Progesterone reprograms γδT cell metabolism and attenuates cytotoxicity to ameliorate recurrent spontaneous abortion

Background Progesterone is essential for pregnancy and immune regulation. Excessive γδT cell cytotoxicity has been implicated in recurrent spontaneous abortion. How progesterone regulates γδT cell function through metabolic pathways remains unclear. Methods Normal pregnancy and abortion-prone murine models were administered the progesterone receptor antagonist and progesterone, respectively, to evaluate pregnancy outcomes. Flow cytometry was employed to assess the proportion of γδT cells in the uterus and spleen and their capacity to secrete cytokines. In vitro assays examined γδT cell proliferation, apoptosis, and cytotoxicity following progesterone treatment. RNA-seq analysis revealed that progesterone modifies metabolic pathways in γδT cells. Which were validated by measurements of glucose uptake, lactate production, extracellular acidification rate (ECAR), and oxygen consumption rate (OCR). Results Progesterone treatment reduced early mouse embryonic resorption rates. It also decreased the proportion and cytotoxicity of uterine γδT cells. Similar phenomena were observed in vitro, where progesterone additionally promoted apoptosis of γδT cells. RNA-seq analysis revealed significant enrichment of gene sets associated with the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) following progesterone treatment, accompanied by upregulation of PCK2 and PPAR signaling pathway in γδT cells. Consistently, progesterone reduced glucose uptake and lactate production, decreased ECAR, and increased OCR in γδT cells. When γδT cells were treated with the PPARγ antagonist GW9662, progesterone failed to diminish their ability to secrete inflammatory cytokines. Conclusions Progesterone upregulates the expression of PCK2 and PPARγ, enhances the TCA cycle and OXPHOS in γδT cells, which reduces cytotoxicity of γδT cells to maintain a successful pregnancy.