Natural killer cells aggravate neuroinflammation through microglial type I interferon pathway activation after traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) compromises blood-brain barrier integrity, facilitating infiltration of peripheral immune cells into cerebral tissue. While natural killer (NK) cells - key innate lymphocytes - have been established as critical mediators of secondary neuroinflammation in acute brain injuries including ischemic stroke and intracerebral hemorrhage, their precise involvement in TBI pathophysiology remains undetermined. This study systematically elucidates the role of NK cells in TBI progression and proposes novel immunomodulatory therapeutic strategies for post-TBI intervention. METHODS: Using the controlled cortical impact (CCI) method to generate TBI models, we quantitatively mapped NK cell infiltration patterns through immunofluorescence imaging and flow cytometric analysis. Single-cell RNA sequencing (scRNA-seq) was employed to characterize intracranial-infiltrating NK cells. To delineate NK cell functionality in post-TBI inflammation, we implemented NK1.1 antibody-mediated NK cells depletion followed by multi-modal assessment including: immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR), flow cytometry, and Western blotting to investigate TBI-induced neuroinflammation; Cortical bulk RNA sequencing to reveal genome-wide transcriptional alterations following NK depletion; And longitudinal evaluation of neurological outcomes using the modified Neurological Severity Score, Morris water maze spatial navigation, and rotarod motor performance tests. Finally, mechanistic insights were further pursued through in vitro neuron-NK cell coculture systems. RESULTS: In the CCI model, NK cells rapidly infiltrated the lesion site, reaching peak infiltration density within 12 h post-TBI. Utilizing single-cell RNA sequencing, we performed the first comprehensive characterization of brain-infiltrating NK cells, revealing a unique regulatory phenotype marked by diminished cytotoxic potential (reduced perforin/granzyme B expression), and enhanced cytokine secretory capacity compared to their peripheral blood counterparts. Pseudotemporal reconstruction delineated NK cell differentiation trajectories originating from their peripheral blood counterparts. Crucially, NK cell depletion significantly attenuated TBI-induced neuroinflammation, as evidenced by transcriptomic profiling showing marked suppression of type I interferon (IFN) signaling in the injured cortex. Microglia-specific isolation and analysis localized this inhibitory effect to the IFNAR-STAT1 axis. This finding suggests that NK cells may regulate microglial IFN-I responses potentially through a pathway involving neuronal interferon-beta (IFN-β) secretion. Mechanistic investigations revealed that NK cells potentiate neuronal ER stress through interferon-gamma (IFN-γ) secretion, thereby triggering neuronal IFN-β release. CONCLUSION: NK cells rapidly infiltrate brain lesions following TBI and adopt a regulatory phenotype. NK cell depletion significantly attenuates neurological deficits by suppressing microglial type I IFN signaling pathway. NK cell-derived IFN-γ is correlated with neuronal IFN-β upregulation, suggesting a potential indirect mechanism for amplifying microglial type I IFN responses. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12974-026-03715-4.