Why Ichthyosis Skin is Scaly Understanding why ichthyosis skin is scaly Update 2010 In Lamellar ichthyosis, skin structure can be compromised, like a defective brick wall, and this sends signals to the skin making cells divide more quickly, resulting in scaling. We refer to this as hyperkeratosis. Although there are several genetic causes of lamellar ichthyosis, it is not known whether the mechanism of scaling is common to all lamellar ichthyosis. We can now accurately model lamellar ichthyosis in the laboratory, producing skin that looks almost identical to patient skin (see below). We have found that increased interleukin-1 alpha may be a master switch responsible for many of the symptoms of this disease. Furthermore, interleukin-1 alpha is increased in all patients with lamellar ichthyosis we have investigated. We have confirmed that increasing interleukin-1 alpha alone is enough to cause skin thickening and that blocking the function of this gene alone is enough to prevent scaling in the disease mimic skin (see below). We are confident that blocking interleukin-1 alpha could be a new therapy for treating the scaling in the disease, in conjunction with, or instead of retinoids. Pump Priming No other grant applications on this subject have been made to date, as we wanted the publication to come out first. We intend to submit a grant application moving this discovery towards a therapy in the autumn. Publications arising O’Shaughnessy RFL, Choudhary I and Harper JI. Interleukin-1 alpha blockade prevents hyperkeratosis in an in vitromodel of lamellar ichthyosis –Human Molecular Genetics 2010; 19(13): 2594-2605 Presentations arising – Poster presentation, British Society of Investigative Dermatology, Edinburgh 2010 “Interleukin-1 alpha blockade prevents hyperkeratosis in an in vitro model of lamellar ichthyosis” – Invited speaker, European Society of Dermatopathology, Helsinki, 2010 “Mechanisms of hyperkeratosis in lamellar ichthyosis, the role of Interleukin-1 alpha” – European Society of Dermatological Research, Helsinki, 2010 “Integration of innate immune signalling pathways in epidermal barrier dysfunction and hyperkeratosis” Intellectual property We have disclosed the invention of using interleukin-1 receptor antagonist as a topical treatment to reduce scaling in lamellar ichthyosis to UCL business. Our future intentions are to determine molecules downstream of IL1A that are responsible for the scaling in ichthyosis, which are more amenable to patenting and are not currently subject to disputes of patent ownership. Beneficiaries of Research The eventual beneficiaries are lamellar ichthyosis patients, if the basic biological discovery can be translated to the clinic. However even now, we are using the phenotypic differences in loricrin cross-linking as a crude diagnostic for Tgm1 or non-Tgm1 mediated lamellar ichthyosis, and interleukin-1 alpha levels as a measure of disease severity. Detailed Report of Research Funded Creation of TGM1 mutants and confirmation of function Unfortunately the idea of transfecting mutagenised TGM1 into TGM1 siRNA expressing keratinocytes was difficult to implement. The mutagenesis worked; however the resultant phenotypes were unchanged. So we decided to extend the concept of genotype-phenotype correlation to other genetic causes of lamellar ichthyosis. We have knocked down Alox12B (Jobard et al., 2002) expression using a plasmid shRNA approach. What was surprising was the similarity with the Tgm1 siRNA expressing cells (O’Shaughnessy et al., 2010). Changes in expression of terminal differentiation markers were identical, except for the expression of loricrin, which was predominantly dimeric in the Alox12b siRNA cells, and predominantly monomeric in the Tgm1 siRNA cells. We observed the same variation in patient scrapings, suggesting that we could use this as a crude diagnostic (O’Shaughnessy et al., 2010) Analysis of barrier, lipids and histology With an improvement in our culture model, we have been able to investigate the changes in lipids, barrier and epidermal differentiation markers in the Tgm1 siRNA expressing organotypic cultures. The experiments are continuing in the Alox12B expressing cells, and we expect organotypic cultures with which to perform comparative studies within the next couple of months. However we determined that although the Tgm1 siRNA expressing cultures were essentially barrier competent, this was due to a large increase in non-polar lipid deposition that we were able to detect by both Oil-red O staining and Nile red staining. (O’Shaughnessy et al., 2010. Interleukin-1 signalling and fatty acid desaturases We have been able to confirm that there is a large up-regulation of interleukin-1 alpha in the Tgm1 siRNA expressing cells, and that by inference by analysis of our patient scrapings, non-TGM1 related lamellar ichthyosis patients have increased interleukin-1 alpha in the epidermis (Figure 1). We examined interleukin-1 alpha related signalling further, and although there was not change in the levels or phosphorylation status of NFkappaBeta, a transcription factor downstream of interleukin-1 alpha, there was constitutive downregulation/degradation of IkBa, which inhibits the transport of NFkappaBeta into the nucleus. Interleukin-1 alpha was increased in Tgm1 siRNA organotypic cultures, confirming the similarities between patient skin and the organotypic culture disease model. We have confirmed that interleukin-1 alpha is both necessary and sufficient for hyperkeratosis, and that treatment of Tgm1 siRNA expressing cultures with interleukin-1 receptor antagonist prevents hyperkeratosis (O’Shaughnessy et al., 2010). From a therapeutic standpoint, this treatment has no effect on the loss of fatty acid desaturases, and non-polar lipid deposition in the cornified layer remained the same (Figure 2). The major implication and conclusion of this work, is that regardless of the genetic cause, up-regulation of interleukin-1 alpha is the principle cause of scaling in lamellar ichthyosis, and that treatment with interleukin-1 receptor antagonist has the potential to reduce scaling while maintaining barrier lipids, which for the patient means less reliance on scrubbing and bathing to control the disease, and less or reduced use of retinoids. References Jobard F, Lefèvre C, Karaduman A, Blanchet-Bardon C, Emre S, Weissenbach J, Ozgüc M, Lathrop M, Prud’homme JF, Fischer J. Lipoxygenase-3 (ALOXE3) and 12(R)-lipoxygenase (ALOX12B) are mutated in non-bullous congenital ichthyosiform erythroderma (NCIE) linked to chromosome 17p13.1. Hum Mol Genet. 2002 Jan 1;11(1):107-13. O’Shaughnessy RFL, Choudhary I and Harper JI. Interleukin-1 alpha blockade prevents hyperkeratosis in an in vitromodel of lamellar ichthyosis –Human Molecular Genetics 2010; 19(13): 2594-2605 Analysis of patient skin scrapings (scales) revealed that increased IL1A expression was common to all lamellar ichthyosis and ARCI tested. A. Western blot analysis of soluble full length TGM1, IL1A, Keratin 1 and loricrin in one control, 4 classical lamellar ichthyosis, 4 other ARCI, including 2 self healing collodion babies and one bathing suit ichthyosis , and 1 patient with ichthyosis vulgaris. Loading was normalised by measuring total soluble protein. Dimeric loricrin in patients 3 and 4, suggesting that the disease was not caused by mutation in TGM1. B. Analysis of IL1A intensity showing a significant increase associated with lamellar ichthyosis and related ARCI (p<0.05, One way ANOVA) C. Table showing patient data, diagnoses and pertinent treatments. Treatment of Tgm1 siRNA expressing organotypic cultures with IL1ra causes a dose dependent reduction in hyperkeratosis but no decrease in neutral lipids. A Histology of organotypic cultures of scrambled, Tgm1 siRNA expressing cells and Tgm1 siRNA expressing cells treated with 0.5 and 2.5 ng/ml IL1RA. No change in neutral lipids (Oil Red O) was seen after IL1RA treatment. FADS2 expression in treated cultures was not increased. . DAPI was used as a nuclear counterstain. Project details Individuals with Lamellar ichthyosis have extensive scaling that requires time consuming skin treatment each and everyday. If we understood why scaly skin occurs in individuals with Lamellar ichthyosis, we could devise better treatments that would reduce the amount of time taken everyday to treat the scaling. We are able to grow Lamellar ichthyosis skin in the laboratory, and using this we have found increased amounts of a protein called interleukin-1 alpha and decreased amounts of proteins that alter the ability of the lipids (fats) of the skin to act as a barrier to water loss. There is a large range of Lamellar ichthyoses, ranging from severe scaling to the much milder self-healing forms of the disease. We intend to model the range of different Lamellar ichthyoses in the lab and determine whether the proteins we are interested in, such as the interleukin-1 alpha protein, are increased in all forms of Lamellar ichthyosis, making it a good potential target for future therapies.