Article Text
Abstract
Objectives TNFAIP3 encodes A20 that negatively regulates nuclear factor kappa light chain enhancer of activated B cells (NF-κB), the major transcription factor coordinating inflammatory gene expression. TNFAIP3 polymorphisms have been linked with a spectrum of inflammatory and autoimmune diseases and, recently, loss-of-function mutations in A20 were found to cause a novel inflammatory disease ‘haploinsufficiency of A20’ (HA20). Here we describe a family with HA20 caused by a novel TNFAIP3 loss-of-function mutation and elucidate the upstream molecular mechanisms linking HA20 to dysregulation of NF-κB and the related inflammasome pathway.
Methods NF-κB activation was studied in a mutation-expressing cell line using luciferase reporter assay. Physical and close-proximity protein–protein interactions of wild-type and TNFAIP3 p.(Lys91*) mutant A20 were analysed using mass spectrometry. NF-κB -dependent transcription, cytokine secretion and inflammasome activation were compared in immune cells of the HA20 patients and control subjects.
Results The protein–protein interactome of p.(Lys91*) mutant A20 was severely impaired, including interactions with proteins regulating NF-κB activation, DNA repair responses and the NLR family pyrin domain containing 3 (NLRP3) inflammasome. The p.(Lys91*) mutant A20 failed to suppress NF-κB signalling, which led to increased NF-κB -dependent proinflammatory cytokine transcription. Functional experiments in the HA20 patients’ immune cells uncovered a novel caspase-8-dependent mechanism of NLRP3 inflammasome hyperresponsiveness that mediated the excessive secretion of interleukin-1β and interleukin-18.
Conclusions The current findings significantly deepen our understanding of the molecular mechanisms underlying HA20 and other diseases associated with reduced A20 expression or function, paving the way for future therapeutic targeting of the pathway.
- inflammation
- autoimmune diseases
- t cells
- behcet's disease
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Footnotes
KR and SK contributed equally.
TH, KKE and MV contributed equally.
Contributors KR and SK designed and performed the experiments, analysed data, prepared the figures and wrote the manuscript. TH identified the index patient. LT and JS performed variant identification. VG organised clinical samples. MS, OK, PV, AV, PK, RK, AJ, NH, PJ, DCN and TH provided medical care and sampling. KKE and MV participated in designing experiments, data analysis, figure preparation, and manuscript writing and submission.
Funding The study was supported by Finska Läkaresällskapet (KKE, DN), The Canadian Institutes of Health Research (THC 135230; KKE), the Stockmann foundation (KKE) and the Paulo foundation (KR). Molecular graphics and analyses were performed with the UCSF Chimera package. Chimera is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311). Exome sequencing of the index patient was conducted in the Institute for Molecular Medicine Finland (FIMM) Technology Centre.
Competing interests None declared.
Patient consent Not required.
Ethics approval Ethical Committee for Clinical Science of Oulu University Hospital, and Coordinating Ethics Committee of The Hospital District of Helsinki and Uusimaa.
Provenance and peer review Not commissioned; externally peer reviewed.