Dominant-negative NFKBIA mutation promotes IL-1β production causing hepatic disease with severe immunodeficiency
Enrica E.K. Tan, … , Woei Kang Liew, John E. Connolly
Enrica E.K. Tan, … , Woei Kang Liew, John E. Connolly
Published August 4, 2020
Citation Information: J Clin Invest. 2020;130(11):5817-5832. https://doi.org/10.1172/JCI98882.
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Research Article Immunology Inflammation

Dominant-negative NFKBIA mutation promotes IL-1β production causing hepatic disease with severe immunodeficiency

Abstract

Although IKK-β has previously been shown as a negative regulator of IL-1β secretion in mice, this role has not been proven in humans. Genetic studies of NF-κB signaling in humans with inherited diseases of the immune system have not demonstrated the relevance of the NF-κB pathway in suppressing IL-1β expression. Here, we report an infant with a clinical pathology comprising neutrophil-mediated autoinflammation and recurrent bacterial infections. Whole-exome sequencing revealed a de novo heterozygous missense mutation of NFKBIA, resulting in a L34P IκBα variant that severely repressed NF-κB activation and downstream cytokine production. Paradoxically, IL-1β secretion was elevated in the patient’s stimulated leukocytes, in her induced pluripotent stem cell–derived macrophages, and in murine bone marrow–derived macrophages containing the L34P mutation. The patient’s hypersecretion of IL-1β correlated with activated neutrophilia and liver fibrosis with neutrophil accumulation. Hematopoietic stem cell transplantation reversed neutrophilia, restored a resting state in neutrophils, and normalized IL-1β release from stimulated leukocytes. Additional therapeutic blockade of IL-1 ameliorated liver damage, while decreasing neutrophil activation and associated IL-1β secretion. Our studies reveal a previously unrecognized role of human IκBα as an essential regulator of canonical NF-κB signaling in the prevention of neutrophil-dependent autoinflammatory diseases. These findings also highlight the therapeutic potential of IL-1 inhibitors in treating complications arising from systemic NF-κB inhibition.

Authors

Enrica E.K. Tan, Richard A. Hopkins, Chrissie K. Lim, Saumya S. Jamuar, Christina Ong, Koh C. Thoon, Mark J.A. Koh, Eun Mong Shin, Derrick W.Q. Lian, Madhushanee Weerasooriya, Christopher Z.W. Lee, Andreas Alvin Pumomo Soetedjo, Chang Siang Lim, Veonice B. Au, Edmond Chua, Hui Yin Lee, Leigh Ann Jones, Sharmy S. James, Nivashini Kaliaperumal, Jeffery Kwok, Ee Shien Tan, Biju Thomas, Lynn Xue Wu, Lena Ho, Anna Marie Fairhurst, Florent Ginhoux, Adrian K.K. Teo, Yong Liang Zhang, Kok Huar Ong, Weimiao Yu, Byrappa Venkatesh, Vinay Tergaonkar, Bruno Reversade, Keh Chuang Chin, Ah Moy Tan, Woei Kang Liew, John E. Connolly

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Figure 2

Molecular analysis of the mutant IκBα.

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Molecular analysis of the mutant IκBα. (A) Pedigree of the family with t...
(A) Pedigree of the family with the immunodeficient patient. Circles denote females, square denotes the male; affected individuals are represented by black symbols. (B) Schematic of IκBα protein. The mutation is indicated in red; letters refer to amino acid usage. PEST, domain consisting of a polypeptide sequence rich in proline (P), glutamic acid (E), serine (S), and threonine (T). (C) EMSA assay of HeLa cells transfected with pBOBI-Vec (pBOBI), pBOBI-IκBα (IκBα WT), or pBOBI-mutant IκBα (IκBα L34P) and treated with TNF-α (100 ng/mL) for the indicated durations. (D) Western blotting to detect Flag-IκBα (IκBα WT) and Flag-mutant IκBα (IκBα L34P) in transduced HeLa cells treated with TNF-α (100 ng/mL). α-Tubulin was used as a loading control. (E) Kinase assay of recombinant GST-IκBα WT and mutant constructs exposed to 2 μg IKK complexes from TNF-α–treated HEK293 cells (top panel). Immunoprecipitated IKK complexes are indicated in the middle panels. Coomassie blue (R-250) staining (bottom panel) was used as a loading control. (F) Western blotting of patient and control primary fibroblasts stimulated with TNF-α (100 ng/mL) for the indicated durations. Cells were lysed and blotted for IκBα and the loading control α-tubulin. (G) Quantification of nuclear colocalization of p65 NF-κB with Hoescht DNA staining in patient and control primary fibroblasts stimulated with TNF-α (100 ng/mL) or LPS (1 μg/mL) for the indicated durations. (H) Luminex cytokine assay for IL-6 production in supernatants of patient and control primary fibroblasts stimulated for 18 hours with IL-1β (100 ng/mL), TNF-α (100 ng/mL), or LPS (1 μg/mL), or left unstimulated. Data are representative of 3 experiments unless otherwise indicated and indicate the mean ± SEM. *P < 0.05, by 2-way ANOVA with Dunnett’s multiple-comparisons test.