UBE2C-TRIM21-driven NOS2 degradation stabilizes Tregs and suppresses pyroptotic signaling in thyrocytes in Hashimoto's thyroiditis.

Abstract

Background: Hashimoto's thyroiditis (HT) features persistent immune intolerance and progressive thyrocyte injury, yet the mechanistic linkage between regulatory T cell (Treg) instability and inflammasome-driven inflammatory damage in thyroid follicular cells remains unclear. We hypothesized that a Treg-intrinsic proteostasis module restrains inflammatory effectors to preserve immune tolerance and limit thyrocyte pyroptotic signaling.

Methods: We integrated peripheral-blood Treg transcriptomics from HT patients and healthy controls with functional perturbation, proteomic interaction screening, ubiquitination reconstitution, immunometabolic profiling, and in vivo validation. Candidate regulators were prioritized using differential expression and network analyses, then interrogated in Tregs using gain/loss-of-function approaches and Transwell co-culture with primary thyroid cells. Protein-protein interactions were identified by Flag immunoprecipitation-mass spectrometry and validated by co-immunoprecipitation and BiFC. NOS2 ubiquitination and degradation were assessed by CHX chase, ubiquitination assays, linkage profiling, and in vitro reconstitution. Mitochondrial and metabolic consequences were evaluated by JC-1 staining and Seahorse OCR/ECAR assays. Translational relevance was tested in an experimental autoimmune thyroiditis (EAT) model with adoptive transfer of engineered Tregs and assessment of thyroid pathology, cytokines, autoantibodies, and pyroptosis-associated markers.

Results: Ubiquitin-conjugating enzyme E2C (UBE2C) emerged as a consistently downregulated hub gene in HT-associated Tregs and was linked to ubiquitin-proteasome and inflammatory programs. UBE2C preserved the Treg suppressive program (FOXP3/CD25, IL-10/TGF-β), improved protection of thyroid cells, and reduced thyrocyte inflammasome/pyroptosis-associated signaling (NLRP3/ASC induction, caspase-1 processing, and GSDMD-N generation). TRIM21 was identified as a UBE2C-interacting E3 ligase partner that cooperated with UBE2C to drive predominantly K48-linked polyubiquitination and proteasomal degradation of NOS2 (with K516 as a key acceptor site). NOS2 accumulation impaired Treg mitochondrial membrane potential and shifted bioenergetics toward glycolysis, undermining Treg stability and restoring thyrocyte inflammasome activation. In EAT, adoptive transfer of UBE2C-enhanced Tregs mitigated thyroid inflammation, reduced TgAb/TPOAb, increased IL-10/TGF-β, and suppressed tissue pyroptosis-associated markers, whereas NOS2 reconstitution counteracted these benefits.

Conclusions: The UBE2C-TRIM21-NOS2 axis defines a proteostasis checkpoint that stabilizes Tregs and disrupts a Treg-thyrocyte inflammasome-GSDMD injury loop, highlighting potentially actionable nodes for future tolerance-restoring strategies in HT.