Wellcome Open Research 2019, 4:134 Last updated: 26 NOV 2019 RESEARCH ARTICLE Functional and proteomic analysis of a full thickness filaggrin-deficient skin organoid model [version 2; peer review: 3 approved] Previously titled: Proteomic analysis of a filaggrin-deficient skin organoid model shows evidence of increased transcriptional-translational activity, keratinocyte-immune crosstalk and disordered axon guidance Martina S. Elias1, Sheila C. Wright1, William V. Nicholson1, Kimberley D. Morrison1, Alan R. Prescott 2, Sara Ten Have3, Phillip D. Whitfield 4, Angus I. Lamond 3, Sara J. Brown 1,5 1Skin Research Group, Division of Molecular and Clinical Medicine, School of Medicine, University of Dundee, Dundee, Scotland, DD1 9SY, UK 2Dundee Imaging Facility, School of Life Sciences, University of Dundee, Dundee, Scotland, DD1 5EH, UK 3Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, Scotland, DD1 5EH, UK 4Lipidomics Research Facility, Division of Biomedical Sciences, University of the Highlands and Islands, Inverness, Scotland, IV2 3JH, UK 5Department of Dermatology, Ninewells Hospital, Dundee, Scotland, DD1 9SY, UK First published: 09 Sep 2019, 4:134 ( Open Peer Review v2 https://doi.org/10.12688/wellcomeopenres.15405.1) Latest published: 26 Nov 2019, 4:134 ( https://doi.org/10.12688/wellcomeopenres.15405.2) Reviewer Status Abstract Invited Reviewers Background: Atopic eczema is an itchy inflammatory disorder 1 2 3 characterised by skin barrier dysfunction. Loss-of-function mutations in the gene encoding filaggrin (FLG) are a major risk factor, but the mechanisms by which filaggrin haploinsufficiency leads to atopic inflammation remain version 2 report incompletely understood. Skin as an organ that can be modelled using published primary cells in vitro provides the opportunity for selected genetic effects to 26 Nov 2019 be investigated in detail. Methods: Primary human keratinocytes and donor-matched primary version 1 fibroblasts from healthy individuals were used to create skin organoid published report report report models with and without siRNA-mediated knockdown of FLG. Biological 09 Sep 2019 replicate sets of organoids were assessed using histological, functional and biochemical measurements. Sabine Werner, ETH Zurich (Swiss Federal Results: FLG knockdown leads to subtle changes in histology and 1 ultrastructure including a reduction in thickness of the stratum corneum and Institute of Technology Zürich), Zürich, smaller, less numerous keratohyalin granules. Immature organoids showed Switzerland some limited evidence of barrier impairment with FLG knockdown, but the Ulrich auf dem Keller, Technical University of mature organoids showed no difference in transepidermal water loss, water Denmark, Kongens Lyngby, Denmark content or dye penetration. There was no difference in epidermal ceramide content. Mass spectrometry proteomic analysis detected >8000 proteins 2 Neil Rajan , Newcastle University, per sample. Gene ontology and pathway analyses identified an increase in Newcastle upon Tyne, UK transcriptional and translational activity but a reduction in proteins contributing to terminal differentiation, including caspase 14, dermokine, 3 Sanja Kezic, University of Amsterdam, AKT1 and TGF-beta-1. Aspects of innate and adaptive immunity were Amsterdam, The Netherlands represented in both the up-regulated and down-regulated protein groups, Page 1 of 28 Wellcome Open Research 2019, 4:134 Last updated: 26 NOV 2019 represented in both the up-regulated and down-regulated protein groups, Any reports and responses or comments on the as was the term ‘axon guidance’. article can be found at the end of the article. Conclusions: This work provides further evidence for keratinocyte-specific mechanisms contributing to immune and neurological, as well as structural, aspects of skin barrier dysfunction. Individuals with filaggrin deficiency may derive benefit from future therapies targeting keratinocyte-immune crosstalk and neurogenic pruritus. Keywords Axon guidance, atopic dermatitis, eczema, filaggrin, gene ontology, keratinocyte-immune crosstalk, organoid, proteomics Corresponding authors: Martina S. Elias ([email protected]), Sara J. Brown ([email protected]) Author roles: Elias MS: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing; Wright SC: Data Curation, Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing – Review & Editing; Nicholson WV: Formal Analysis, Methodology, Visualization, Writing – Review & Editing; Morrison KD: Formal Analysis, Methodology, Validation, Visualization, Writing – Review & Editing; Prescott AR: Data Curation, Investigation, Methodology, Resources, Supervision, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing; Ten Have S: Data Curation, Formal Analysis, Investigation, Methodology, Resources, Software, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing; Whitfield PD: Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Resources, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing; Lamond AI: Funding Acquisition, Methodology, Project Administration, Resources, Supervision, Writing – Review & Editing; Brown SJ: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests: No competing interests were disclosed. Grant information: This work was supported by the Wellcome Trust [106865], a Senior Research Fellowship in Clinical Science awarded to SJB; the Brown laboratory has also received financial support from the Manknell Charitable Trust, the Tayside Dermatology Research Charity and the British Skin Foundation. The mass spectrometry proteomic analysis was supported by grants to AIL from the Wellcome Trust [105024]. Dundee Imaging Facility is supported by a Wellcome Trust Technology Platform award [097945]. UHI Lipidomics Research Facility acknowledges the support of European Regional Development Fund, Scottish Funding Council and Highlands and Islands Enterprise. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2019 Elias MS et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite this article: Elias MS, Wright SC, Nicholson WV et al. Functional and proteomic analysis of a full thickness filaggrin-deficient skin organoid model [version 2; peer review: 3 approved] Wellcome Open Research 2019, 4:134 ( https://doi.org/10.12688/wellcomeopenres.15405.2) First published: 09 Sep 2019, 4:134 (https://doi.org/10.12688/wellcomeopenres.15405.1) Page 2 of 28 Wellcome Open Research 2019, 4:134 Last updated: 26 NOV 2019 Filaggrin is a marker of keratinocyte differentiation; it is REVISED Amendments from Version 1 expressed in the granular layer of the upper epidermis as a polymer – profilaggrin – which undergoes post-translational The main aim of this publication is to facilitate sharing of the global mass spectrometry data generated from the skin organoid modification and stepwise proteolysis to release monomeric models with FLG knockdown (described in detail) and functional filaggrin14. Profilaggrin and filaggrin have multiple functions, analyses (described and reported); this main aim has been each of which have been described as contributing to the mechani- clarified. cal, biochemical, immunological and microbiological aspects In response to the interest from Reviewers 1 and 3, we have of skin barrier function15. Clinical studies have shown that filag- provided an explanation as to why knockdown of FLG at mRNA grin haploinsufficiency is associated with xerosis and ichthyo- level is demonstrated to a lesser degree than the knockdown at protein level, reflecting the prolonged duration (10 days) of sis (dry and scaly skin), keratosis pilaris (increased keratin organoid culture. within skin follicles), palmar hyperlinearity16 and increased 17 We have emphasised that increased dye penetration was transepidermal water loss . Transcriptomic analysis of full thick- observed in some but not all of the immature organoids, as ness skin biopsies have shown evidence of an abnormal defence requested by Reviewer 1. response in FLG haploinsufficient atopic skin18; proteomic Additional details have been provided to explain how the protein analysis to assess the effect of FLG knockdown in an epider- false discovery rate was determined for the removal of false mal organoid has also shown features of inflammation and stress positives, as requested by Reviewer 1. protease activity19. However, in vitro studies have not shown We have included a fuller explanation of the analysis approach, consistent histological or functional effects of filaggrin defi- in which we focus on consistent differences observed across the biological replicate experiments and use thresholds for increased ciency in the various different skin organoid models published and decreased expression to include roughly equal numbers of to date20 and the multiple mechanisms by which filaggrin