MACROPHAGE VITAMIN D RECEPTOR DEFICIENCY IMPAIRS MITOCHONDRIAL FUNCTION AND WEAKENS HOST DEFENSE AGAINST CANDIDA ALBICANS-RELATED INFLAMMATION

Dysbiosis microbiota can drive chronic inflammation by disrupting immune regulation, especially in macrophages. Mitochondria, vital for energy production, play a central role in macrophage metabolic and immune functions. The vitamin D receptor (VDR), a nuclear receptor activated by Vitamin D, modulates macrophage activity. Our prior research demonstrated that myeloid-specific VDR knockout (VDR^ΔLYZ) mice showed elevated levels of Candida Albicans, a phenomenon observed in human inflammatory bowel disease (IBD). However, the role of VDR in regulating macrophage mitochondrial function and the inflammatory response remains unclear. We hypothesize that mitochondrial dysfunction in VDR-deficient macrophages impairs host defense mechanisms.

VDR^ΔLYZ mice were generated by crossing VDR^LoxP mice with Lyz-cre mice. Colitis was induced using 5% Dextran Sulfate Sodium (DSS). Bone marrow-derived macrophages were generated by stimulating cells from mouse femurs and tibias with 20 ng/mL M-CSF. To assess host-microbiota interactions, VDR-deficient and wild-type macrophages were treated with Mito-Q, a mitochondria-targeted antioxidant, followed by an infection with Candida Albicans. Mitochondrial dynamics (fusion/fission), reactive oxygen species (ROS) production, cytochrome C release, and cell death were measured using Mito-track immunofluorescence, MitoSox Red staining, flow cytometry, and Western blotting.

VDR^-/- macrophages from VDR^ΔLYZ mice exhibited pronounced mitochondrial dysfunction, with increased fusion/fission, elevated mitochondrial ROS, cytochrome C and mtDNA release, and heightened apoptosis and pyroptosis (via Caspase 3 and Caspase 1). Macrophages from mice with DSS-induced colitis displayed severe mitochondrial abnormalities: elevated fusion/fission, cytochrome C and mtDNA release, decreased Tomm20 expression, and increased mitophagy. These defects led to increased cell death, confirmed by flow cytometry and increased cleaved caspase 3 and BAX/Bcl-2 ratio. Functionally, VDR^-/- macrophages showed impaired fungal clearance post Candida Albicans infection. Mito-Q treatment significantly restored mitochondrial function by reducing ROS, inhibiting cytochrome C and mtDNA release, and preventing macrophage death. It also normalized immune function, decreasing the expression of CD206, CD163, and IL-10 in macrophages of VDR^ΔLYZ mice.

VDR deficiency in macrophages results in profound mitochondrial dysfunction, increased oxidative stress, and impaired immune responses, leading to ineffective pathogen clearance and chronic inflammation. Mito-Q effectively restored mitochondrial integrity and reduced cell death. This study underscores the critical role of VDR in maintaining macrophage homeostasis and suggests therapeutic potential for addressing mitochondrial dysfunction in inflammation-related diseases.