Evaluating EV as a therapeutic target in Graves’ disease

Project Description

Evaluating EV as a therapeutic target in Graves’ disease

Graves’ disease is an autoimmune disease characterised by elevated circulating autoantibodies to the thyrotropin receptor (TRAB), resulting in hyperthyroidism. Inflammation, characteristic of Graves’ disease can lead to thyroid associated orbitopathy/opthalmopathy, an eye disease, which occurs in 30-50% of Graves’ disease patients, with 5% leading to blindness (1). Despite extensive research the pathogenesis of these diseases remains unclear.
Exosomes and microvesicles (collectively known as extracellular vesicles; EV) are membrane bound vesicles, released from all cells. They are found in all biological fluids, have a molecular profile representative of the cellular source and are elevated in the blood of patients with Graves’ disease(2). EV are thought to be key players in intercellular communication both locally and systemically and have been shown to activate the immune system either by direct stimulation of CD4+ T cells or via phagocytosis and presentation by dendritic cells (3). In addition, EV are able to inhibit the apoptosis of activated T cells exacerbating the inflammatory response, as shown in patients with rheumatoid arthritis whose EV have higher levels of membrane bound TNF compared with those from healthy controls and could prevent apoptosis in activated T cells (4).

The main treatment for Graves’ disease is the anti-thyroidal drug methimazole, which reduces inflammation, but the drug does not target the pathogenic process and relapse rate is very high after withdrawal (40-60%), thus adjunct treatment with the anti-inflammatory corticosteroid dexamethasone has been trialled (5). The results of this trial showed a reduction in serum TRAB, thyroid volume and relapse rate following treatment (5). Patients with active, moderate/severe Graves’ opthalmopathy are commonly treated with dexamethasone (6), but the use of dexamethasone as a treatment for Graves’ disease per se remains controversial as the side effects can be severe (acute/severe liver damage) (5).
It is postulated that EV (containing specific miRNAs) released from Graves’ tissue may stimulate the inflammatory immune response, aggravating the disease further and contributing to Graves’ opthalmopathy as increased miRNA-155 (a single stranded, small, non-coding RNA) has the potential to promote ocular inflammation and proliferation in this disease (7,8). The mechanism of action of methimazole and dexamethasone may be to reduce the release of exosomes making it imperative to understand the role of exosomes in these diseases so that therapies, alternative to dexamethasone with its associated side effects, which target EV and/or their contents, can be pursued.
The majority of EV work to date has been carried out on either cell lines or serum/plasma. Although serum and plasma are a rich source of EV, they contain a mix of vesicles derived from various cell types of both healthy and diseased origin making characterisation complicated and those derived from cell lines are a single cell source, which may not be representative of the multicellular nature of the tissue. The use of microfluidics to culture small (3mm3) pieces of human tissue (9,10), provides the distinct advantage of being able to isolate EV known to originate from the diseased thyroid and associated cells. The isolation of EV from HNSCC tissue maintained on the microfluidic device has already been demonstrated as has the maintenance of Graves’ disease tissue and the response to corticosteroids (11).
Hypothesis: Graves’ tissue maintained on a microfluidic culture device release more exosomes than healthy tissue with a unique profile. Treatment of Graves’ tissue with methimazole and dexamethasone reduces the release of EV.
Aims:
1) To isolate and characterise EV released from Graves’ disease and healthy thyroid tissue maintained on a microfluidic device
2) To investigate the effect of treatment on EV release.

EV generation & isolation: Graves’ disease tissue samples will be obtained from patients undergoing total thyroidectomy under existing ethical approval (LREC 17_LO_0209). Tissue specimens will be divided into separate microfluidic devices and perfused medium, containing 10% EV-free foetal bovine serum, at a rate of 2μl min-1. The device will be maintained at 37oC for up to 4 days, with and without treatment with methimazole and/or dexamethasone. Effluent will be collected throughout the culture and EV fractions isolated using the Total Exosome Isolation kit™ (Invitrogen). The concentration and size of EV will be determined using the Malvern®nanosight LM10.
EV characterisation: EV protein will be prepared from isolated vesicles and surface marker expression characterised using immunoblotting techniques for common EV surface markers, including tetraspanins (CD9, CD63, CD81 and CD82) and TSG10112. In addition RNA will be extracted from the EV, reverse transcribed and analysed using quantitative RT-PCR for the presence of miRNA-155 (TaqMan miRNA kit;Applied Biosystems)
Outcome: The results of this study will determine if EV can be isolated and characterised from healthy and Graves’ disease tissue maintained on a microfluidic device and whether treatment schedules alter EV release. The study will provide the basis for future research into the role Graves’-derived EV may play in immune cell modulation and thus alternative therapeutic avenues.

References:
1) Tanda ML et al. Prevalence and natural history of Graves' orbitopathy in a large series of patients with newly diagnosed graves' hyperthyroidism seen at a single center. J Clin Endocrinol Metab. 2013;98(4):1443-9.
2) Mobarrez F et al. The expression of microvesicles in the blood of patients with Graves’ disease and its relationship to treatment. Clin Endocrinol 2016;84,729-735.
3) Robbins PD and Morelli AE. Regulation of Immune Responses by Extracellular Vesicles. Nat Rev Immunol 2014;14(3):195-208
4) Zhang HG et al. A membrane form of TNF-alpha presented by exosomes delays T cell activation-induced cell death. J Immunol. 2006; 176(12):7385-93.
5) Mao XM et al. Prevention of Relapse of Graves’ Disease by Treatment with an Intrathyroid Injection of Dexamethasone J Clin Endocrinol Metab 2009;94(12):4984–4991.
6) Zang S et al. Intravenous glucocorticoids for Graves’ orbitopathy:efficacy and morbidity. J Clin Endocrinol Metab. 2011;96:320-332.
7) Li K et al. Increased microRNA-155 and decreased microRNA-146a may promote ocular inflammation and proliferation in Graves’ opthalmopathy. Med Sci Monit. 2014;20:639-643.
8) Ma XD et al. MicroRNAs in NF-kappa B signalling. J Mol Cell Biol. 2011;3:159-166.
9) Cheah R et al. Measuring the response of human head and neck squamous cell carcinoma to irradiation in a microfluidic model allowing customized therapy. Int J Oncol. 2017;51(4):1227-1238
10) Bower R et al. Maintenance of head and neck tumor on-chip: gateway to personalized treatment? Future Sci OA. 2017;3(2): FSO174.
11) McKenzie G et al. The effect of corticosteroids on the release of immune modulating factors from Graves' disease tissue maintained using microfluidic culture. European Journal of Surgical Oncology. 2017;43:2395.
12) Théry C et al. Membrane vesicles as conveyors of immune responses. Nat. Rev. Immunol. 2009;9:581-593.