Mechanistic characterization of the auto-inflammatory pathology caused by NFκB RelB deficiency
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Mechanistic characterization of the auto-inflammatory pathology caused by NFκB RelB deficiency

Abstract

Autoimmune and inflammatory diseases are leading causes of morbidity and death and arise from highly unique etiologies. Many autoimmune disorders involve dysregulation of the transcription factor NFκB/RelA, termed relopathies, and the interferon signaling pathway, termed interferonopathies. Previous reports have demonstrated that the loss of RelB results in autoimmunity and inflammatory pathology in both pediatric patients and mice. While recent studies reported immune sentinel cells to be critical mediators of the loss of RelB pathology, it remains unknown what dysregulated immune response pathways exist within these cells that drive the loss of RelB pathology.In these studies, I took an unbiased approach to characterize innate responses of immune sentinel cells to determine both the functional role of dysregulated gene programs in the RelB- deficient pathology and the mechanistic regulation of these gene programs by RelB. I found that loss of RelB in patient-derived fibroblasts and mouse myeloid cells results in elevated induction of hundreds of interferon-stimulated genes. To examine their functional role in RelB-deficient pathology, I generated compound mutant mice in which IFN signaling was genetically ablated. Removing hyper-expression of the interferon stimulated gene program did not ameliorate the 3 autoimmune pathology of RelB knockout mice. Instead, I found that RelB suppresses a smaller set of pro-inflammatory response genes sharing a common NFκB binding motif in a manner that is independent of interferon signaling. Therefore, while transcriptomic profiling would describe the loss of RelB pathology as an interferonopathy, the functional genetic evidence indicates that the pathology in mice is interferon-independent. To further determine the mechanism by which these pro-inflammatory genes were dysregulated by the loss of RelB, I performed biochemical and genome-wide analysis of RNA- Seq and ChIP-Seq data sets from innate immune cells derived from WT and RelB-/- mice. I found that the loss of RelB results in the hyper-binding by RelA to κB sites at or near the TSS of these IFN-independent pro-inflammatory genes, suggesting RelB may inhibit pro-inflammatory gene expression via competition with RelA for target gene promoters. To test this, I generated a novel RelB-DNA binding mutant mouse with three specific mutations in amino acids that contact the κB site. Indeed, while other cytoplasmic functions of RelB remained intact, the directed loss of RelB DNA binding function resulted in the hyper-expression of both IFN-dependent and independent gene expression and phenocopied the loss of RelB-/- inflammatory pathology. These results together suggest a key regulatory mechanism by the NFκB system in innate immune cells, in which RelB functions as a critical regulator of RelA pro-inflammatory gene expression and suppressor of autoimmune pathology.

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