Regulation of type i interferon signaling by A20 (TNFAIP3) in autoimmune and autoinflammatory disease

The National Institute of Allergy and Infectious Diseases awarded the University of Pittsburgh $110,228 on December 22, 2026, under the Allergy and Infectious Diseases Research program (CFDA 93.855). The award funds research into the regulation of Type I interferon signaling by A20 (TNFAIP3) in autoimmune and autoinflammatory disease. The research investigates how mutations in the TNFAIP3 gene, which encodes the ubiquitin-editing enzyme A20, underlie inflammatory diseases including systemic lupus erythematosus and rheumatoid arthritis, and how A20 deficiency produces a Type I interferon signature that may explain treatment-refractory disease in haploinsufficiency of A20 patients. Preliminary findings indicate that interferon signaling molecules STAT1, TBK1, and IRF7 are direct binding targets of A20, and that monocytes may be the primary responders to enhanced interferon signaling in A20 deficiency. The research seeks to determine whether this interferon-stimulated gene overexpression represents a novel pathway regulated by A20 independently of established NF-κB and inflammasome signaling pathways, with implications for new therapeutic targets. Performance occurs at Pittsburgh, Pennsylvania, with a period of performance through August 31, 2029. The assistance type is a Project Grant.

Abstract

Mutations in the gene TNFAIP3, which encodes the ubiquitin-editing enzyme A20, underlie a spectrum of inflammatory diseases. For example, genome-wide association studies revealed that single nucleotide polymorphisms in TNFAIP3 confer a two-fold increased risk of developing systemic lupus erythematosus and rheumatoid arthritis. Furthermore, individuals born with loss-of-function TNFAIP3 variants develop haploinsufficiency of A20 (HA20), a systemic disease characterized by autoinflammation, autoimmunity, lymphoproliferation, and immunodeficiency. This phenotypic heterogeneity is due to A20's multiple known functions. A20 is a negative regulator of signaling molecules in the NF-KB and NLRP3 inflammasome signaling pathways, and its functional domains have differential impacts on whether signaling molecules are degraded or stabilized in the cytosol. A20 can remove ubiquitin tags to inactivate signaling scaffolds or add ubiquitin tags to mark a protein for proteasomal degradation. Although therapies targeting A20-regulated pathways (i.e., TNF/NF-KB and IL-1B/NLRP3 blockade) show efficacy in some HA20 patients, many exhibit partial or no response. Recently, our group discovered that HA20 patients develop a type i interferon (IFN-i) signature using a validated IFN gene score, which quantifies the constitutive induction of IFN-stimulated genes (isgs). Given A20's pleiotropic roles, we questioned if IFN-i is a novel pathway regulated by A20 independently of NF-KB/inflammasome signaling - a mechanism that could explain treatment refractory disease and offer new therapeutic targets. In preliminary studies, we found that the IFN-i signaling molecules STAT1, TBK1, and IRF7 are direct binding targets of A20, and that monocytes may be the primary responders to enhanced IFN-i signaling in A20 deficiency. However, we do not know whether this HA20-linked ISG overexpression reflects a causal mechanism driven by mutant A20 or a downstream byproduct of systemic inflammation. Therefore, the objective of this proposal is to identify the cell types responsible for pathological IFN-i overactivation in HA20 and to define how this dysregulation disrupts signaling pathways and organ function. We hypothesize that A20 restricts IFN-i signaling in monocytes by promoting STAT1 degradation and inhibiting TBK1 and IRF7 activation. In aim 1, we will map IFN-i-hypersensitive cells using cellular indexing of transcriptomes and epitopes by sequencing (cite-SEQ) in HA20 patient samples and in vivo imaging system (ivis) optical imaging in A20-haploinsufficient interferon-stimulated response element (isre)-reporter mice (MX1GFP/GFP/A20-/+ mice). In aim 2, we will dissect the mechanisms by which A20 regulates STAT1, TBK1, and IRF7, three signaling molecules that initiate and amplify IFN-i signaling. Findings from these studies will provide mechanistic understanding of how A20 regulates IFN-i signaling and whether dysregulated IFN-i underlies specific pathology in HA20. These data will accelerate clinical translation by linking molecular mechanisms with targeted therapies (i.e., TBK1 or JAK inhibitors) for HA20 and related autoimmune diseases.