Abstract
BACKGROUND: Severe aplastic anemia (SAA) is a life-threatening hematologic disorder characterized by bone marrow failure and impaired immunity. PURPOSE: Investigating the role of Toll-like receptor 4 (TLR4) highly expressing macrophages in the immunopathogenesis of SAA. METHODS: Macrophage TLR4 expression levels were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting (WB). Knocked down or inhibited macrophage TLR4 expression, detected the pyroptosis (IL-1β, IL-18, NLRP3, caspase-1, gasdermin D) levels by RT-qPCR and WB. Using RNA sequencing to find differential genes and pathways. Co-cultured CD8+ T cells with macrophages that knocked down or inhibited TLR4, and the levels of perforin and granzyme B expression in CD8+ T cells were detected by flow cytometry. CD8+ T cells were further co-cultured with K562, and the apoptosis rate of K562 was detected. RESULTS: The TLR4 in the bone marrow macrophages of patients with untreated SAA were significantly higher than those in the remission and control groups, and were negatively correlated with clinical indicators. RNA sequencing of macrophages with TLR4 knockdown showed that differentially expressed genes were enriched in the innate immune and inflammatory chemotaxis signaling pathways. After TLR4 knockout or TLR4 inhibitor addition in bone marrow macrophages of patients with untreated SAA, the mRNA and protein expression levels of pyroptosis markers interleukin (IL)-1β, IL-18, NLRP3, caspase-1, and gasdermin D were significantly lower than those in the control group. When CD8+ T cells were co-cultured with TLR4-knocked-down or inhibitor-added macrophages, the expression of perforin and granzyme B in CD8+ T cells was significantly reduced, and CD8+ T cell cytotoxicity decreased. CONCLUSIONS: Inhibition of macrophage TLR4 expression in SAA patients could alleviate the over-activated cellular immune response in SAA patients by decreasing the level of pyroptosis.