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 3

Increased NF-κB activation and type I IFN production in T cells and monocytes expressing NEMO-Δex5.

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Increased NF-κB activation and type I IFN production in T cells and mono...
(A) Relative gene expression by whole-blood RNA-Seq from P1, P2, and P3 obtained at serial clinic visits; patients with systemic lupus erythematosus (SLE) with SLEDAI greater than 6 (SLE hi, n = 22); SLEDAI less than 6 (SLE lo, n = 76), and from healthy controls (n = 13). (B) IFN signature gene score (see Methods) from whole-blood RNA-Seq data, ordinary 1-way ANOVA, Dunnett’s correction for multiple comparisons, group mean indicated by red bar with SD. (C) IFN and NF-κB scores plotted versus NEMO-Δex5 splicing index, least squares linear regression fit, R2 shown. (D) P1 and HC PBMCs cultured in media or stimulated with poly(I:C) 10 μg/mL for 60 minutes and treated with brefeldin for 4 hours, stained with antibodies against IFN-α, IFN-β, p-STAT1, CD14, and CD4. (E) THP1 were stably transduced to express FL or mutant forms of NEMO. NF-κB p65 phosphorylation was measured by intracellular flow cytometry. (F) PMA-differentiated reconstituted THP1 cells were stimulated with LPS 10 ng/mL for 60 minutes, fixed, and stained with anti–NF-κB p65 (red) and nuclear dye (green). Scale bars: 70 μm. Areas of nuclear p65 overlap (pink) were quantitated (below). Means compared by 2-way ANOVA with Dunnett’s correction. (G) Secreted cytokines in cell culture supernatant from NEMO-reconstituted THP1 cells (black symbols) after 72-hour PMA differentiation (red). (H) P1, P4, and HC blasting T cells were infected with GFP-expressing human parainfluenza virus 3 (hPIV3-GFP) for 48 hours; n = 6, 2, and 10 replicates, respectively. Frequency of hPIV3-GFP+ cells, right. P value calculated with Mann-Whitney unpaired, 2-tailed U test. All data except from Figures 3A–C are representative of at least 2 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.