Genetically programmed alternative splicing of NEMO mediates an autoinflammatory disease phenotype
Younglang Lee, … , Richard M. Siegel, Eric P. Hanson
Younglang Lee, … , Richard M. Siegel, Eric P. Hanson
Published March 15, 2022
Citation Information: J Clin Invest. 2022;132(6):e128808. https://doi.org/10.1172/JCI128808.
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Research Article Genetics Immunology Inflammation

Genetically programmed alternative splicing of NEMO mediates an autoinflammatory disease phenotype

Abstract

Host defense and inflammation are regulated by the NF-κB essential modulator (NEMO), a scaffolding protein with a broad immune cell and tissue expression profile. Hypomorphic mutations in inhibitor of NF-κB kinase regulatory subunit gamma (IKBKG) encoding NEMO typically present with immunodeficiency. Here, we characterized a pediatric autoinflammatory syndrome in 3 unrelated male patients with distinct X-linked IKBKG germline mutations that led to overexpression of a NEMO protein isoform lacking the domain encoded by exon 5 (NEMO-Δex5). This isoform failed to associate with TANK binding kinase 1 (TBK1), and dermal fibroblasts from affected patients activated NF-κB in response to TNF but not TLR3 or RIG-I–like receptor (RLR) stimulation when isoform levels were high. By contrast, T cells, monocytes, and macrophages that expressed NEMO-Δex5 exhibited increased NF-κB activation and IFN production, and blood cells from these patients expressed a strong IFN and NF-κB transcriptional signature. Immune cells and TNF-stimulated dermal fibroblasts upregulated the inducible IKK protein (IKKi) that was stabilized by NEMO-Δex5, promoting type I IFN induction and antiviral responses. These data revealed how IKBKG mutations that lead to alternative splicing of skipping exon 5 cause a clinical phenotype we have named NEMO deleted exon 5 autoinflammatory syndrome (NDAS), distinct from the immune deficiency syndrome resulting from loss-of-function IKBKG mutations.

Authors

Younglang Lee, Alex W. Wessel, Jiazhi Xu, Julia G. Reinke, Eries Lee, Somin M. Kim, Amy P. Hsu, Jevgenia Zilberman-Rudenko, Sha Cao, Clinton Enos, Stephen R. Brooks, Zuoming Deng, Bin Lin, Adriana A. de Jesus, Daniel N. Hupalo, Daniela G.P. Piotto, Maria T. Terreri, Victoria R. Dimitriades, Clifton L. Dalgard, Steven M. Holland, Raphaela Goldbach-Mansky, Richard M. Siegel, Eric P. Hanson

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Figure 5

IKKi can be induced by TNF in hPIV3-infected P1 dermal fibroblasts, Ih rescues innate antiviral immunity.

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IKKi can be induced by TNF in hPIV3-infected P1 dermal fibroblasts, Ih r...
(A) Venn diagram indicates the intersection of TNF-inducible, TLR3/RLR/IRF3 pathway genes that interact with NEMO, both directly (red) and indirectly (black). (B) IKKi expression in primary dermal fibroblasts from HCs in response to TNF 20 ng/mL for 24 hours or TNF 20 ng/mL, LPS 10 ng/mL, or viral infection for 72 hours detected by Western blot. TBK1 and TUBULIN served as loading controls. (C) P1 and HC skin fibroblasts were stimulated with poly(I:C) 10 μg/mL for 3 hours with or without TNF 20 ng/mL costimulation, and IFNB1 gene expression was determined by qPCR, 2-tailed Mann-Whitney U test. (D) P1, P4, and HC dermal fibroblasts were infected with hPIV3-GFP and treated with TNF 20 ng/mL as indicated by red shaded histograms. At 72 hours, cells were fixed and stained intracellularly with antibody to detect IKKi expression (quantitated on far right). (E) NDAS, NEMO control, and HC dermal fibroblasts (or iPSC-derived fibroblast-like cells from P5) were infected with hPIV3-GFP with or without TNF 20 ng/mL costimulation and imaged in time course experiments of 48 to 72 hours duration to measure virus protein expression. AUC of total GFP intensity per cell was measured and within-experiment normalization to HC samples was performed. For HC and NDAS-P1 samples, n = 7 independent experiments, for all other samples n = 3 independent experiments, with total numbers of conditions shown; “+/- TNF” in figure table underneath indicates mean hPIV3-GFP AUC ratio of TNF-stimulated versus unstimulated infected cells. Means compared by 2-way ANOVA with Tukey’s multiple-comparison test. (F) iPSC-derived fibroblast-like cells were transduced with lentiviral overexpression vector encoding IKKi-IRES-mCherry and infected with hPIV3-GFP. Scale bars: 400 μm. Two-way ANOVA with Bonferroni’s correction for multiple comparisons. Data are representative of at least 2 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.