3D model of harlequin ichthyosis reveals inflammatory therapeutic targets
Florence Enjalbert, … , Anton J. Enright, Edel A. O’Toole
Florence Enjalbert, … , Anton J. Enright, Edel A. O’Toole
Published June 16, 2020
Citation Information: J Clin Invest. 2020;130(9):4798-4810. https://doi.org/10.1172/JCI132987.
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Research Article Dermatology Genetics

3D model of harlequin ichthyosis reveals inflammatory therapeutic targets

Abstract

The biology of harlequin ichthyosis (HI), a devastating skin disorder caused by loss-of-function mutations in the gene ABCA12, is poorly understood, and to date, no satisfactory treatment has been developed. We sought to investigate pathomechanisms of HI that could lead to the identification of new treatments for improving patients’ quality of life. In this study, RNA-Seq and functional assays were performed to define the effects of loss of ABCA12 using HI patient skin samples and an engineered CRISPR/Cas9 ABCA12 KO cell line. The HI living skin equivalent (3D model) recapitulated the HI skin phenotype. The cytokines IL-36α and IL-36γ were upregulated in HI skin, whereas the innate immune inhibitor IL-37 was strongly downregulated. We also identified STAT1 and its downstream target inducible nitric oxide synthase (NOS2) as being upregulated in the in vitro HI 3D model and HI patient skin samples. Inhibition of NOS2 using the inhibitor 1400W or the JAK inhibitor tofacitinib dramatically improved the in vitro HI phenotype by restoring the lipid barrier in the HI 3D model. Our study has identified dysregulated pathways in HI skin that are feasible therapeutic targets.

Authors

Florence Enjalbert, Priya Dewan, Matthew P. Caley, Eleri M. Jones, Mary A. Morse, David P. Kelsell, Anton J. Enright, Edel A. O’Toole

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Figure 4

Decrease in antiinflammatory response and activation of NOS2 pathway in the in vitro ABCA12 KO 3D model.

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Decrease in antiinflammatory response and activation of NOS2 pathway in ...
(A) qPCR analysis of IL37 in ABCA12 WT and KO 3D models. n = 3. Data are represented as mean ± SD. NS, P = 0.068, unpaired t test. qPCR analysis of (B) SOCS1 and (C) SOCS3 in ABCA12 WT and KO 2D model cell lysates. Each dot represents the mean of 3 technical replicates. n = 3. Data are represented as mean ± SD. **P ≤ 0.01, unpaired t test. (D) Representative immunoblot of p-STAT1 (Y701), total STAT1, and GAPDH proteins in untreated (–) or stimulated (+) with IFN-γ ABCA12 WT and KO cell lysates and (E) associated p-STAT1 quantitative analysis. n = 4. Data are represented as mean ± SD. *P ≤ 0.05, unpaired t test. The p-STAT1 blot was run in parallel, contemporaneously, with total STAT1 and GAPDH blots. (F) Representative NOS2 (green channel) and DAPI (blue channel) staining images of in vitro WT and HI 3D models, and (G) associated quantitative NOS2 analysis. Each dot represents the mean of relative NOS2 intensity from 3 independent images. Scale bars: 100 μm. n = 3. Data are represented as mean ± SD. *P ≤ 0.05, unpaired t test.