TY - JOUR
T1 - Investigations into the FLG Null Phenotype:
T2 - showcasing the methodology for CRISPR/Cas9 editing of human keratinocytes
AU - Smits, Jos P H
AU - van den Brink, Noa J M
AU - Meesters, Luca D
AU - Hamdaoui, Hadia
AU - Niehues, Hanna
AU - Jansen, Patrick A M
AU - van Vlijmen-Willems, Ivonne M J J
AU - Rodijk-Olthuis, Diana
AU - Evrard, Céline
AU - Poumay, Yves
AU - van Geel, Michel
AU - Hendriks, Wiljan J A J
AU - Schalkwijk, Joost
AU - Zeeuwen, Patrick L J M
AU - van den Bogaard, Ellen H
N1 - Funding Information:
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 – Original Draft Preparation: JPHS; Writing – Review and Editing: JPHS, HN, JS, EHVDB, PLJMZ
Funding Information:
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 .
Publisher Copyright:
© 2023 The Authors
PY - 2023/8/1
Y1 - 2023/8/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85151279501&partnerID=8YFLogxK
U2 - 10.1016/j.jid.2023.02.021
DO - 10.1016/j.jid.2023.02.021
M3 - Article
C2 - 36893939
SN - 0022-202X
VL - 143
SP - 1520-1528.e5
JO - Journal of Investigative Dermatology
JF - Journal of Investigative Dermatology
IS - 8
ER -