A Preclinical State of Graves' Ophthalmopathy Characterized by Hypoxia of T-cells Identified via Multiomics Analysis

Abstract Context A preclinical state of Graves' ophthalmopathy (pre-GO) exists during the progression from Graves' hyperthyroidism (GH) to GO. Objective To distinguish the pre-GO state and identify key pathways of T-cell immunity. Methods Twenty-four GH (without ophthalmopathy within 6-month follow-up), 10 pre-GO (ophthalmopathy occurred within 6-month follow-up), and 21 GO patients were enrolled, and the transcription and DNA methylation profiles of peripheral blood mononuclear cells were generated. The differentially expressed genes (DEGs), differentially methylated CpG sites (DMCs), and differentially methylated genes (DMGs) were identified. Cluster analysis, functional analysis, and data integration analysis using latent components (DIABLO) were performed to distinguish pre-GO and identify key pathways. Flow cytometry was performed for in vitro verification. Results In total, 731, 1214, and 372 DEGs and 1583, 277, and 555 DMCs were detected via pairwise comparisons of GH vs GO, pre-GO vs GO, and GH vs pre-GO, respectively. DIABLO accurately discriminated the pre-GO state via 17 DMC and 11 DEG features ( receiver operating characteristic = 0.9975 and 0.9407, respectively). The functional analysis revealed that the DMGs and DEGs were enriched in T-cell differentiation pathways and related cytokine pathways, respectively. Further cluster analysis revealed a cluster of pre-GO-specific DEGs enriched in the hypoxia pathway. Flow cytometry confirmed that hypoxia promoted Th1, Th17, and antigen-specific CD4+ cytotoxic T-cell differentiation. Conclusion The pre-GO state was identified from GH and GO and characterized by upregulation of the hypoxia pathway that may promote effector CD4+ T-cells differentiation. These findings provide new insight into the pathogenesis and prevention of GO.