Investigations into the FLG Null Phenotype: showcasing the methodology for CRISPR/Cas9 editing of human keratinocytes

Jos P H Smits, Noa J M van den Brink, Luca D Meesters, Hadia Hamdaoui, Hanna Niehues, Patrick A M Jansen, Ivonne M J J van Vlijmen-Willems, Diana Rodijk-Olthuis, Céline Evrard, Yves Poumay, Michel van Geel, Wiljan J A J Hendriks, Joost Schalkwijk, Patrick L J M Zeeuwen, Ellen H van den Bogaard

Résultats de recherche: Contribution à un journal/une revueArticleRevue par des pairs

Résumé

Ever since the association between FLG loss-of-function variants and ichthyosis vulgaris and atopic dermatitis disease onset was identified, FLGs function has been under investigation. Intraindividual genomic predisposition, immunological confounders, and environmental interactions complicate the comparison between FLG genotypes and related causal effects. Using CRISPR/Cas9, we generated human FLG-knockout (ΔFLG) N/TERT-2G keratinocytes. FLG deficiency was shown by immunohistochemistry of human epidermal equivalent cultures. Next to (partial) loss of structural proteins (involucrin, hornerin, keratin 2, and transglutaminase 1), the stratum corneum was denser and lacked the typical basket weave appearance. In addition, electrical impedance spectroscopy and transepidermal water loss analyses highlighted a compromised epidermal barrier in ΔFLG human epidermal equivalents. Correction of FLG reinstated the presence of keratohyalin granules in the stratum granulosum, FLG protein expression, and expression of the proteins mentioned earlier. The beneficial effects on stratum corneum formation were reflected by the normalization of electrical impedance spectroscopy and transepidermal water loss. This study shows the causal phenotypical and functional consequences of FLG deficiency, indicating that FLG is not only central in epidermal barrier function but also vital for epidermal differentiation by orchestrating the expression of other important epidermal proteins. These observations pave the way to fundamental investigations into the exact role of FLG in skin biology and disease.

langue originaleAnglais
Pages (de - à)1520-1528.e5
journalJournal of Investigative Dermatology
Volume143
Numéro de publication8
Date de mise en ligne précoce7 mars 2023
Les DOIs
Etat de la publicationPublié - 1 août 2023

Financement

This work was supported by a LEO foundation grant LF18068 (PZ and EB), PAST4FUTURE grant LSHM20043-HSGF (EB), and Innovative Medicines Initiative 2 Joint Undertaking (JU) grant under grant agreement (No. 821511, EB). The JU receives support from the European Union's Horizon 2020 research and innovation program and EFPIA. The Graphical abstract was created with Biorender.com. Conceptualization: JPHS, WJAJH, JS, PLJMZ, EHVDB; Data Curation: JPHS; Formal Analysis: JPHS; Funding Acquisition: EHVDB, PLJMZ, JS; Investigation: JPHS, NJMVDB, LDM, HH, HN, PLJMJ, IMJJVVW, DRO, MVG; Methodology: JPHS, NJMVDB, HH, CE, YP, WJAJH; Project Administration: EHVDB; Software: JPHS; Supervision: PZ, EHVDB; Validation: JPHS; Visualization: JPHS; Writing \u2013 Original Draft Preparation: JPHS; Writing \u2013 Review and Editing: JPHS, HN, JS, EHVDB, PLJMZ This work was supported by a LEO foundation grant LF18068 (PZ and EB), PAST4FUTURE grant LSHM20043-HSGF (EB), and Innovative Medicines Initiative 2 Joint Undertaking (JU) grant under grant agreement (No. 821511, EB). The JU receives support from the European Union\u2019s Horizon 2020 research and innovation program and EFPIA. The Graphical abstract was created with Biorender.com .

Bailleurs de fondsNuméro du bailleur de fonds
JPHS
LDM
European Federation of Pharmaceutical Industries and Associations
European commission
NJMVDB
EHVDB
Innovative Medicines Initiative
Horizon 2020 Framework Programme821511
LEO FondetLF18068, LSHM20043-HSGF

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