Abstract

Objective: Metformin, which targeting respiratory chain complex I, was the first-line medication for type 2 diabetes. Recent studies showed that metformin can improve several autoimmune diseases through a metabolic-immunological pattern. CD4+ cytotoxicity T cells (CTLs) have been reported in the pathogenesis of various autoimmune inflammatory diseases, including Graves' ophthalmopathy (GO). However, the effect of metformin on these CD4+CTLs remains unclear. This study explores the metabolic changes of CD4+ T cell subtypes in GO and investigates the effect of metformin on the pathogenic CD4+ T cell.

Methods: Single-cell transcriptome sequencing of CD4+ T cells from GO patients was performed to systematically describe the status of different T cell subtypes.

In vitro culture and 48h intervention were performed to explore the effect of metformin and the mechanism of respiratory chain complex I. Flow cytometry was used to detect T cell subtypes and functions.

Results: Compared to healthy controls (HC), the ratio of CD4+GZMB+ cells (CD4+CTLs) of GO patients was significantly increased, as well as the effector molecules PRF and IFNγ. The ratio of Th2 and Treg cells in GO patients were significantly decreased, while the ratio of Th1 and Th17 cells didn’t change. Single-cell sequencing revealed that CD4+CTLs exhibited significantly upregulated pathways related to mitochondrial metabolism, respiratory chain, ATP production, and ROS, compared to Th1 and Th17. Further analysis focused on the genes of subunits of respiratory chain complex I, including NduFB10, NduFB2, NduFB7, NduFC2, and NduFS6. After inhibiting respiratory chain complex I, the ratio of CD4+CTLs in the peripheral blood of GO patients significantly decreased, accompanied by improvements in effector molecules PRF and IFNγ, with no significant changes in the ratio of Th1, Th2, Th17, and Treg cells. Metformin intervention significantly decreased the the ratio of CD4+CTLs of GO patients, as well as the effector molecules PRF.

Conclusions: This study revealed that CD4+CTLs, as disease-specific T cell subtypes in GO, exhibited a significant upregulation of genes related to respiratory chain complex I.  metformin can improve the ratio and function of CD4+CTLs through respiratory chain complex I. This result systematically describes the metabolic characteristics of CD4+CTLs and provides the first evidence that metformin regulates CD4+CTLs, laying the foundation for the metabolic-immunological regulation of metformin in improving immunity.