2 resultados para PSORIASIS
em Glasgow Theses Service
The phosphodiesterase 4 (PDE4) family are cAMP specific phosphodiesterases that play an important role in the inflammatory response and is the major PDE type found in inflammatory cells. A significant number of PDE4 specific inhibitors have been developed and are currently being investigated for use as therapeutic agents. Apremilast, a small molecule inhibitor of PDE 4 is in development for chronic inflammatory disorders and has shown promise for the treatment of psoriasis, psoriatic arthritis as well as other inflammatory diseases. It has been found to be safe and well tolerated in humans and in March 2014 it was approved by the US food and drug administration for the treatment of adult patients with active psoriatic arthritis. The only other PDE4 inhibitor on the market is Roflumilast and it is used for treatment of respiratory disease. Roflumilast is approved in the EU for the treatment of COPD and was recently approved in the US for treatment to reduce the risk of COPD exacerbations. Roflumilast is also a selective PDE4 inhibitor, administered as an oral tablet once daily, and is thought to act by increasing cAMP within lung cells. As both (Apremilast and Roflumilast) compounds selectively inhibit PDE4 but are targeted at different diseases, there is a need for a clear understanding of their mechanism of action (MOA). Differences and similarity of MOA should be defined for the purposes of labelling, for communication to the scientific community, physicians, and patients, and for an extension of utility to other diseases and therapeutic areas. In order to obtain a complete comparative picture of the MOA of both inhibitors, additional molecular and cellular biology studies are required to more fully elucidate the signalling mediators downstream of PDE4 inhibition which result in alterations in pro- and anti-inflammatory gene expression. My studies were conducted to directly compare Apremilast with Roflumilast, in order to substantiate the differences observed in the molecular and cellular effects of these compounds, and to search for other possible differentiating effects. Therefore the main aim of this thesis was to utilise cutting-edge biochemical techniques to discover whether Apremilast and Roflumilast work with different modes of action. In the first part of my thesis I used novel genetically encoded FRET based cAMP sensors targeted to different intracellular compartments, in order to monitor cAMP levels within specific microdomains of cells as a consequence of challenge with Apremilast and Roflumilast, which revealed that Apremilast and Roflumilast do regulate different pools of cAMP in cells. In the second part of my thesis I focussed on assessing whether Apremilast and Roflumilast cause differential effects on the PKA phosphorylation state of proteins in cells. I used various biochemical techniques (Western blotting, Substrate kinase arrays and Reverse Phase Protein array and found that Apremilast and Roflumilast do lead to differential PKA substrate phosphorylation. For example I found that Apremilast increases the phosphorylation of Ribosomal Protein S6 at Ser240/244 and Fyn Y530 in the S6 Ribosomal pathway of Rheumatoid Arthritis Synovial fibroblast and HEK293 cells, whereas Roflumilast does not. This data suggests that Apremilast has distinct biological effects from that of Roflumilast and could represent a new therapeutic role for Apremilast in other diseases. In the final part of my thesis, Phage display technology was employed in order to identify any novel binding motifs that associate with PDE4 and to identify sequences that were differentially regulated by the inhibitors in an attempt to find binding motifs that may exist in previously characterised signalling proteins. Petide array technology was then used to confirm binding of specific peptide sequences or motifs. Results showed that Apremilast and Roflumilast can either enhance or decrease the binding of PDE4A4 to specific peptide sequences or motifs that are found in a variety of proteins in the human proteome, most interestingly Ubiquitin-related proteins. The data from this chapter is preliminary but may be used in the discovery of novel binding partners for PDE4 or to provide a new role for PDE inhibition in disease. Therefore the work in this thesis provides a unique snapshot of the complexity of the cAMP signalling system and is the first to directly compare action of the two approved PDE4 inhibitors in a detailed way.
Psoriasis is a common, debilitating systemic inflammatory disorder that is characterised by sharply demarcated, thick, erythematous scaly skin plaques. Such plaques commonly appear on skin that is subjected to repeated tensile trauma, such as elbows, knees and flexures. The mechanism by which these inflammatory lesions are spatially restricted is not known and yet knowledge of this could be of critical importance for our understanding of this disease. Chemokines are the principal regulators of leukocyte migration and play a critical role in the initiation and maintenance of inflammation. The atypical chemokine receptor ACKR2 (formerly D6) binds inflammatory CC-chemokines, but does not signal upon ligand binding; instead ACKR2 internalises and helps degrade such chemokines, after which it continues to cycle back to the cell surface. ACKR2 acts, through this mechanism, as a high-capacity scavenger of chemokines, and plays an important role in regulating inflammation. It is known that ACKR2 expression is high in unaffected skin in patients with psoriasis (remote from inflammatory plaques) and concurrently deficient in the plaques themselves. Additionally, human studies have shown that simple skin trauma in psoriasis patients causes a reduction in cutaneous ACKR2 expression at the site of trauma. However, the functional significance and the molecular mechanism by which it occurs are not understood. This thesis explored the role of ACKR2 in the spatial restriction of psoriasiform inflammation and the molecular mechanisms for its differential regulation. Through the use of disease relevant mouse models, primary human cell cultures and novel cell migration assays, the results presented here show that localised psoriasiform inflammation upregulates ACKR2 in remote tissues through the systemic release of cytokines. This remotely upregulated ACKR2 expression protects tissues from the further spread of inflammation. This protective effect is mediated by stromally expressed ACKR2 that acts to control inflammatory T-cell positioning within the skin. Tensile trauma of keratinocytes however, acted to reduce ACKR2 expression in the context of inflammation, which in turn provides a novel mechanism for the well-characterised phenomenon that occurs in psoriasis (and a range of skin condition) termed ‘koebnerisation’. Koebnerisation refers to the phenomenon by which relatively simple skin trauma induces the development of disease-specific skin lesions. Furthermore, this thesis defines novel disease-relevant regulators of ACKR2 expression. In silico analyses identified psoriasis-associated microRNAs that bound to the 3’-UTR of ACKR2, and reduced its expression at transcriptional and protein level. Importantly, trauma of keratinocytes induced ACKR2 downregulation concurrent with a substantial and significant increase in the expression of the identified ACKR2 targeting microRNAs. Together, this thesis defines a novel mechanism by which ACKR2-mediated regulation of chemokine function, cutaneous trauma, microRNAs and systemic cytokines, co-ordinately modulate the predisposition of remote tissue sites to the development of new lesions. Importantly, the results presented here have profound implications for how spatial restriction is imposed on inflammation. The data also highlight therapeutic ACKR2 induction as a plausible novel strategy for the limitation and treatment of psoriasiform- and potentially other forms of inflammation.