Genetic and Functional Associations with Decreased Anti-inflammatory Tumor Necrosis Factor Alpha Induced Protein 3 in Macrophages from Subjects with Axial Spondyloarthritis

OBJECTIVE Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) is an anti-inflammatory protein implicated in multiple autoimmune and rheumatologic conditions. We hypothesized that lower levels of TNFAIP3 contributes to excessive cytokine production in response to inflammatory stimuli in axial spondyloarthritis (AxSpA). A further aim was to determine the immune signaling and genetic variation regulating TNFAIP3 expression in individual subjects. METHODS Blood-derived macrophages from 50 AxSpA subjects and 30 healthy controls were assessed for TNFAIP3 expression. Cell lysates were also analyzed for NF-κB, mitogen-activated protein (MAP) kinase and STAT3 phosphorylation, and supernatants for cytokine production. Coding and regulatory regions in the TNFAIP3 gene and other auto-inflammation-implicated genes were sequenced by next-generation sequencing and variants identified. RESULTS Mean TNFAIP3 was significantly lower in spondyloarthritis macrophages than controls (p = 0.0085). Spondyloarthritis subject macrophages correspondingly produced more TNF-α in response to lipopolysaccharide (LPS, p = 0.015). Subjects with the highest TNFAIP3 produced significantly less TNF-α in response to LPS (p = 0.0023). Within AxSpA subjects, those on TNF blockers or with shorter duration of disease expressed lower levels of TNFAIP3 (p = 0.0011 and 0.0030, respectively). TNFAIP3 expression correlated positively with phosphorylated IκBα, phosphorylated MAP kinases, and unstimulated phosphorylated STAT3, but negatively with LPS or TNF-α-stimulated fold induction of phosphorylated STAT3. Further, subjects with specific groups of variants within TNFAIP3 displayed differences in TNFAIP3 (p = 0.03-0.004). Nominal pQTL associations with genetic variants outside TNFAIP3 were identified. CONCLUSION Our results suggest that both immune functional and genetic variations contribute to the regulation of TNFAIP3 levels in individual subjects. Decreased expression of TNFAIP3 in AxSpA macrophages correlated with increased LPS-induced TNF-α, and thus, TNFAIP3 dysregulation may be a contributor to excessive inflammatory responses in spondyloarthritis subjects.