Estrogen deficiency impairs double-negative T cell function and activates NF-κB in postmenopausal osteoporosis

Postmenopausal osteoporosis (PMOP), which is driven primarily by estrogen deficiency, is characterized by excessive bone resorption and disrupted immune homeostasis. Although immune system contributions to bone loss are well-documented, the roles of specific regulatory subsets, such as double-negative T (DNT) cells (CD3⁺TCRαβ⁺CD4^-CD8^-NK1.1^-), remain unclear. Herein, by using an ovariectomized (OVX) mouse model of PMOP, we integrated transcriptomic profiling, in vivo functional assays, and in vitro studies to investigate DNT cell alterations. The OVX mice presented significant reductions in both DNT cell frequency and immunoregulatory capacity, which were accompanied by their increased apoptosis and suppressed proliferation. RNA sequencing revealed concurrent upregulation of NF-κB signaling and apoptotic pathways with downregulation of estrogen receptor (ER) signaling and cytotoxicity. In vitro, estrogen stimulation enhanced DNT cell immunoregulatory function and estrogen receptor Esr1 expression, while inhibiting NF-κB activation. These results demonstrated that estrogen deficiency impaired DNT cell viability and function, potentially through NF-κB pathway activation, thereby exacerbating immune dysregulation in PMOP. Our study suggests that DNT cells may represent important immune players involved in osteoimmunology and potential targets for immunomodulatory therapies of osteoporosis.