关键词: 自然杀伤细胞, 免疫衰老, 皮肌炎, 炎性反应, 抗感染

Abstract: Objective To explore the intrinsic mechanism of the contradictory phenotype “high inflammation and weak anti-infection” in MDA5 antibody-positive in patients with dermatomyositis (MDA5⁺ DM), and to clarify the senescent characteristics and functional status of peripheral blood natural killer (NK) cells, thus provide experi- mental basis for developing new DM therapeutic strategy. Methods Single-cell RNA sequencing (scRNA-seq) was used to analyze the Senescence-Associated Secretory Phenotype (SASP) score and function-related differential genes of peripheral blood NK cells in 6 MDA5⁺ DM patients and 2 healthy controls (HC). Peripheral blood was collected from 83 patients (38 in active phase, 45 in stable phase) and 44 HCs. C12FDG staining combined with flow cytometry was performed to detect the senescent phenotype of NK cells. The secretion level of interferon-γ(IFN-γ) and granzyme B (GZMB) in NK cells were measured under basal conditions and after IL-12/IL-15/IL-18 stimulation. Spearman correlation analysis was applied to evaluate the association between NK cell senescence degree and function. Results ScRNA-seq showed that the SASP score of NK cells in DM patients was significantly higher than that in HC(P＜0.001). The mean fluorescence intensity (MFI) of C12FDG in peripheral blood NK cells of DM patients in both active and stable phases was significantly higher than that in HCs(P＜0.05 and P＜0.001, respectively), with no significant difference between different disease stages. Functional tests revealed that the basal secretion level of IFN-γ and GZMB in DM patients' NK cells were significantly higher than those in HCs (P＜0.01 or P＜0.000 1). Incubation with IL-12/IL-15/IL-18, reduced IFN-γ secretion level and inhibited its increase in DM patients' NK cells as compared to the situation in HCs (P＜0.05 or P＜0.000 1), while GZMB secretion remained at a high level, and like HCs, it showed almost no response to cytokines. Correlation analysis suggested a moderate negative correlation between NK cell C12FDG accumulation and IFN-γ stress response ability (r=-0.398 1, P=0.002 9). Conclusions dPeripheral blood NKf cells in DM patients show obvious senescent characteristics, which are closely related to the defective stress response of IFN-γ secretion. This may contribute to the formation of the “high inflammation and weak anti-infection” contradictory phenotype in DM patients. This result provides experimental basis for developing therapeutic strategies targeting at NK cell senescence and for identification of related biomarkers in DM patients.

Key words: natural killer cell, immunosenescence, dermatomyositis, inflammation, anti-infection