Congenital deficiency reveals critical role of ISG15 in skin homeostasis
Muhammad Nasir Hayat Malik, Syed Fakhar-ul-Hassnain Waqas, Jana Zeitvogel, Jingyuan Cheng, Robert Geffers, Zeinab Abu-Elbaha Gouda, Ahmed Mahrous Elsaman, Ahmed R. Radwan, Matthias Schefzyk, Peter Braubach, Bernd Auber, Ruth Olmer, Mathias Müsken, Lennart M. Roesner, Gisa Gerold, Sven Schuchardt, Sylvia Merkert, Ulrich Martin, Felix Meissner, Thomas Werfel, Frank Pessler
Muhammad Nasir Hayat Malik, Syed Fakhar-ul-Hassnain Waqas, Jana Zeitvogel, Jingyuan Cheng, Robert Geffers, Zeinab Abu-Elbaha Gouda, Ahmed Mahrous Elsaman, Ahmed R. Radwan, Matthias Schefzyk, Peter Braubach, Bernd Auber, Ruth Olmer, Mathias Müsken, Lennart M. Roesner, Gisa Gerold, Sven Schuchardt, Sylvia Merkert, Ulrich Martin, Felix Meissner, Thomas Werfel, Frank Pessler
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Research Article Autoimmunity

Congenital deficiency reveals critical role of ISG15 in skin homeostasis

Abstract

Ulcerating skin lesions are manifestations of human ISG15 deficiency, a type I interferonopathy. However, chronic inflammation may not be their exclusive cause. We describe two siblings with recurrent skin ulcers that healed with scar formation upon corticosteroid treatment. Both had a homozygous nonsense mutation in the ISG15 gene, leading to unstable ISG15 protein lacking the functional domain. We characterized ISG15–/– dermal fibroblasts, HaCaT keratinocytes, and human induced pluripotent stem cell–derived vascular endothelial cells. ISG15-deficient cells exhibited the expected hyperinflammatory phenotype, but also dysregulated expression of molecules critical for connective tissue and epidermis integrity, including reduced collagens and adhesion molecules, but increased matrix metalloproteinases. ISG15–/– fibroblasts exhibited elevated ROS levels and reduced ROS scavenger expression. As opposed to hyperinflammation, defective collagen and integrin synthesis was not rescued by conjugation-deficient ISG15. Cell migration was retarded in ISG15–/– fibroblasts and HaCaT keratinocytes, but normalized under ruxolitinib treatment. Desmosome density was reduced in an ISG15–/– 3D epidermis model. Additionally, there were loose architecture and reduced collagen and desmoglein expression, which could be reversed by treatment with ruxolitinib/doxycycline/TGF-β1. These results reveal critical roles of ISG15 in maintaining cell migration and epidermis and connective tissue homeostasis, whereby the latter likely requires its conjugation to yet unidentified targets.

Authors

Muhammad Nasir Hayat Malik, Syed Fakhar-ul-Hassnain Waqas, Jana Zeitvogel, Jingyuan Cheng, Robert Geffers, Zeinab Abu-Elbaha Gouda, Ahmed Mahrous Elsaman, Ahmed R. Radwan, Matthias Schefzyk, Peter Braubach, Bernd Auber, Ruth Olmer, Mathias Müsken, Lennart M. Roesner, Gisa Gerold, Sven Schuchardt, Sylvia Merkert, Ulrich Martin, Felix Meissner, Thomas Werfel, Frank Pessler

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Figure 4

Validation of dysregulated collagen homeostasis, increased type I IFN activity, and increased oxidative stress in ISG15–/– fibroblasts.

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Validation of dysregulated collagen homeostasis, increased type I IFN ac...
WT or ISG15–/– immortalized fibroblasts were stimulated with IFN-α (1000 IU/mL) for the indicated times and analyzed by RT-qPCR, immunoblot, and flow cytometry. (A) Immunoblots showing increased IFIT1, MX1, MMP1, and ALDH2 but decreased phosphorylated AKT and near-absent TGF-β1 in ISG15–/– cells. The MX1 and corresponding β-actin blots were obtained from a separate experiment. (B–G) RT-qPCR analysis revealing decreased expression of collagens (B–D), increased expression of MMP1 but decreased TIMP1 expression in ISG15–/– cells, but no apparent change in TGF-β1 mRNA (E–G). ΔΔCt analysis, with expression in unstimulated WT cells arbitrarily assigned the reference value of 1. (H and I) Determination of mitochondrial and cellular ROS (mtROS, cROS) by flow cytometry. Total numbers and percentages of mtROS- and cROS-positive cells are higher among the ISG15–/– cells. (J–M) Addition of the PI3K inhibitor PKI-402 phenocopies reduced COL1A1, TIMP1, and IFIT1 (J, L, and M) and increased MMP1 (K) expression in human fibroblasts. RT-qPCR, ΔΔCt analysis; reference = expression in untreated cells. n = 3, ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 (B–I, Student’s t test; J–M, 1-way ANOVA with Dunnett’s post hoc test).