Advertisement

Archives of Dermatological Research

, Volume 305, Issue 6, pp 501–512 | Cite as

Identifying targets for topical RNAi therapeutics in psoriasis: assessment of a new in vitro psoriasis model

  • S. Bracke
  • E. Desmet
  • S. Guerrero-Aspizua
  • S. G. Tjabringa
  • J. Schalkwijk
  • M. Van Gele
  • M. Carretero
  • J. Lambert
Original Paper

Abstract

Diseases of the skin are amenable to RNAi-based therapies and targeting key components in the pathophysiology of psoriasis using RNAi may represent a successful new therapeutic strategy. We aimed to develop a straightforward and highly reproducible in vitro psoriasis model useful to study the effects of gene knockdown by RNAi and to identify new targets for topical RNAi therapeutics. We evaluated the use of keratinocytes derived from psoriatic plaques and normal human keratinocytes (NHKs). To induce a psoriatic phenotype in NHKs, combinations of pro-inflammatory cytokines (IL-1α, IL-17A, IL-6 and TNF-α) were tested. The model based on NHK met our needs of a reliable and predictive preclinical model, and this model was further selected for gene expression analyses, comprising a panel of 55 psoriasis-associated genes and five micro-RNAs (miRNAs). Gene silencing studies were conducted by using small interfering RNAs (siRNAs) and miRNA inhibitors directed against potential target genes such as CAMP and DEFB4 and miRNAs such as miR-203. We describe a robust and highly reproducible in vitro psoriasis model that recapitulates expression of a large panel of genes and miRNAs relevant to the pathogenesis of psoriasis. Furthermore, we show that our model is a powerful first step model system for testing and screening RNAi-based therapeutics.

Keywords

Psoriasis In vitro model RNA interference siRNA miRNA 

Notes

Acknowledgments

We thank Martine De Mil for help with cell culture, and Marie-Chantal Herteleer and Els Van Maelsaeke for technical assistance. Dr. S. Bracke is funded by an IWT grant (091208) (‘Flemish government agency for Innovation by Science and Technology’).

Conflict of interest

None declared.

Supplementary material

403_2013_1379_MOESM1_ESM.doc (164 kb)
Supplementary material 1 (DOC 163 kb)

References

  1. 1.
    Amigo M, Schalkwijk J, Olthuis D, De Rosa S, Paya M, Terencio MC, Lamme E (2006) Identification of avarol derivatives as potential antipsoriatic drugs using an in vitro model for keratinocyte growth and differentiation. Life Sci 79(25):2395–2404PubMedCrossRefGoogle Scholar
  2. 2.
    Bando M, Hiroshima Y, Kataoka M, Shinohara Y, Herzberg MC, Ross KF, Nagata T, Kido J (2007) Interleukin-1alpha regulates antimicrobial peptide expression in human keratinocytes. Immunol Cell Biol 85(7):532–537PubMedCrossRefGoogle Scholar
  3. 3.
    Banno T, Gazel A, Blumenberg M (2004) Effects of tumor necrosis factor-alpha (TNF alpha) in epidermal keratinocytes revealed using global transcriptional profiling. J Biol Chem 279(31):32633–32642PubMedCrossRefGoogle Scholar
  4. 4.
    Bernerd F, Magnaldo T, Darmon M (1992) Delayed onset of epidermal differentiation in psoriasis. J Invest Dermatol 98(6):902–910PubMedCrossRefGoogle Scholar
  5. 5.
    Bigler J, Rand HA, Kerkof K, Timour M, Russell CB (2013) Cross-study homogeneity of psoriasis gene expression in skin across a large expression range. PLoS One 8(1):e52242PubMedCrossRefGoogle Scholar
  6. 6.
    Bumcrot D, Manoharan M, Koteliansky V, Sah DW (2006) RNAi therapeutics: a potential new class of pharmaceutical drugs. Nat Chem Biol 2(12):711–719PubMedCrossRefGoogle Scholar
  7. 7.
    Capon F, Burden AD, Trembath RC, Barker JN (2012) Psoriasis and other complex trait dermatoses: from Loci to functional pathways. J Invest Dermatol 132(3 Pt 2):915–922. doi: 10.1038/jid.2011.395 PubMedCrossRefGoogle Scholar
  8. 8.
    Chiricozzi A, Guttman-Yassky E, Suarez-Farinas M, Nograles KE, Tian S, Cardinale I, Chimenti S, Krueger JG (2011) Integrative responses to IL-17 and TNF-alpha in human keratinocytes account for key inflammatory pathogenic circuits in psoriasis. J Invest Dermatol 131(3):677–687PubMedCrossRefGoogle Scholar
  9. 9.
    Coimbra S, Figueiredo A, Castro E, Rocha-Pereira P, Santos-Silva A (2012) The roles of cells and cytokines in the pathogenesis of psoriasis. Int J Dermatol 51(4):389–395 quiz 395-388PubMedCrossRefGoogle Scholar
  10. 10.
    Davidson BL, McCray PB Jr (2011) Current prospects for RNA interference-based therapies. Nat Rev Genet 12(5):329–340. doi: 10.1038/nrg2968 PubMedCrossRefGoogle Scholar
  11. 11.
    Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391(6669):806–811PubMedCrossRefGoogle Scholar
  12. 12.
    Frohm Nilsson M, Sandstedt B, Sorensen O, Weber G, Borregaard N, Stahle-Backdahl M (1999) The human cationic antimicrobial protein (hCAP18), a peptide antibiotic, is widely expressed in human squamous epithelia and colocalizes with interleukin-6. Infect Immun 67(5):2561–2566PubMedGoogle Scholar
  13. 13.
    Geusens B, Sanders N, Prow T, Van Gele M, Lambert J (2009) Cutaneous short-interfering RNA therapy. Expert Opin Drug Deliv 6(12):1333–1349. doi: 10.1517/17425240903304032 PubMedCrossRefGoogle Scholar
  14. 14.
    Geusens B, Strobbe T, Bracke S, Dynoodt P, Sanders N, Van Gele M, Lambert J (2011) Lipid-mediated gene delivery to the skin. Eur J Pharm Sci 43(4):199–211PubMedCrossRefGoogle Scholar
  15. 15.
    Girolomoni G, Mrowietz U, Paul C (2012) Psoriasis: rationale for targeting interleukin-17. Br J Dermatol 167(4):717–724PubMedCrossRefGoogle Scholar
  16. 16.
    Grossman RM, Krueger J, Yourish D, Granelli-Piperno A, Murphy DP, May LT, Kupper TS, Sehgal PB, Gottlieb AB (1989) Interleukin 6 is expressed in high levels in psoriatic skin and stimulates proliferation of cultured human keratinocytes. Proc Natl Acad Sci USA 86(16):6367–6371PubMedCrossRefGoogle Scholar
  17. 17.
    Gudjonsson JE, Ding J, Johnston A, Tejasvi T, Guzman AM, Nair RP, Voorhees JJ, Abecasis GR, Elder JT (2010) Assessment of the psoriatic transcriptome in a large sample: additional regulated genes and comparisons with in vitro models. J Invest Dermatol 130(7):1829–1840PubMedCrossRefGoogle Scholar
  18. 18.
    Guttman-Yassky E, Lowes MA, Fuentes-Duculan J, Zaba LC, Cardinale I, Nograles KE, Khatcherian A, Novitskaya I, Carucci JA, Bergman R, Krueger JG (2008) Low expression of the IL-23/Th17 pathway in atopic dermatitis compared to psoriasis. J Immunol 181(10):7420–7427PubMedGoogle Scholar
  19. 19.
    Hollox EJ, Huffmeier U, Zeeuwen PL, Palla R, Lascorz J, Rodijk-Olthuis D, van de Kerkhof PC, Traupe H, de Jongh G, den Heijer M, Reis A, Armour JA, Schalkwijk J (2008) Psoriasis is associated with increased beta-defensin genomic copy number. Nat Genet 40(1):23–25PubMedCrossRefGoogle Scholar
  20. 20.
    Ichihara A, Jinnin M, Yamane K, Fujisawa A, Sakai K, Masuguchi S, Fukushima S, Maruo K, Ihn H (2011) microRNA-mediated keratinocyte hyperproliferation in psoriasis vulgaris. Br J Dermatol 165(5):1003–1010PubMedCrossRefGoogle Scholar
  21. 21.
    Iizuka H, Takahashi H, Honma M, Ishida-Yamamoto A (2004) Unique keratinization process in psoriasis: late differentiation markers are abolished because of the premature cell death. J Dermatol 31(4):271–276PubMedGoogle Scholar
  22. 22.
    Joyce CE, Zhou X, Xia J, Ryan C, Thrash B, Menter A, Zhang W, Bowcock AM (2011) Deep sequencing of small RNAs from human skin reveals major alterations in the psoriasis miRNAome. Hum Mol Genet 20(20):4025–4040. doi: 10.1093/hmg/ddr331 PubMedCrossRefGoogle Scholar
  23. 23.
    Kupper TS, Fuhlbrigge RC (2004) Immune surveillance in the skin: mechanisms and clinical consequences. Nat Rev Immunol 4(3):211–222PubMedCrossRefGoogle Scholar
  24. 24.
    Lande R, Gregorio J, Facchinetti V, Chatterjee B, Wang YH, Homey B, Cao W, Wang YH, Su B, Nestle FO, Zal T, Mellman I, Schroder JM, Liu YJ, Gilliet M (2007) Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature 449(7162):564–569PubMedCrossRefGoogle Scholar
  25. 25.
    Leigh IM, Navsaria H, Purkis PE, McKay IA, Bowden PE, Riddle PN (1995) Keratins (K16 and K17) as markers of keratinocyte hyperproliferation in psoriasis in vivo and in vitro. Br J Dermatol 133(4):501–511PubMedCrossRefGoogle Scholar
  26. 26.
    Lerman G, Avivi C, Mardoukh C, Barzilai A, Tessone A, Gradus B, Pavlotsky F, Barshack I, Polak-Charcon S, Orenstein A, Hornstein E, Sidi Y, Avni D (2011) MiRNA expression in psoriatic skin: reciprocal regulation of hsa-miR-99a and IGF-1R. PLoS One 6(6):e20916PubMedCrossRefGoogle Scholar
  27. 27.
    Mee JB, Johnson CM, Morar N, Burslem F, Groves RW (2007) The psoriatic transcriptome closely resembles that induced by interleukin-1 in cultured keratinocytes: dominance of innate immune responses in psoriasis. Am J Pathol 171(1):32–42PubMedCrossRefGoogle Scholar
  28. 28.
    Nestle FO, Kaplan DH, Barker J (2009) Psoriasis. The New England journal of medicine 361(5):496–509. doi: 10.1056/NEJMra0804595 PubMedCrossRefGoogle Scholar
  29. 29.
    Nograles KE, Zaba LC, Guttman-Yassky E, Fuentes-Duculan J, Suarez-Farinas M, Cardinale I, Khatcherian A, Gonzalez J, Pierson KC, White TR, Pensabene C, Coats I, Novitskaya I, Lowes MA, Krueger JG (2008) Th17 cytokines interleukin (IL)-17 and IL-22 modulate distinct inflammatory and keratinocyte-response pathways. Br J Dermatol 159(5):1092–1102PubMedGoogle Scholar
  30. 30.
    Oka A, Mabuchi T, Ozawa A, Inoko H (2012) Current understanding of human genetics and genetic analysis of psoriasis. J Dermatol 39(3):231–241. doi: 10.1111/j.1346-8138.2012.01504.x PubMedCrossRefGoogle Scholar
  31. 31.
    Pol A, Bergers M, van Ruissen F, Pfundt R, Schalkwijk J (2002) A simple technique for high-throughput screening of drugs that modulate normal and psoriasis-like differentiation in cultured human keratinocytes. Skin Pharmacol Appl Skin Physiol 15(4):252–261PubMedCrossRefGoogle Scholar
  32. 32.
    Pol A, van Ruissen F, Schalkwijk J (2002) Development of a keratinocyte-based screening model for antipsoriatic drugs using green fluorescent protein under the control of an endogenous promoter. J Biomol Screen 7(4):325–332PubMedCrossRefGoogle Scholar
  33. 33.
    Reich K, Bewley A (2011) What is new in topical therapy for psoriasis? J Eur Acad Dermatol Venereol 25 Suppl 4:15–20PubMedCrossRefGoogle Scholar
  34. 34.
    Sonkoly E, Wei T, Janson PC, Saaf A, Lundeberg L, Tengvall-Linder M, Norstedt G, Alenius H, Homey B, Scheynius A, Stahle M, Pivarcsi A (2007) MicroRNAs: novel regulators involved in the pathogenesis of psoriasis? PLoS One 2(7):e610PubMedCrossRefGoogle Scholar
  35. 35.
    Suarez-Farinas M, Lowes MA, Zaba LC, Krueger JG (2010) Evaluation of the psoriasis transcriptome across different studies by gene set enrichment analysis (GSEA). PLoS One 5(4):e10247PubMedCrossRefGoogle Scholar
  36. 36.
    Svensson L, Ropke MA, Norsgaard H (2012) Psoriasis drug discovery: methods for evaluation of potential drug candidates. Expert Opin Drug Discov 7(1):49–61PubMedCrossRefGoogle Scholar
  37. 37.
    Taganov KD, Boldin MP, Chang KJ, Baltimore D (2006) NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc Natl Acad Sci USA 103(33):12481–12486PubMedCrossRefGoogle Scholar
  38. 38.
    Tian S, Krueger JG, Li K, Jabbari A, Brodmerkel C, Lowes MA, Suarez-Farinas M (2012) Meta-analysis derived (MAD) transcriptome of psoriasis defines the “core” pathogenesis of disease. PLoS One 7(9):e44274PubMedCrossRefGoogle Scholar
  39. 39.
    Tjabringa G, Bergers M, van Rens D, de Boer R, Lamme E, Schalkwijk J (2008) Development and validation of human psoriatic skin equivalents. Am J Pathol 173(3):815–823PubMedCrossRefGoogle Scholar
  40. 40.
    Van Ruissen F, de Jongh GJ, Zeeuwen PL, Van Erp PE, Madsen P, Schalkwijk J (1996) Induction of normal and psoriatic phenotypes in submerged keratinocyte cultures. J Cell Physiol 168(2):442–452PubMedCrossRefGoogle Scholar
  41. 41.
    Vandesompele J, De Paepe A, Speleman F (2002) Elimination of primer-dimer artifacts and genomic coamplification using a two-step SYBR green I real-time RT-PCR. Anal Biochem 303(1):95–98PubMedCrossRefGoogle Scholar
  42. 42.
    Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):RESEARCH0034PubMedCrossRefGoogle Scholar
  43. 43.
    Wilson NJ, Boniface K, Chan JR, McKenzie BS, Blumenschein WM, Mattson JD, Basham B, Smith K, Chen T, Morel F, Lecron JC, Kastelein RA, Cua DJ, McClanahan TK, Bowman EP, de Waal MalefytR (2007) Development, cytokine profile and function of human interleukin 17-producing helper T cells. Nat Immunol 8(9):950–957PubMedCrossRefGoogle Scholar
  44. 44.
    Xu N, Brodin P, Wei T, Meisgen F, Eidsmo L, Nagy N, Kemeny L, Stahle M, Sonkoly E, Pivarcsi A (2011) MiR-125b, a microRNA downregulated in psoriasis, modulates keratinocyte proliferation by targeting FGFR2. J Invest Dermatol 131(7):1521–1529PubMedCrossRefGoogle Scholar
  45. 45.
    Yi R, Poy MN, Stoffel M, Fuchs E (2008) A skin microRNA promotes differentiation by repressing ‘stemness’. Nature 452(7184):225–229PubMedCrossRefGoogle Scholar
  46. 46.
    Zeeuwen PL, de Jongh GJ, Rodijk-Olthuis D, Kamsteeg M, Verhoosel RM, van Rossum MM, Hiemstra PS, Schalkwijk J (2008) Genetically programmed differences in epidermal host defense between psoriasis and atopic dermatitis patients. PLoS One 3(6):e2301PubMedCrossRefGoogle Scholar
  47. 47.
    Zeng Y, Yi R, Cullen BR (2003) MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proc Natl Acad Sci USA 100(17):9779–9784PubMedCrossRefGoogle Scholar
  48. 48.
    Zibert JR, Lovendorf MB, Litman T, Olsen J, Kaczkowski B, Skov L (2010) MicroRNAs and potential target interactions in psoriasis. J Dermatol Sci 58(3):177–185PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • S. Bracke
    • 1
  • E. Desmet
    • 1
  • S. Guerrero-Aspizua
    • 2
  • S. G. Tjabringa
    • 4
  • J. Schalkwijk
    • 4
  • M. Van Gele
    • 1
  • M. Carretero
    • 3
  • J. Lambert
    • 1
  1. 1.Department of Dermatology 2K4Ghent University HospitalGhentBelgium
  2. 2.Regenerative Medicine Unit and the Cutaneous Diseases Modeling UnitMedioambientales y Tecnológicas (CIEMAT) and Centre for Biomedical Research on Rare Diseases (CIBERER)MadridSpain
  3. 3.Epithelial Biomedicine Division, Basic Research Department, Centro de Investigaciones EnergéticasMedioambientales y Tecnológicas (CIEMAT) and Centre for Biomedical Research on Rare Diseases (CIBERER)MadridSpain
  4. 4.Department of DermatologyRadboud University Nijmegen Medical CentreNijmegenThe Netherlands

Personalised recommendations