DIFFERENTIAL STING ACTIVATION IN DENDRITIC CELLS SHAPES DISTINCT CD4 T CELL RESPONSES AND OPPOSING COLITIS OUTCOMES

Abstract The stimulator of interferon genes (STING), an intracellular DNA sensor, regulates immune responses and contributes to various diseases. While STING activation has shown both protective and pathogenic roles in colitis, the underlying mechanisms remain unclear. We hypothesized that the strength of STING activation contributes to its dual role in colitis. Using a murine dextran sulfate sodium (DSS) colitis model, we investigated how different levels of STING stimulation affect CD4+ T cell responses and intestinal inflammation. Mice were administered low (0.5 mg/kg) or high (2.5 mg/kg) doses of 2′3′-cGAMP, a STING agonist active in both mice and humans. Low-dose cGAMP ameliorated colitis, increased IL-10 production, elevated Foxp3+ regulatory T cell frequencies, and reduced IFN-γ expression in colonic CD4+ T cells. In contrast, high-dose cGAMP exacerbated colitis, increased IFN-γ, and reduced Foxp3+ Tregs, without affecting IL-10 or IL-17 levels in CD4+ T cells. To dissect the cellular mechanisms underlying these dose-dependent effects, we first examined the direct impact of STING agonists on CD4+ T cells in vitro. DMXAA, a murine-specific STING agonist, showed a narrow working range in T cells, with higher doses inducing apoptosis and lower doses failing to alter cytokine profiles. In contrast, dendritic cells (DCs) tolerated higher doses without loss of viability. Given this differential sensitivity, we focused on STING activation in DCs as a key upstream regulator of T cell modulation and colitis outcomes. DMXAA treatment of bone marrow–derived dendritic cells (DCs) induced dose-dependent Ifnb1 and CD80 expression. When cocultured with CD4+ T cells, both DMXAA-pretreated DCs promoted IL-10 and IFN-γ production, but high-dose DMXAA-pretreated DCs did not further enhance IL-10 while significantly increasing IFN-γ. Under TGF-β–induced Treg conditions, low-dose DMXAA-pretreated DCs promoted Foxp3+ Treg differentiation, whereas high-dose DCs impaired it. Under Th17-polarizing conditions, STING activation in DCs did not affect IL-17 production. To further understand how STING activation strength shapes DC function, we profiled cytokine expression in DCs treated with low or high doses of DMXAA. DMXAA increased IL-12, IL-23, IL-10, and IL-6 expression in a dose-dependent manner, while TGF-β remained unchanged. Interestingly, both doses induced similar levels of IL-27. Blocking IL-27 with a neutralizing antibody suppressed IL-10 production in CD4+ T cells cocultured with DMXAA-pretreated DCs, suggesting that DC-derived IL-27 contributes to STING-mediated IL-10 induction. Together, these findings reveal that STING activation strength in DCs differentially regulates cytokine output and CD4+ T cell polarization, providing mechanistic insight into the dual immunomodulatory effects of STING in colitis.