Final Report Summary - PSORIASIS IL17 (Investigating the role of dysregulated Interleukin-17 signalling in the pathogenesis of psoriasis)
• Summary description of the project objectives
Psoriasis is a common chronic inflammatory condition of the skin, characterised by inflamed scaly red psoriatic plaques on the epidermis of sufferers. It is highly prevalent, affecting 1-3% of the global population (approximately 125 million people). As psoriasis can affect all parts of the body, including the face, scalp, trunk, limbs, the genital area, the soles of the feet and the palms of the hands, it can affect a patient’s ability to work, their daily lives and their self-esteem and many sufferers report a loss of quality of life, which correlates with severity of disease. 20% of patients develop severe psoriasis, which is also associated with an increased risk of cardiovascular disease and metabolic syndrome. 25% of sufferers also develop incapacitating psoriatic arthritis. However, to date, the link between psoriasis and associated co-morbidities is not well understood.
The Interleukin-17 (IL-17) cytokines are an important family of chemical messengers that orchestrate immune responses to bacterial and fungal infection. The prototypical member of the IL-17 family is IL-17A and is primarily produced by a subset of T helper cells called Th17 cells. Despite the important immune function of Th17 cells, they have been implicated in the pathogenesis of many autoimmune and chronic inflammatory diseases, including multiple sclerosis, chronic inflammatory bowel disease, rheumatoid arthritis and psoriasis. Cytokines function by binding to their specific receptors on target cells and initiating a cascade of intracellular events that carries the signal from the cell surface to the nucleus of the cell, where transcription factors regulate pro-inflammatory gene expression. These signalling pathways utilise a multitude of molecules to relay this message and these are subject to feedback inhibition and protein modifications, such as phosphorylation, that fine-tune the signal to tailor specific responses to different cytokine stimuli. How IL-17 cytokines mediate the inflammatory effects in the skin that are associated with psoriasis remains incompletely understood and it was the aim of this project, PsoriasisIL17, to identify new signalling pathways initiated by IL-17 cytokines that may be responsible for their contribution to inflammatory diseases including psoriasis.
One such regulator that we previously described, is an orphan receptor of the IL-17 receptor family, IL-17 Receptor D (IL-17RD). We have shown that IL-17RD dampens immune responses triggered by IL-17A, by interfering with downstream signalling events. It was our aim to investigate the role of IL-17RD in psoriasis pathogenesis and whether it played an important protective role against developing the disease.
While IL-17A is mostly produced by immune cells, another IL-17 cytokine, IL-17C, is actually secreted by epithelial cells including skin keratinocytes. It was also our objective to determine how IL-17C was regulated in psoriasis pathogenesis and how it contributed to early events of disease initiation.
• Description of the work performed since the beginning of the project
To answer these questions we cultured human primary keratinocytes in vitro as studying cells alone without influence from infiltrating immune cells or other skin resident cells such as fibroblasts and Langerhan cells, allowed us to assess the response of these cells to pro-inflammatory stimuli, including IL-17A and IL-17C. We were able to dissect the effects of the IL-17A cytokine and the signalling pathways it induced. In particular, we were able to characterise the initiation of a Syk-dependent pathway utilised by IL-17 cytokines in keratinocytes. Using siRNA technology to “silence” Syk expression we were able to confirm that it is an important regulator of inflammation in this context. Using pharmacological inhibiton of Syk and other kinases we were able to map out which kinase activated the next in the signalling cascade.
Mimicking stress in keratinocytes by means of infection, chemical and mechanical stress we were able to determine that IL-17C is quickly upregulated in response to these stimuli and we were also able to assess the autocrine effects of IL-17C on keratinocytes. We established that stressed keratinocytes produced IL-17C and this subsequently prompted these cells to produce pro-inflammatory cytokines. This gives us hints into the initiation phase of psoriasis as previously this hasn’t been well understood. This effect also mirrors the Koebner phenonomen in psoriasis patients, where a scratch on unaffected skin would develop into a psoriatic lesion. Additionally, we generated a transgenic mouse that develops psoriatic-like disease from an early age. This mouse harbours a mutation in a gene expressed in keratinocytes. Characterisation of this mouse has enabled us to determine the early triggers of psoriasis initiated by keratinocytes.
We were also able to determine a protective role of IL-17RD in psoriatic skin disease. We observed, using immunohistochemistry and quantititative PCR, that expression of IL-17RD was diminished in psoriatic tissue and this correlated with an increased expression of pro-inflammatory molecules. Interestingly, we also found IL-17RD expression on a specialised set of macrophages. While macrophages are key players in combating microbial infection and inducing inflammation, they can show very different cytokine signatures both pro- and anti-inflammatory and IL-17RD was exclusively expressed on a subset of anti-inflammatory macrophages. This would suggest that IL-17RD has an endogenous ligand that activates these macrophages to counteract inflammation. Discovering such an agonist would be of keen interest. The capability to activate an anti-inflammatory pathway rather than blocking inflammation (which is necessary for proper immune system function) may represent a more therapeutic attractive route for the future.
• Description of the main results achieved so far
The aim of this project was to further our understanding of the pro-inflammatory effects initiated by IL-17 by investigating how IL-17 can activate other molecules and how these contribute to inflammation and to psoriasis pathogenesis. We have characterised an orphan receptor of the IL-17 family of a tight regulator of IL-17 functional effects. Moreover expression of this receptor negatively correlates with psoriasis disease severity and is expressed on a specialist cell-type of the immune system in chronic inflammatory disease. Identifying an agonist for such a receptor will be a favourable therapeutic approach for the future.
We identified a new target of IL-17, a protein that is encoded by a gene that has been previously shown to be mutated in some psoriasis patients. We have generated mutants of this gene and used in in vitro experimental approaches and have identified a number of transcription factors that this protein activates and subsequent pro-inflammatory cytokine and chemokine (which act as chemoattractants to immune cells) readouts induced by these mutants. With this knowledge, we have identified a mechanism of action of why psoriasis can be initially triggered by stress and infection in keratinocytes but also perpetuated by infiltrating immune cells, such as Th17 cells that migrate to the skin in response to chemokines produced by keratinocytes.
• Expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far)
Identification of new players in the initiation of psoriasis offers alternative therapeutic avenues to explore and actually there are already therapies available targeting these molecules that are used as cancer treatments. We believe we have made a considerable advancement on the state of the art in the field of psoriasis and we plan to exploit some of our findings for commercialisation gains in the near future. We have also developed a mouse model of psoriatic disease that highlights the central role of keratinocytes in initiating disease progression. This model, would also be a useful test-bed for current and future therapeutics. We believe that these discoveries will have far reaching consequences for human health in the area of skin diseases such as psoriasis. The psoriasis drug market is estimated to be worth $7.6 billion by 2022 and it is imperative that the European Research Area develops new intellectual property and therapies in this area. New therapeutics will also contribute to a decrease in hospital consultations and lost working hours within the European workforce.
Psoriasis is a common chronic inflammatory condition of the skin, characterised by inflamed scaly red psoriatic plaques on the epidermis of sufferers. It is highly prevalent, affecting 1-3% of the global population (approximately 125 million people). As psoriasis can affect all parts of the body, including the face, scalp, trunk, limbs, the genital area, the soles of the feet and the palms of the hands, it can affect a patient’s ability to work, their daily lives and their self-esteem and many sufferers report a loss of quality of life, which correlates with severity of disease. 20% of patients develop severe psoriasis, which is also associated with an increased risk of cardiovascular disease and metabolic syndrome. 25% of sufferers also develop incapacitating psoriatic arthritis. However, to date, the link between psoriasis and associated co-morbidities is not well understood.
The Interleukin-17 (IL-17) cytokines are an important family of chemical messengers that orchestrate immune responses to bacterial and fungal infection. The prototypical member of the IL-17 family is IL-17A and is primarily produced by a subset of T helper cells called Th17 cells. Despite the important immune function of Th17 cells, they have been implicated in the pathogenesis of many autoimmune and chronic inflammatory diseases, including multiple sclerosis, chronic inflammatory bowel disease, rheumatoid arthritis and psoriasis. Cytokines function by binding to their specific receptors on target cells and initiating a cascade of intracellular events that carries the signal from the cell surface to the nucleus of the cell, where transcription factors regulate pro-inflammatory gene expression. These signalling pathways utilise a multitude of molecules to relay this message and these are subject to feedback inhibition and protein modifications, such as phosphorylation, that fine-tune the signal to tailor specific responses to different cytokine stimuli. How IL-17 cytokines mediate the inflammatory effects in the skin that are associated with psoriasis remains incompletely understood and it was the aim of this project, PsoriasisIL17, to identify new signalling pathways initiated by IL-17 cytokines that may be responsible for their contribution to inflammatory diseases including psoriasis.
One such regulator that we previously described, is an orphan receptor of the IL-17 receptor family, IL-17 Receptor D (IL-17RD). We have shown that IL-17RD dampens immune responses triggered by IL-17A, by interfering with downstream signalling events. It was our aim to investigate the role of IL-17RD in psoriasis pathogenesis and whether it played an important protective role against developing the disease.
While IL-17A is mostly produced by immune cells, another IL-17 cytokine, IL-17C, is actually secreted by epithelial cells including skin keratinocytes. It was also our objective to determine how IL-17C was regulated in psoriasis pathogenesis and how it contributed to early events of disease initiation.
• Description of the work performed since the beginning of the project
To answer these questions we cultured human primary keratinocytes in vitro as studying cells alone without influence from infiltrating immune cells or other skin resident cells such as fibroblasts and Langerhan cells, allowed us to assess the response of these cells to pro-inflammatory stimuli, including IL-17A and IL-17C. We were able to dissect the effects of the IL-17A cytokine and the signalling pathways it induced. In particular, we were able to characterise the initiation of a Syk-dependent pathway utilised by IL-17 cytokines in keratinocytes. Using siRNA technology to “silence” Syk expression we were able to confirm that it is an important regulator of inflammation in this context. Using pharmacological inhibiton of Syk and other kinases we were able to map out which kinase activated the next in the signalling cascade.
Mimicking stress in keratinocytes by means of infection, chemical and mechanical stress we were able to determine that IL-17C is quickly upregulated in response to these stimuli and we were also able to assess the autocrine effects of IL-17C on keratinocytes. We established that stressed keratinocytes produced IL-17C and this subsequently prompted these cells to produce pro-inflammatory cytokines. This gives us hints into the initiation phase of psoriasis as previously this hasn’t been well understood. This effect also mirrors the Koebner phenonomen in psoriasis patients, where a scratch on unaffected skin would develop into a psoriatic lesion. Additionally, we generated a transgenic mouse that develops psoriatic-like disease from an early age. This mouse harbours a mutation in a gene expressed in keratinocytes. Characterisation of this mouse has enabled us to determine the early triggers of psoriasis initiated by keratinocytes.
We were also able to determine a protective role of IL-17RD in psoriatic skin disease. We observed, using immunohistochemistry and quantititative PCR, that expression of IL-17RD was diminished in psoriatic tissue and this correlated with an increased expression of pro-inflammatory molecules. Interestingly, we also found IL-17RD expression on a specialised set of macrophages. While macrophages are key players in combating microbial infection and inducing inflammation, they can show very different cytokine signatures both pro- and anti-inflammatory and IL-17RD was exclusively expressed on a subset of anti-inflammatory macrophages. This would suggest that IL-17RD has an endogenous ligand that activates these macrophages to counteract inflammation. Discovering such an agonist would be of keen interest. The capability to activate an anti-inflammatory pathway rather than blocking inflammation (which is necessary for proper immune system function) may represent a more therapeutic attractive route for the future.
• Description of the main results achieved so far
The aim of this project was to further our understanding of the pro-inflammatory effects initiated by IL-17 by investigating how IL-17 can activate other molecules and how these contribute to inflammation and to psoriasis pathogenesis. We have characterised an orphan receptor of the IL-17 family of a tight regulator of IL-17 functional effects. Moreover expression of this receptor negatively correlates with psoriasis disease severity and is expressed on a specialist cell-type of the immune system in chronic inflammatory disease. Identifying an agonist for such a receptor will be a favourable therapeutic approach for the future.
We identified a new target of IL-17, a protein that is encoded by a gene that has been previously shown to be mutated in some psoriasis patients. We have generated mutants of this gene and used in in vitro experimental approaches and have identified a number of transcription factors that this protein activates and subsequent pro-inflammatory cytokine and chemokine (which act as chemoattractants to immune cells) readouts induced by these mutants. With this knowledge, we have identified a mechanism of action of why psoriasis can be initially triggered by stress and infection in keratinocytes but also perpetuated by infiltrating immune cells, such as Th17 cells that migrate to the skin in response to chemokines produced by keratinocytes.
• Expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far)
Identification of new players in the initiation of psoriasis offers alternative therapeutic avenues to explore and actually there are already therapies available targeting these molecules that are used as cancer treatments. We believe we have made a considerable advancement on the state of the art in the field of psoriasis and we plan to exploit some of our findings for commercialisation gains in the near future. We have also developed a mouse model of psoriatic disease that highlights the central role of keratinocytes in initiating disease progression. This model, would also be a useful test-bed for current and future therapeutics. We believe that these discoveries will have far reaching consequences for human health in the area of skin diseases such as psoriasis. The psoriasis drug market is estimated to be worth $7.6 billion by 2022 and it is imperative that the European Research Area develops new intellectual property and therapies in this area. New therapeutics will also contribute to a decrease in hospital consultations and lost working hours within the European workforce.