Functional and proteomic analysis of a full thickness filaggrin-deficient skin organoid model

Research output: Contribution to journalArticle

26 Downloads (Pure)

Abstract

Background: Atopic eczema is an itchy inflammatory disorder 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 incompletely understood. Skin as an organ that can be modelled using primary cells in vitro provides the opportunity for selected genetic effects to be investigated in detail. 
Methods: Primary human keratinocytes and donor-matched primary fibroblasts from healthy individuals were used to create skin organoid models with and without siRNA-mediated knockdown of FLG. Biological replicate sets of organoids were assessed using histological, functional and biochemical measurements. 
Results: FLG knockdown leads to subtle changes in histology and ultrastructure including a reduction in thickness of the stratum corneum and smaller, less numerous keratohyalin granules. Immature organoids showed some limited evidence of barrier impairment with FLG knockdown, but the mature organoids showed no difference in transepidermal water loss, water content or dye penetration. There was no difference in epidermal ceramide content. Mass spectrometry proteomic analysis detected >8000 proteins per sample. Gene ontology and pathway analyses identified an increase in transcriptional and translational activity but a reduction in proteins contributing to terminal differentiation, including caspase 14, dermokine, AKT1 and TGF-beta-1. Aspects of innate and adaptive immunity were represented in both the up-regulated and down-regulated protein groups, as was the term 'axon guidance'. 
 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.
Original languageEnglish
Pages (from-to)134
Number of pages28
JournalWellcome Open Research
Volume4
DOIs
Publication statusE-pub ahead of print - 9 Sep 2019

Fingerprint

Organoids
Proteomics
Skin
Keratinocytes
Caspase 14
Haploinsufficiency
Transforming Growth Factor beta1
Gene Ontology
Proteins
Histology
Gene encoding
Water
Ceramides
Adaptive Immunity
Atopic Dermatitis
Fibroblasts
Pruritus
Crosstalk
Innate Immunity
Cornea

Keywords

  • Axon guidance
  • atopic dermatitis
  • eczema
  • filaggrin
  • gene ontology
  • keratinocyte-immune crosstalk
  • organoid
  • proteomics

Cite this

@article{09c678a634c6458d8d10893a6116b930,
title = "Functional and proteomic analysis of a full thickness filaggrin-deficient skin organoid model",
abstract = "Background: Atopic eczema is an itchy inflammatory disorder 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 incompletely understood. Skin as an organ that can be modelled using primary cells in vitro provides the opportunity for selected genetic effects to be investigated in detail. Methods: Primary human keratinocytes and donor-matched primary fibroblasts from healthy individuals were used to create skin organoid models with and without siRNA-mediated knockdown of FLG. Biological replicate sets of organoids were assessed using histological, functional and biochemical measurements. Results: FLG knockdown leads to subtle changes in histology and ultrastructure including a reduction in thickness of the stratum corneum and smaller, less numerous keratohyalin granules. Immature organoids showed some limited evidence of barrier impairment with FLG knockdown, but the mature organoids showed no difference in transepidermal water loss, water content or dye penetration. There was no difference in epidermal ceramide content. Mass spectrometry proteomic analysis detected >8000 proteins per sample. Gene ontology and pathway analyses identified an increase in transcriptional and translational activity but a reduction in proteins contributing to terminal differentiation, including caspase 14, dermokine, AKT1 and TGF-beta-1. Aspects of innate and adaptive immunity were represented in both the up-regulated and down-regulated protein groups, as was the term 'axon guidance'.  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",
author = "Elias, {Martina S.} and Wright, {Sheila C.} and Nicholson, {William V.} and Morrison, {Kimberley D.} and Prescott, {Alan R.} and {Ten Have}, Sara and Whitfield, {Phillip D.} and Lamond, {Angus I.} and Brown, {Sara J.}",
note = "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.",
year = "2019",
month = "9",
day = "9",
doi = "10.12688/wellcomeopenres.15405.2",
language = "English",
volume = "4",
pages = "134",
journal = "Wellcome Open Research",
issn = "2398-502X",
publisher = "F1000Research",

}

TY - JOUR

T1 - Functional and proteomic analysis of a full thickness filaggrin-deficient skin organoid model

AU - Elias, Martina S.

AU - Wright, Sheila C.

AU - Nicholson, William V.

AU - Morrison, Kimberley D.

AU - Prescott, Alan R.

AU - Ten Have, Sara

AU - Whitfield, Phillip D.

AU - Lamond, Angus I.

AU - Brown, Sara J.

N1 - 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.

PY - 2019/9/9

Y1 - 2019/9/9

N2 - Background: Atopic eczema is an itchy inflammatory disorder 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 incompletely understood. Skin as an organ that can be modelled using primary cells in vitro provides the opportunity for selected genetic effects to be investigated in detail. Methods: Primary human keratinocytes and donor-matched primary fibroblasts from healthy individuals were used to create skin organoid models with and without siRNA-mediated knockdown of FLG. Biological replicate sets of organoids were assessed using histological, functional and biochemical measurements. Results: FLG knockdown leads to subtle changes in histology and ultrastructure including a reduction in thickness of the stratum corneum and smaller, less numerous keratohyalin granules. Immature organoids showed some limited evidence of barrier impairment with FLG knockdown, but the mature organoids showed no difference in transepidermal water loss, water content or dye penetration. There was no difference in epidermal ceramide content. Mass spectrometry proteomic analysis detected >8000 proteins per sample. Gene ontology and pathway analyses identified an increase in transcriptional and translational activity but a reduction in proteins contributing to terminal differentiation, including caspase 14, dermokine, AKT1 and TGF-beta-1. Aspects of innate and adaptive immunity were represented in both the up-regulated and down-regulated protein groups, as was the term 'axon guidance'.  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.

AB - Background: Atopic eczema is an itchy inflammatory disorder 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 incompletely understood. Skin as an organ that can be modelled using primary cells in vitro provides the opportunity for selected genetic effects to be investigated in detail. Methods: Primary human keratinocytes and donor-matched primary fibroblasts from healthy individuals were used to create skin organoid models with and without siRNA-mediated knockdown of FLG. Biological replicate sets of organoids were assessed using histological, functional and biochemical measurements. Results: FLG knockdown leads to subtle changes in histology and ultrastructure including a reduction in thickness of the stratum corneum and smaller, less numerous keratohyalin granules. Immature organoids showed some limited evidence of barrier impairment with FLG knockdown, but the mature organoids showed no difference in transepidermal water loss, water content or dye penetration. There was no difference in epidermal ceramide content. Mass spectrometry proteomic analysis detected >8000 proteins per sample. Gene ontology and pathway analyses identified an increase in transcriptional and translational activity but a reduction in proteins contributing to terminal differentiation, including caspase 14, dermokine, AKT1 and TGF-beta-1. Aspects of innate and adaptive immunity were represented in both the up-regulated and down-regulated protein groups, as was the term 'axon guidance'.  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.

KW - Axon guidance

KW - atopic dermatitis

KW - eczema

KW - filaggrin

KW - gene ontology

KW - keratinocyte-immune crosstalk

KW - organoid

KW - proteomics

U2 - 10.12688/wellcomeopenres.15405.2

DO - 10.12688/wellcomeopenres.15405.2

M3 - Article

C2 - 31641698

VL - 4

SP - 134

JO - Wellcome Open Research

JF - Wellcome Open Research

SN - 2398-502X

ER -