Inclusion and exclusion criteria were strict to control for confounding factors suspected of altering transcriptional activity in orbital adipose tissue in TED or normal controls. All cases and controls were Caucasian and female to control for gender and racial transcriptional variations. Any patients with significant current or recent tobacco smoking history were excluded, since smoking is well known to increase the incidence and severity of TED and induce numerous gene expression changes [3]. Factors that influence adipogenesis and inflammation were also controlled. Patients were excluded if they were overweight or obese (BMI over 25 kg/m²), had diabetes or metabolic syndrome, or were currently or recently taking any systemic steroids, immunomodulatory agents, or had undergone orbital radiation. All controls had no major medical problems, history of thyroid abnormalities or clinical evidence of TED. For detailed clinical characteristics, see Table (1).

Table 1.

-	Case 1		Case 2	Case 3		Control 1	Control 2		Control 3
Age (years)	68		81	62		60	58		80
Gender	F		F	F		F	F		F
Race	Caucasian		Caucasian	Caucasian		Caucasian	Caucasian		Caucasian
Duration of Grave's disease prior to surgery (mo)	12		96	20		N/A	N/A		N/A
Duration of TED prior to surgery (mo)	6		8	20		N/A	N/A		N/A
Previous treatment for Grave's disease	Methimazole, radioactive iodine		Methimazole	Methimazole		N/A	N/A		N/A
Previous treatment for TED	Selenium supplements		Peribulbar steroid injection to OS, fat specimen from OD	Selenium supplements		N/A	N/A		N/A
Smoking history	10 pack-years, quit 40 years prior		7.5 pack-years, quit 44 years prior	0.2 pack-years, quit 3 years prior		None	None		None
Body mass index (kg per m^2)	22.9		24.2	24.1		22.7	22.8		22.3
Exophthalmometry, Naugle (mm)	24.5 OD, 24.5 OS		26 OD, 30 OS	23 OD, 22 OS		N/A	N/A		N/A
Thyroid stimulating immunoglobulin (TSI) level at time of surgery	Not available		>500	530		N/A	N/A		N/A
Presence of compressive optic neuropathy	Yes		No	Yes		No	No		No
CAS Score (Range 0-10)	7		7	8		N/A	N/A		N/A

“Pack-years” refers to the number of packs of cigarettes smoked per day multiplied by the number of years a person has smoked.

3. RESULT

3.1. Differentially Expressed Genes Between Severe, Active TED Patients and Healthy Controls

RNA-Seq yielded a total of 57,736 genes tested, and 352 genes were identified having adjusted p-values < 0.05 after excluding outliers. After filtering for a minimum read count of 5 for all cases and controls, 328 genes comprised the final differential expression signature of which 52 were downregulated and 276 were upregulated relative to controls Fig. (2). There were 44 genes with very low FDRs (< 0.0001), including 5 downregulated genes (-2.3 to -4.3 fold change) and 39 upregulated genes (+2.0 to +8.8 fold change) Table (2).

3.4. qRT-PCR Analysis of CCL2, S100A9, VCAN, and SERPINA1

Four upregulated genes were selected among the top-ranked differentially expressed genes in the signature for validation by qRT-PCR. S100A9 showed a 9.40-fold increased expression (p < 0.0001), CCL2 showed a 7.25-fold increased expression (p < 0.0001), VCAN showed a 1.68-fold increased expression (p = 0.0018), and SERPINA1 showed a 1.24-fold increased expression (p = 0.0811) among the cases group relative to the controls Fig. (3).

4. DISCUSSION

In this prospective case-control study, we sought to identify differences in the transcriptome between active, severe TED patients and healthy controls using NGS. Cases with active rather than quiescent TED were selected to ensure that differential gene expression findings were associated with an actively worsening clinical phenotype of TED. This study selected severe TED cases with a CAS of 7-8 and actively worsening signs and symptoms, with the goal of maximizing the yield of differentially expressed genes relevant to TED.

Understanding the pathogenesis of TED has been greatly facilitated by the study of cultured orbital fibroblasts from TED patients [12]. Previously published studies utilized the “candidate” gene approach to identify differential gene expression using cultured cell models and have identified multiple transcripts related to the inflammatory cascade [13]. These studies created primary cell cultures of orbital fibroblasts isolated from orbital adipose tissue, but the validity of such experiments conducted in tissue culture media without the in vivo environment and intact immune system remains unclear. Linquist et al studied fresh orbital tissues to assay gene expression for eight pre-selected cytokines [14]. While more physiological, this study was limited by the candidate gene approach, since a limited number of pre-selected transcripts were assayed. Moreover, the study included patients in the quiescent stage of TED with no active inflammation.

To enhance the validity of our findings, we eliminated confounding factors, such as tobacco smoking, use of systemic steroids or immunomodulatory medications, and orbital radiation therapy, all of which modulate the immune system and likely alter the transcription of genes involved in TED pathogenesis [2]. Previous studies have not controlled for these confounding factors [7, 8, 11, 14] or did not provide these data [9, 10]. We chose cases and controls of the same gender and racial ancestry and excluded potential subjects who were overweight or obese-due to their tendency toward systemic increased adipogenesis—or who had diabetes or metabolic syndrome. Diabetes medications like thiazolidinediones are known to activate Peroxisome Proliferator-Activated Receptors (PPARs), which have been linked to thyroid eye disease on a clinical basis as well as in previously published differential gene expression studies [9, 15, 16]. Not surprisingly, rosiglitazone, a thiazolidinedione, was found to be highly linked to differentially expressed genes in our study Table (5).

This is also the first study to use NGS technology in the form of RNA-Seq using the Illumina HiSeq2000 platform on in vivo orbital adipose tissue in TED. RNA-Seq provides a broader dynamic range, since unlike microarray technology, its gene expression measurement is not limited by background at the low end and signal saturation at the high end. It is able to yield absolute rather than relative expression values, and it offers superior sensitivity and specificity in differential expression studies [17].

Perhaps due to the central role of orbital fat expansion in TED, previous studies have focused on Wnt signaling genes [7, 9, 11], which regulate adipogenesis, as well as the IGF-1 pathway genes [9], which are implicated in adipogenesis and induce HA synthesis. Other studies have focused on upregulation of the immediate early genes, which are induced during the initial proliferative phase of preadipocytes and other adipogenesis-related genes [7, 8, 10]. Our study found several genes in the differential expression signature that were reported in prior studies, such as IGF1 (insulin-like growth factor 1), FADS1 (fatty acid desaturase 1), and immediate early genes BTG2 (BTG anti-proliferation factor 2). Moreover, 26 genes in the differential expression signature were involved in lipid binding and 12 genes were involved in glycosaminoglycan binding. However, our study selected different types of cases and controls from prior studies and looked broadly at the differential expression signature for enriched pathways and functions.

Among the biological functions and pathways enriched in the differential expression signature, the top functions and processes expectedly included immunoglobulin binding, leukocyte activation and migration, cell adhesion and small molecule binding, and various other immune-regulated functions. Previous studies have documented specific cytokine-dependent fibroblast activation [18]. IFN-gamma, TNF-alpha, and IL-1-alpha have been detected in orbital fat from patients with TED [19], and cytokines such as IL-4, IL-6, and IL-10 have been detected in affected extraocular muscle and orbital fat [20].

In addition to specifically identifying 20 genes involved in the chemokine pathway that were upregulated Table (4), our enrichment analysis identified the IL-5 signaling pathway as among the most enriched pathways with 12 upregulated genes identified Table (4). IL-5 is a cytokine produced by type-2 T helper cells and mast cells, is involved in stimulating B cell growth and immunoglobulin secretion, and is a key mediator of eosinophil production and activation [21]. Mepolizumab and reslizumab are IL-5 inhibitors that are FDA-approved for severe refractory asthma and eosinophil-mediated inflammation [22, 23]. Given the predominance of this pathway, these medications deserve further study for their potential role in treating TED.

Our analysis also demonstrated marked enrichment for the very-Low-Density Lipoprotein (vLDL) receptor activity function, with apolipoprotein B receptor and low density lipoprotein receptor both showing significant upregulation. Orbital adipogenesis is central to TED, and upregulation of the LDL receptor allows increased cholesterol and fatty acid uptake by cells that is necessary for lipid storage, cell division, and production of cholesterol-derived steroid hormones [24]. The Sterol-Regulatory Element Binding Protein (SREBP) pathway is the master regulator of the lipid biosynthetic pathway and mediates the increase in lipid biosynthesis [25]. Post-translational regulation of lipid biosynthesis would not be captured by RNA-Seq analysis, and follow-up functional studies at the protein level are planned to confirm these initial RNA-Seq findings.

Our enrichment analysis showed that simvastatin was linked to 35 genes in the signature, while atherosclerosis was linked with 6 genes in the signature Table (5). This finding is even more significant as none of our patients were taking hydroxymethylglutaryl-CoA reductase inhibitors (statins), which are known to upregulate the LDL receptor [24]. Statins have been shown to improve cholesterol levels, reduce cardiovascular events, and have anti-inflammatory actions independent of their cholesterol-lowering role [26]. A large, longitudinal cohort study of patients with Graves’ disease found that statin use was associated with reduced incidence of TED, although no similar associations were found for non-statin cholesterol lowering medications [27]. Given this epidemiological association and our preliminary gene expression data showing upregulation of APOBR and LDLR in TED and statins’ links to numerous upregulated genes in the signature, further molecular studies and clinical trials are warranted to evaluate the possible therapeutic benefits of statin therapy in TED.

The primary limitation of our study is its small sample size. The stringent inclusion and exclusion criteria for cases and controls resulted in limited patients eligible for the study. While the qRT-PCR data largely support the RNA-Seq data for differentially expressed genes, a greater sample of cases and controls for qRT-PCR experiments would further confirm the differential expression of genes identified in this study. Despite a small sample size, this study used NGS technology and obtained results with robust p-values and false discovery rates. Moreover, the prospective case-control study design and exclusion of potential confounders affecting gene expression enhance the validity of this study’s findings.

Another limitation of this study is that cases selected for our study had predominantly Type II TED with marked extraocular muscle enlargement. Thus, the differential expression signature in this study may not apply for milder cases, quiescent disease, or Type I TED cases mainly affected by orbital fat enlargement.

Finally, the value of RNA-Seq studies lies in their ability to generate hypotheses and candidate genes and pathways for further studies. While the qRT-PCR validation studies largely support the RNA-Seq data for differentially expressed genes, further studies with larger samples of cases and controls are needed to test the hypotheses generated from this study via qRT-PCR and Western Blot experiments. They might provide a more definitive answer on the involvement or lack of involvement of genes in the differential expression signature implicated in TED.

CONCLUSION

In conclusion, this study is the first prospective case-control study to investigate differential gene expression in orbital adipose tissue from active, severe TED patients and healthy controls. It is also the first to use RNA-Seq technology in profiling the transcriptome. The differential expression signature and enrichment analysis were concordant with prior differential expression studies and identified numerous genes involved in inflammation, cytokine signaling, adipogenesis, the IGF-1 signaling pathway, and glycosaminoglycan binding. Notably, it identified vLDL receptor activity, the LDL receptor, and the Apolipoprotein B receptor along with statins as being implicated in TED, thereby providing a molecular corroboration of epidemiological data linking statin used to reduced incidence of TED.⁴⁹ Finally, while our study findings are biologically plausible with robust statistical significance, larger confirmatory studies are needed with further exploration of the idiosyncrasies and diverse presentations of TED.

REFERENCES