Role of IRF-1 in Regulating the Proliferation and Function of Bone Marrow NK Cells in Patients with Severe Aplastic Anemia

Objective: To investigate the expression level of IRF1 in bone marrow NK cells of patients with severe aplastic anemia (SAA), and to explore its effect on NK cell proliferation and function and its molecular biological mechanism. Methods: The expression level of IRF1 and its correlation with clinical characteristic indexes were explored by analyzing bone marrow NK cells of SAA patients. The effects of IRF1 on NK cell proliferation and cycle were preliminarily analyzed by in vitro experiments. We searched for the downstream target proteins and related signaling pathways of IRF1 by high-throughput sequencing of transcriptome (RNA sequencing (RNA-seq)) combined with personalized analysis of PPI. Finally, the therapeutic effect of IRF1 inhibitor Rottlerin on SAA was further explored by constructing a mouse model of bone marrow failure. Results: By testing the bone marrow NK cells of SAA patients, it was found that IRF1 expression was significantly elevated in bone marrow NK cells of SAA patients, and was associated with the severity of the patient's condition. After sh-IRF1 lentivirus knockdown in YT-NK cells, the apoptosis rate of NK cells was higher than that of control. And five consecutive time points showed that the proliferative ability of YT-NK cells was significantly weakened after knockdown of the IRF1 gene. 72h after shIRF1 virus infection, flow assay revealed that the cell cycle of the knockdown group was slightly increased in the G1 phase and slightly decreased in the S phase. NKG2D, perforin was significantly lower in the shRNA-IRF1 group than in the NC group. The knockdown of IRF1 resulted in significant alterations in the NFkB signaling pathway. RNA-seq results of knockdown IRF1 showed that all differentially downregulated protein functions were enriched in RNA metabolism, multicellular biological processes, signaling molecules and interactions, NF-κB pathway, JAK/STAT pathway and IFN-α-mediated related pathways. Using string11.5 database for protein-protein interaction (PPI) analysis of genes enriched in the NF-κB signaling pathway with IRF1, we screened the top 10 core targets, which were IL-1β, CXCL8, CXCL1, CCL4, CXCL2, NFKBIA, VCAM1, MYD88, LY96, CD14. And then by protein-protein interaction study (CO-IP) technique, it was found that IRF1 might interact with IL-1β in the nucleus, and the rescue assay of overexpression of IL-1β after knocking down IRF1 showed that the proliferative activity of the cells was significantly weakened and apoptosis rate was significantly increased. In addition, the WB results showed that the NF-κB signaling pathway was phosphorylated and inhibited after IRF1 was knocked down, whereas overexpression of IL-1β could activate the NF-κB signaling pathway, and down-regulation of IRF1 and simultaneous up-regulation of IL-1β partially restored the inhibition of phosphorylation of the NF-κB signaling pathway. We applied Rotterin to treat mice with bone marrow failure and found that after injection of Rotterin, the blood count, bone marrow hematopoietic function, peripheral blood CD4/CD8 ratio, and IFN-γ expression level in CD8+T lymphocytes of mice were significantly restored compared to SAA mice. Conclusions: IRF1 may promote the phosphorylation level expression of NF - κ B pathway related proteins by interacting with IL-1 β, affecting the proliferation and function of bone marrow NK cells in SAA patients, and participating in the immunopathogenesis of SAA.