Background/Purpose: The NF-kB family of transcription factors regulate innate and adaptive immunity, in addition to driving pro-inflammatory-disease states.  The type I interferon response acts in parallel, and is mediated by IRF3 and IRF7 transcription factors.  The IRFs are triggered by the RIG-I-like family of receptors (RLRs) in fibroblasts and Toll-like receptors (TLRs) in dendritic cells and macrophages.  Both NF-kB activation and the Type I IFN response require the NF-kB essential modulator (NEMO) for normal function. The mechanisms by which NEMO is regulated to integrate various upstream signals to direct transcription in antiviral immune defense and inflammatory disease is unknown. We present the molecular mechanism by which a naturally occurring NEMO mutation associated with inflammatory disease leads to aberrant cell responses.

Methods: Genomic DNA and cDNA from dermal fibroblasts and peripheral blood cells from a patient with optic neuritis, panniculitis and physical characteristics consistent with NEMO mutation were analyzed to determine the genotype and effect on NEMO protein product.  Patient cells were stimulated with TNF, anti-CD3/CD28,viral nucleic acid analogs, and RSV and HPIV3 infection.  Gene expression profiling, cytokine production and biochemical analysis was performed to characterize and determine the mechanism of aberrant signaling produced by the mutant form of NEMO.

Results: We identified an individual with Ectodermal Dysplasia, a history of in utero cytomegalovirus exposure, and persistent inflammatory disease.  Sanger sequencing revealed a de novo synonymous mutation in IKBKG, which encodes NEMO, the NF-kB essential modulator.  The mutation results in an mRNA splicing defect which leads to the production of a mutant protein containing an in-frame deletion of 51 residues, lacking a previously identified TANK (TRAF family member-associated NFKB activator) interaction domain.  Co-immunoprecipitation studies revealed that the mutant form of NEMO from patient T cells is unable to recruit the IRF3 kinase TANK-binding kinase-1 (TBK1), due to impaired association with TANK. Patient fibroblasts stimulated with the RLR ligand poly(I:C) led to impaired induction of IRF3 response genes IFNB1, CCL5, CXCL10, IP10, OASL, and DDX58.  Consequently, dermal fibroblasts infected with RSV or hPIV3 demonstrated increased virus propagation relative to healthy control cells. Impaired gene expression occurred despite enhanced nuclear translocation of IRF3, suggesting that the mechanism of impaired activation is not a simple defect in nuclear localization of the transcription factor. In contrast to the impaired type I IFN response, canonical IKK activation and gene induction in dermal fibroblasts appeared relatively normal following TNF treatment. 

Conclusion: These results illustrate a novel mechanism of autoinflammatory disease due to aberrant expression of an alternately spliced NEMO and deficient IRF3-mediated response.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/inflammatory-disease-due-to-dysregulated-nuclear-factor-%ce%bab-activation-and-impaired-type-i-interferon-response-resulting-from-a-de-novo-human-nemo-hypomorphic-mutation/