Cholesterol 25‐Hydroxylase Enhances Myeloid‐Derived Suppressor Cell (MDSC) Immunosuppression via the Stimulator of Interferon Genes (STING)‐Tank‐Binding Kinase 1 (TBK1)‐Receptor‐Interacting Protein Kinase 3 (RIPK3) Pathway in Colorectal Cancer

ABSTRACT Myeloid‐derived suppressor cells (MDSCs) represent a significant immunosuppressive population within the tumor microenvironment of colorectal cancer (CRC). Their activity has been strongly associated with the reprogramming of cholesterol metabolism, although the underlying mechanisms remain unclear. To investigate this, we generated myeloid‐specific cholesterol 25‐hydroxylase (CH25H) knockdown mice and differentiated bone marrow cells from wild‐type (WT) or Ch25h f/f Lyz2 Cre mice into MDSCs, subsequently treating them with 25‐hydroxycholesterol (25HC). Immune function was evaluated using flow cytometry, Western blotting, and real‐time polymerase chain reaction (PCR). Our findings indicated that CH25H and its metabolite 25HC were significantly upregulated in CRC‐associated MDSCs. The loss of CH25H impaired their immunosuppressive capacity by reducing arginase‐1 (ARG1) expression, an effect that was restored by 25HC supplementation. Mechanistically, 25HC suppressed the activation of the cyclic guanosine monophosphate–adenosine monophosphate synthase–stimulator of interferon genes (cGAS–STING) pathway and the downstream tank‐binding kinase 1 (TBK1). TBK1 formed a complex with receptor‐interacting protein kinase 3 (RIPK3), thereby repressing ARG1 expression through phosphorylation‐dependent signaling. Collectively, these findings reveal a previously unrecognized CH25H–25HC–STING axis in MDSC‐mediated immune regulation and suggest that targeting cholesterol metabolism may provide a promising therapeutic strategy for CRC immunotherapy.