The Role of TLR4-Induced Macrophage Pyroptosis in the Pathogenesis of Severe Aplastic Anemia

Objective: By studying the level of macrophage pyroptosis in patients with severe aplastic anemia (SAA) and its effect on CD8+ T cells, we investigated the mechanisms by which Toll-like receptor 4 (TLR4) induces macrophage pyroptosis and regulates the immune status in SAA. Methods: We derived macrophages in vitro from bone marrow samples collected from SAA treatment-naive patients, remission patients, and healthy controls and cultured the macrophages. Flow cytometry, quantitative reverse transcription polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay were used to detect the levels of pyroptosis and the expression levels of TLR4. Knockdown of TLR4 or the addition of TLR4 inhibitors in bone marrow-derived macrophages of SAA treatment-naive patients was performed to explore the effects of changes in TLR4 levels on the pyroptosis level of macrophages in SAA patients and on the killing effect of CD8+ T cells. Results: 1. The relative mRNA and protein levels of interleukin (IL)-1β, IL-18, NLRP3, caspase-1, and gasdermin D (GSDMD) in macrophages in the SAA treatment-naive group were significantly higher than those in the healthy control group, and the concentrations of IL-1β and IL-18 were significantly increased in the supernatants of macrophage cultures. IL-1β, IL-18, NLRP3, caspase-1, and GSDMD were significantly negatively correlated with the severity of the disease(Fig.A). 2. The relative mRNA and protein expression levels of TLR4 in macrophages were significantly higher in the SAA treatment-naive group than in the remission group or the control group. In addition, the relative mRNA expression level of TLR4 was positively correlated with the expression levels of IL-1β, IL-18, NLRP3, caspase-1, and GSDMD. After TLR4 knockdown or the addition of TLR4 inhibitors, the mRNA and protein expression levels of IL-1β, IL-18, NLRP3, caspase-1, and GSDMD were significantly lower than those in the control group, and the concentrations of IL-1β and IL-18 in the culture supernatant were significantly lower than those in the control group. 3. After CD8+ T cells were cocultured with macrophages with TLR4 knockdown or with the addition of inhibitors, the expression of perforin and granzyme B in CD8+ T cells was significantly reduced. Further coculture of CD8+ T cells with K562 reduced the apoptosis level of K562. Conclusion: In SAA patients, the level of macrophage pyroptosis was increased, and the expression levels of TLR4 in macrophages were increased and positively correlated with the level of macrophage pyroptosis. Inhibiting TLR4 expression reduced the level of macrophage pyroptosis. TLR4 might induce pyroptosis of macrophages in SAA patients and enhance the killing effect of CD8+ T cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal