Phil's Web Sight

The eye has evolved many mechanisms to prevent significant inflammation, that can lead to visual impairment. We have been investigating why in patients with uveitis, the eye cannot control the inflammation. Our recent studies showed that apoptosis (cell death) of inflammatory T cells is inhibited during uveitis by the cytokine IL-6.

The pathway used is peculiar in that the receptor for IL-6, rather than being expressed on the surface of the T cells, is present as a soluble molecule in inflammatory ocular fluids. The combination of IL-6 and sIL-6R results in trans-signalling and subsequent inhibition of apoptosis in uveitis. Our current studies are focused on understanding how T cells, such as those that infiltrate the eye in uveitis, regulate their proliferation, differentiation and survival. In particular we are studying the regulation of cytokine receptors, including the IL-6R. Our initial results suggest that as T cells differentiate towards effector T cells they down-regulate their responses to a number of cytokines. We hope that once we understand the functioning of these molecules on T cells we will be able to examine their potential role in inflammatory diseases including uveitis.

The number of inflammatory cells accumulating within the eye in uveitis will be controlled by a number of factors. As well as our studies of apoptosis we have examined the role of chemokines and their receptors that play a major role in recruiting and positioning cells within tissues. We determined that one receptor, CXCR4, was expressed at increased levels and that the very high levels of CXCR4 were directly due to the treatment with glucocorticoids. These studies will have a profound impact on how lymphocyte numbers are regulated both under resting conditions and at sites of inflammation.

Over the past few years we have been using a new technology, Luminex, to analyse the aqueous fluid from the anterior chamber of the eye. We were able to measure many cytokines and chemokines produced during inflammatory episodes. We have studied the data using the latest bioinformatic analysis. Our latest results identified the key molecules in the eye that define the inflammatory environment in uveitis, distinct from the resting non-inflammatory situation. We have recently adapted this technique to looking at the vitreous fluid in the eye.

We are focused on understanding how the inflammatory environment that we have identified in uveitis, affects the immunoregulatory properties of the eye. We are now looking at the activation and function of dendritic cells (DCs), cells that are central to the control of immune responses. We propose that the ocular microenvironment during health and disease has differential effects on the maturation and function of DCs, thereby maintaining immune privilege or leading to an active inflammatory process.

Glucocorticoids (natural steroid hormones) are pivotal to the regulation of the inflammatory mechanisms of the ocular microenvironment.

We have a specific interest in the expression of a bidirectional isozyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) that inter-converts active cortisol and inactive cortisone. Over the last decade, 11beta-HSD1 has emerged as a critical determinant of glucocorticoid function in tissues such as the liver, adipose and bone. Interest in the isozyme has escalated primarily because of its putative role in diseases such as human obesity, insulin resistance and osteoporosis, and also its role in the regulation of immune-cell function and inflammation.

The eye represents an important corticosteroid target tissue and our descriptive studies have localised 11beta-HSD1 to the human corneal epithelium and ciliary body epithelium. We believe that this enzyme may be a feature of a number of blinding conditions. Our ongoing studies focus on several areas:

Glaucoma - This is a leading cause of blindness in the Western world resulting from high pressure inside the eye causing permanent damage to the main nerve of sight (optic nerve). We have already shown that 11beta-HSD1 is involved in keeping the pressure inside the eye normal, and that the pressure can be reduced if the action of 11beta-HSD1 is prevented by a tablet called carbenoxolone, a non-specific inhibitor of 11beta-HSD1. Through a material transfer agreement with Pfizer Inc we are now examining specific 11beta-HSD1 inhibitors in the laboratory. We hope that our results may pave the way for a new treatment for glaucoma.

The Ocular Surface - Corneal scarring where the clear window of the eye becomes damaged from infection or inflammation is a leading cause of blindness worldwide. The cornea provides both protective and refractive properties essential for sight. The corneal epithelium is the most superficial layer formed from highly specialised cells that are rapidly proliferating from a peripheral (limbal) stem cell population, replenishing the ocular surface. Our data have demonstrated the expression of 11beta-HSD1 to the basal cells of the corneal epithelium. Our current studies are exploring the role of this isozyme in ocular surface renewal, combating inflammation via the generation of local cortisol, and fighting infection through activation of sentinel receptors called toll-like receptors. We believe that 11beta-HSD1 is of paramount importance to the ocular surface.

Thyroid hormone related eye problems (‘bulgy’ eyes) - This is a common condition where 5% of patients can go blind. Thyroid-associated ophthalmopathy is a condition where the tissues behind the eyes become inflamed and swollen often associated with an overactive thyroid gland in the neck. This not only causes the eyes to be pushed forwards (proptosis) becoming red and sore, but the swelling squeezes the optic nerve behind the eye (optic neuropathy) and this can result in complete blindness. The two most common reasons for the eye becoming bulgy are firstly, too much fat is made in the eye socket ad secondly, inflammation causes swelling. As 11beta-HSD1 appears to be involved in increasing the amount of fat in some obese patients, and is a key player in inflammation, we are currently investigating how 11beta-HSD1 influences orbital fat physiology and inflammation within the socket of patients with thyroid-associated ophthalmopathy providing us with a better understanding of the disease.

Intracranial pressure (ICP) - Cerebrospinal fluid (CSF) is a clear fluid that surrounds the brain and spinal cord. It is crucial for normal brain function and provides a very important role in protecting the brain from injury. If too much CSF accumulates in the space around the brain, the intracranial pressure (the pressure of CSF surrounding the brain) increases and causes visual loss by compression of the optic nerve. We believe that the ICP may be regulated 11beta-HSD1 in the same way it regulates aqueous humour within the eye and intraocular pressure. By conducting a range of laboratory and clinical-based studies we hope to define 11beta-HSD1 as a novel determinant of CSF dynamics and ICP balance. If this is the case, 11beta-HSD1 could be targeted for the treatment of patients with raised ICP thereby preventing visual loss.

The cornea is the transparent window at the front of the eye, forming a vital barrier preventing pathogens, such as bacteria and viruses from entering the eye. Disruption of ocular defence mechanisms following contact-lens wear, corneal trauma, herpes simplex virus, corneal anaesthesia, corneal exposure, and ocular surface disease predisposes the eye to severe corneal infection known as microbial keratitis (one of the commonest causes of worldwide blindness). The patient complains of a painful red eye, associated with loss of vision and sticky discharge. Many cases are sudden in onset, whereas others are insidious. Over one third of patients require hospital admission. Corneal ulceration, abscess formation, perforation and loss of eye are recognised sequelae. Visual morbidity of hospitalised cases is inevitable and ranges from reduction in quality of vision to complete blindness. Binocularity is compromised thus impacting upon the patient’s ability to undertake common everyday tasks involving stereopsis, such as picking-up objects, driving and crossing roads.

The cornea must be able to protect the eye from infection, but at the same time control the immune response to eliminate the pathogens. Protective cells recognise invading pathogens through molecules called Toll-like receptors (TLR). Naturally occurring hormones such as Vitamin D and cortisol are known to reduce inflammation. We believe that the organisation of immune responses to infections in the cornea is, in part, mediated through a complex interaction between TLR and hormonal signalling. We are investigating the presence, function and cross-talk between both these pathways in the cornea. Understanding how the cornea deals with infection and the resulting immune response will provide new insights into corneal biology and will lead to novel therapies for treatment of this sight-threatening disease.

In studies on patients with retinal vasculitis we have identified two changes in genes (polymorphisms) that relate to progression of the disease. Polymorphisms that encode for high production of a proinflammatory cytokine and low production of an anti-inflammatory cytokine, when found in the same individual were associated with a poor outcome. This finding suggests that these patients may need different forms of treatment to allow an improved outcome. Similarly, in patients with Behçet’s disease we have confirmed the association of a particular form of the stress induced molecule MICA. Current studies are underway to determine the functional significance of the link between expression of this molecule and HLA-B51, which together help to control recognition and clearance of stressed (infected) cells. However, analysis of molecules that recognise bacterial products, Toll-like receptors and CARD15, that have been linked to susceptibility to bacterial infection and Crohn’s disease respectively, did not associate with Behçet’s disease in our patient cohorts. The search for polymorphisms in these general control molecules is continuing.

Macrophages are scavenger cells that under normal circumstances engulf and destroy damaged or dying cells. Macrophages also act as sentinel cells and rapidly respond to bacterial and viral infection. The ocular microenvironment is regarded as suppressive to the immune response via various secreted molecules, a mechanism thought to protect the eye from immune mediated pathology. However, when infection occurs this forms a paradox as the ocular tissues need to eliminate the pathogen at the same time as they are inhibiting the immune response. The cell central to this paradox is the macrophage. Current studies are using human macrophages grown in environments that mimic the ocular tissue and analysing responses to pathogens. It is envisaged that these studies will identify switch mechanisms that determine the type of response macrophages will produce to bacterial infection in conditions such as endophthalmitis. This work forms a natural link with the 11-beta HSD research outlined above, as this enzyme may be a crucial regulator of macrophage function.

MLST is a specific method of typing bacteria. It has recently been applied to a number of bacterial species. We are performing MLST on bacteria that cause sight threatening infections of the eye - keratitis (infection of the cornea) commonly caused by Pseudomonas, particularly in contact lens wearers, and endophthalmitis (infection inside the eye that can follow cataract surgery) and is caused by Staphylococcal and Propionibacter species. We are developing our own Propionibacter MLST database. Once these bacteria have been typed we will try to develop a rapid molecular diagnostic test for these potentially blinding infections.

Metabolomics involves running a biological sample (serum, plasma, urine) through a nuclear magnetic resonance (NMR) machine. A whole array of small metabolites is measured and forms a specific profile, which can be measured by a computer programme, principal component analysis, which studies the area under each peak. Therefore one profile can be compared with another. We have shown that vitreous humour samples from patients undergoing retinal surgery can be separated by metabolomics based on the disease type. Although preliminary, the data points to the possibility that metabolomic analysis will provide a simple diagnostic method for ocular disease.