Background/Purpose: Systemic juvenile idiopathic arthritis (sJIA) is severe and poorly understood inflammatory condition. Tumor necrosis factor receptor 1 (TNFR1) is activated by TNF and is found on most cell types. TNFR1 signaling triggers complex formation via its death domain (DD), activating proinflammatory processes through NFκB and various cell death pathways. Using a combination of genetic, in vitro and ex vivo studies, we have discovered a novel class of TNFRSF1A variation in children with sJIA.

Methods: We performed trio exome sequencing in a child with sJIA and targeted sequencing of TNFRSF1A in 525 sJIA cases. TNFR1 DD interactions were examined by modeling, Förster resonance energy transfer (FRET) and immunoprecipitation. TNFR1 variants were assayed in vitro for NFkB and cell death, and receptor localization was analyzed by superresolution microscopy (SRM). Patient peripheral blood mononuclear cells (PBMC) were compared to age matched control PBMC using single cell RNA sequencing (scRNAseq) and SRM. Resting/LPS stimulated monocyte phenotypes, cell death and cytokine secretion were compared to age matched controls ex vivo.

Results: Trio exome sequencing identified a novel, de novo nonsense variant of TNFRSF1A that excludes the transmembrane domain and DD (K203X), while targeted sequencing found 2 subjects with ultra rare variants in the DD (P367S, E398K). All variants showed defective NFκB signaling and attenuated cell death in vitro, attributable to their impaired ability to nucleate the TNFR1 signaling complex, while SRM found reduced surface expression and enhanced Golgi localization of all 3 variants. scRNAseq of K203X PBMC revealed a paucity of classical and nonclassical monocytes. Instead, most monocytes formed a unique patient specific cluster that exhibited suppressed TNF signaling with paradoxical overactivation of NFκB signaling, resulting in a distinct inflammatory signature. Functional enrichment analysis revealed markedly reduced vesicular trafficking and autophagy related pathways, accompanied by activation of stress responses and innate immune pathways. Key regulators of Golgi and endosomal function were consistently downregulated across multiple pathways, indicating widespread disruption of Golgi homeostasis in K203X monocytes. Indeed, K203X PBMC displayed larger areas positive for a Golgi marker, indicative of fragmentation and damage. K203X monocytes also displayed a strongly M1 skewed transcriptional program, adopting a proinflammatory M1 like phenotype and producing abundant inflammatory cytokines even under steady state conditions. Finally, K203X monocytes showed downregulation of cell death related genes and demonstrated striking resistance to apoptosis upon stimulation with LPS or Fas ligand ex vivo, consistent with a hyperinflammatory, antiapoptotic state.

Conclusion: By investigating a novel loss of function variant of TNFR1, we identified a unique mechanism of immune dysregulation. This is characterized by dysfunctional trafficking and chronic accumulation of K203X TNFR1 in the Golgi, which leads to endosomal and autophagic failure, disruption of Golgi homeostasis, and ultimately cascading cellular stress that leads to hyperactive immune function.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/retention-of-variant-forms-of-tnfr1-in-the-golgi-induces-stress-responses-and-monocyte-activation-in-systemic-juvenile-idiopathic-arthritis-sjia/