CCAR2 Dictates tTreg Instability and iTreg-Driven Dendritic Cell Tolerance via Divergent AKT/mTOR Modulation in High-Salt Microenvironments

Purpose A high-salt environment serves as a pro-inflammatory milieu that induces autoimmune responses by triggering self-reactive immune activation. While thymus-derived regulatory T cells (tTregs) exhibit significantly impaired immunosuppressive function under high-salt diet (HSD) conditions, the TGF-β-induced Treg subset (iTregs) retains full stability and functional integrity in high-salt environments. Despite these findings, endogenous salt-resistant molecular mechanisms that preserve Treg-mediated immunosuppression remain unidentified. Therefore, to address this gap, we propose to investigate the therapeutic potential of Treg cell adoptive transfer in experimental autoimmune encephalomyelitis (EAE) mouse models. By systematically analyzing the differential capacity of tTregs and iTregs to reprogram pro-inflammatory dendritic cells (DCs) into tolerogenic DCs under high-salt conditions, this study aims to identify the mechanistic distinctions that confer resistance to salt-induced inflammatory perturbations in iTregs, while tTregs remain susceptible. Methods Both Treg cell subsets generated from Foxp3-GFP mice were transferred into naïve Rag1-/- mice, GFP frequency were dynamically detected and compared within each time point. Subsequently, an EAE mouse model was established, and either iTregs or tTregs were intravenously administrated. Clinical scores were continuously recorded, while brain inflammation was evaluated using hematoxylin and eosin (H&E) staining. Additionally, brain-infiltrating Th1/Th17 cells and the presence of splenic CD11c + dendritic cells (DCs) were analyzed by flow cytometry. A DC-T co-culture assay was then conducted, followed by mechanistic studies using western blotting and FACS. Finally, CCAR2-deficient tTregs and iTregs were generated and co-cultured with DCs with or without NaCl addition. The expression of antigen-presenting molecules and the activation of the AKT/mTOR signaling pathway were then systematically evaluated. Results iTregs demonstrate superior efficacy over tTregs in alleviating brain inflammation in both EAE and high-salt diet (HSD)-exacerbated EAE. Unlike tTregs, iTregs suppress pro-inflammatory dendritic cells (DCs) and promote their conversion to an anti-inflammatory phenotype, primarily via membrane-bound TGF-β signaling rather than IL-10R signaling. This functional transformation of DCs is likely mediated by iTreg-induced inhibition of the AKT/mTOR signaling pathway. Notably, under high-salt conditions, this regulatory crosstalk appears specific to iTregs, as tTregs conversely upregulate AKT/mTOR in DCs. Furthermore, CCAR2 contributes to tTreg instability, and its knockdown restores tTreg functionality. In contrast, iTregs enhance DC tolerogenic phenotypes independently of CCAR2. Conclusion This study delineates a previously unrecognized functional dichotomy between Treg subsets, revealing that iTregs uniquely endow DC tolerance in high-salt environments through membrane-bound TGF-β-dependent suppression of AKT/mTOR signaling, whereas tTregs exacerbate DC immunogenicity via CCAR2-mediated pathway activation. By identifying CCAR2 as a critical destabilizing factor in tTregs and demonstrating the salt-resistant mechanistic signature of iTregs, our findings not only redefine microenvironment-specific regulatory paradigms in autoimmune pathogenesis but also establish iTregs as a superior therapeutic modality for inflammation-dominated disorders, particularly under metabolically stressful conditions such as high-salt exposure.