Role of Macrophage TLR4 Expression in the Immunopathogenesis of Severe Aplastic Anemia

By studying the expression level of TLR4 in macrophages of patients with severe aplastic anemia (SAA) and its impact on CD8+ T cells, we explored the role of macrophages with high TLR4 expression in the immunopathogenesis of SAA. Mononuclear cells were isolated from the bone marrow samples of untreated and remissive SAA patients as well as healthy controls, and macrophages were cultured in vitro. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western Blot were used to detect the expression level of TLR4 in macrophages. The effect of TLR4 level changes on macrophages and CD8+ T cell cytotoxicity was studied by knocking down TLR4 or using TLR4 inhibitors in the bone marrow macrophages of untreated SAA patients. The results showed that: 1. The relative expression level of TLR4 mRNA and protein in the bone marrow macrophages of untreated SAA patients was significantly higher than that in the remission group and control group, and was negatively correlated with clinical indicators. 2. RNA-seq of macrophages with knocked-down TLR4 showed that differential genes were enriched in innate immune and inflammatory chemotaxis signaling pathways. 3. After TLR4 knock-down or TLR4 inhibitor addition in bone marrow macrophages of untreated SAA patients, the mRNA and protein expression levels of pyroptosis markers IL-1β, IL-18, NLRP3, caspase-1, and GSDMD were significantly lower than those in the control group. 4. 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. Therefore, the high expression of TLR4 in macrophages of SAA patients is positively correlated with pyroptosis levels, and inhibiting TLR4 expression can reduce macrophage pyroptosis levels, thereby alleviating the overactivated cellular immune response in SAA patients.