Tony Day, MA DPhil
Inflammation and innate immunity

Localisation of CFH-binding sites in retinal tissue from a human eye

Inflammation is part of our body’s natural defence against infection and is also required to successfully heal wounds. However, if inflammation continues unchecked (and becomes chronic) then damage to our tissues occurs; e.g., as is seen in the joints of individuals with arthritis. It is our aim to better understand the molecular processes that occur during inflammatory diseases and how this is altered during normal ageing. This includes investigating the role of the innate immune system in a major form of blindness (age-related macular degeneration or AMD) and how an intrinsic protective mechanism, triggered by acute inflammation, may serve to prevent cartilage and bone breakdown. Here our goal is to translate our research findings into the development of novel therapeutics and diagnostics for AMD and musculoskeletal disease.

3D structure of CUB module from human TSG-6A major focus for our studies is a type of sugar that is found on all cell surfaces and in the matrix of every tissue. These so called, GAGs, determine the location and functions of protein molecules and therefore play a key role in human biology. For example, we have found that a mutation in an immune system protein, which increases the risk of AMD, alters its binding to GAGs found in a particular part of the human retina. A reduced amount of this immune regulator at this site (caused in part by an age-related loss of GAGs) is likely to lead to local chronic inflammation and thus directly contribute to the AMD disease process and loss of central vision. In addition, we are determining how a GAG-binding protein, called TSG-6, can be harnessed for the treatment of osteoarthritis and osteoporosis.

Mouse “egg” before ovulation: cumulus-oocyte complex showing cumulus cells (blue) and ECM (red) surrounding the oocyte (black)We also have had a long-term interest in the role of GAGs and GAG-binding proteins in mammalian ovulation – a process that has things in common with inflammation. Before the egg is released from the ovary a jelly-like matrix forms rapidly round the egg; this protects the egg during ovulation and is also necessary for the attachment of sperm to the egg during fertilisation. Thus the formation of this matrix is critical for female fertility. Recent work has characterised several of the molecular interactions that organise and stabilise this essential matrix, providing insight into similar matrices made during inflammatory processes.

For research professionals

Glycosaminoglycan-protein interactions in inflammatory processes

3D structure of CD44 in complex with hyaluronan

 

Our main area of interest is the role of glycosaminoglycan-binding proteins in inflammatory disease (e.g. arthritis and age-related macular degeneration (AMD)) and in inflammation-like processes (e.g. ovulation). Glycosaminoglycans (GAGs) are linear polysaccharides that are key components of matrix as well as being found ubiquitously on cell surfaces. For example, the interactions of hyaluronan (HA) – a high molecular weight GAG – with specific HA-binding proteins (hyaladherins) are responsible for the mechanical properties of cartilage and many other tissues. They also play a key role in the formation of an elastic ‘cumulus’ matrix around the oocyte required for ovulation/fertilisation as well as mediating immune cell trafficking. 

 

One of the long-standing aims of our research is to determine the structural basis and molecular regulation of GAG-protein interactions. We have made significant progress on this, for example, through X-ray crystallographic and NMR analysis of TSG-6 and CD44; CD44 is the major cell surface receptor for HA, and TSG-6 is a secreted protein often associated with inflammation that has been implicated in mediating many of immunomodulatory and tissue protecting effects of mesenchymal stem/stromal cells. Our recent work has also used biophysical techniques such as AUC, QCM-D, SAXS, SEC-MALLS and SPR to help characterise protein/GAG complexes/interactions. 

In addition to HA, TSG-6 binds to sulphated GAGs (i.e. chondroitin-4-sulphate (C4S), dermatan sulphate (DS), heparan sulphate (HS) and heparin) as well as a growing list of proteins (e.g. including 10 chemokines, 7 BMPs and RANKL) where these interactions likely underpin many of TSG-6’s anti-inflammatory and tissue protective activities. For example, we have shown that the interaction of TSG-6 with CXCL8 prevents the binding of this chemokine to HS on the endothelial surface and thereby inhibits the transendothelial migration of neutrophils; recent studies have identified TSG-6 as the first soluble mammalian chemokine-binding protein with the potential to modulate the migration/activity of a wide variety leukocyte subsets. TSG-6 also interacts with inter-a-inhibitor (IaI) and catalyses the covalent transfer of heavy chains from this serum protein onto HA to form HC•HA complexes that are essential for the formation/stability of the cumulus matrix and where this modification of HA can also occur at sites of inflammation; our recent studies have determined the role of metal ions in TSG-6-mediated HC•HA formation and shown that PTX3 plays a critical role in cross-linking the HC•HA complexes to form a stable HA matrix. Furthermore, our work has determined that TSG-6 can protect cartilage and bone in models of arthritis and osteoporosis. Research is in progress to develop a TSG-6-based biologic for the treatment of osteoarthritis, where our drug target may also have utility for a wide range of other inflammatory diseases. 

Schematic showing TSG-6-mediated heavy chain (HC) transfer from II onto hyaluronan (HA) and 2 possible mechanisms of HA cross-linking

 

Another key area of interest is the investigation of the role of complement factor H (FH) in AMD, which is the predominant cause of blindness in the industrialised world; the Y402H polymorphism in the FH gene (first described by Tony in 1988) has been implicated as a major risk factor for developing AMD. We have shown that this Tyr to His coding change has a large effect on the binding of FH to sulphated GAGs, e.g. HS and DS present in the Bruch’s membrane of the human retina, a matrix structure that is the site of AMD pathology. In addition, we have recently found that there is a large reduction in the amount of HS present in the Bruch's membrane as a consequence of normal ageing. We have hypothesised that the poorer binding of the disease-associated 402H variant could lead to chronic local inflammation (due to complement dysregulation), contributing directly to the development and/or progression of AMD. We are now investigating how other age-related changes in Bruch’s membrane composition may drive AMD pathogenesis.

Recent discoveries

 

Recent key publications

Dyer, D.P., Salanga, C.L., Johns, S.C., Valdambrini, E., Fuster, M.L., Milner, C.M.*, Day, A.J.* and Handel, T.M.* (2016) The anti-inflammatory protein TSG-6 regulates chemokine function by inhibiting chemokine-glycosaminoglycan interactions. J Biol Chem. In press. Published as a Paper in Press on 4 April. *Co-corresponding authors.

Briggs, D.C., Birchenough, H.L., Ali, T., Rugg, M.R., Waltho, J.P., Ievoli, E., Jowitt, T.A., Enghild, J.J., Richter, R.P., Salustri, A., Milner, C.M. and Day, A.J.* (2015). Metal ion-dependent heavy chain transfer activity of TSG-6 mediates assembly of the cumulus oocyte matrix. J Biol Chem. 290, 28708-23. PubMed *Corresponding author. Featured on Front Cover on 27th November 2015.

Baranova, N.S., Inforzato, A., Briggs, D.C., Tilakaratna, V., Enghild, J.J., Thakar, D., Milner, C.M., Day, A.J.* and Richter, R.P.* Incorporation of pentraxin 3 into hyaluronan matrices is tightly regulated and promotes matrix cross-linking. J Biol Chem. 289, 30481-30498. *Co-corresponding authors. PubMed

Clark, S.J., Ridge, L.A., Herbert, A.P., Hakobyan, S., Mulloy, B., Lennon, R., Wurzner, R., Morgan, B.P., Urhin, D., Bishop, P.N. and Day, A.J. (2013). Tissue-specific host recognition by complement factor H is mediated by differential activities of its glycosaminoglycan-binding regions. J. Immunol. 190, 2049-2057.PubMed

Full list of publications