烟曲霉感染通过γδT细胞抑制IFN-γ产生介导肺部免疫逃逸

Aspergillus fumigatus orchestrates pulmonary immune evasion through γδT cell-mediated suppression of interferon-gamma production

摘要:

目的揭示烟曲霉通过γδT细胞调控γ干扰素(IFN-γ)信号通路介导免疫逃逸的关键机制。 方法建立烟曲霉标准株AF293压舌法肺部感染模型。采用流式细胞术动态监测野生型(WT)小鼠感染后2、4d肺组织γδT细胞亚群动态；通过TCRδ基因敲除(TCRδ-/-)小鼠及WT全脾细胞过继转输模型，评估γδT细胞对肺真菌负荷的影响；体外以烟曲霉膨胀态孢子(MOI为1和5)梯度刺激γδT细胞，ELISA检测PBS对照组(0h)和刺激后3、6、24h细胞上清液中IFN-γ、IL-17A及IL-4的分泌水平；流式细胞术定量分析WT与TCRδ-/-小鼠肺中巨噬细胞及中性粒细胞浸润差异。 结果WT小鼠感染后肺组织γδT细胞比例显著升高(P=0.0001)，其中Vγ1+亚群占主导。TCRδ-/-小鼠肺真菌负荷显著低于WT组(P=0.0112)，而在WT全脾细胞过继转输后显著恢复(P=0.0001)。体外实验显示，烟曲霉膨胀态孢子(MOI=5)刺激γδT细胞24h后，IFN-γ产生水平显著下降(P=0.0487)。此外，TCRδ-/-小鼠肺部巨噬细胞比例较WT显著降低(P=0.0054)，而中性粒细胞的募集无显著差异。 结论烟曲霉通过γδT细胞负向调控IFN-γ信号通路，抑制巨噬细胞募集及清除真菌，从而介导肺部免疫逃逸。研究揭示了γδT细胞-IFN-γ通路在真菌免疫逃逸中的功能作用，为侵袭性肺曲霉病的免疫靶向治疗提供了新的理论依据。

Abstract:

ObjectiveTo elucidate the key mechanisms that Aspergillus fumigatus(A. fumigatus) orchestrates pulmonary immune evasion through γδT cell-mediated regulation of the interferon-gamma(IFN-γ) signaling pathway. MethodsA mouse model of A. fumigatus lung infection with tongue pressure infection was contructed: wild type(WT) mice were infected with A. fumigatus AF293 and the lung tissues were collected on 2 d, 4 d of infection for flow cytometry to analyze the proportion of γδT cells and their subsets. Infection of AF293 was performed on TCRδ KO(TCRδ-/-) mice, and the whole spleen cells of WT mice were transferred to TCRδ-/- mice for infection to detect changes in lung bacterial load. Using the swollen conidia of A. fumigatus, the γδT cells were stimulated in vitro with multiplicity of infection(MOI)=1 and 5, and the levels of interferon(IFN)-γ, interleukin(IL)-17A, and interleukin(IL)-4 were detected by enzyme-linked immunosorbent assay in the cell supernatant of the PBS control group(0h) and at 3h, 6h, and 24h after stimulation, respectively. Finally, flow cytometry was used to analyze and compare the differences in the proportion of neutrophils and macrophages in the lung of WT and TCRδ-/- mice before and after infection. ResultsAfter infection with A. fumigatus(AF293), wild-type(WT) mice showed a significant increase in the proportion of pulmonary γδT cells(P=0.0001), with the expansion of the Vγ1 subgroup being the main one. The lung bacterial load of TCRδ-/- mice was significantly lower than that in WT mice (P=0.0112), while the adoptive transfer of whole spleen cells in WT mice restored the susceptibility of TCRδ -/- mice(P=0.0001). In vitro experiments indicated that the level of IFN-γ was significantly downregulated(P=0.0487) when stimulated by swollen conidia of AF293 on γδT cells for 24 hours. In addition, flow cytometry analysis showed that compared with WT mice, the proportion of pulmonary macrophages in TCRδ-/- mice was significantly reduced(P=0.0054), while no significant difference was observed in neutrophil recruitment. ConclusionA. fumigatus mediates pulmonary immune evasion by negatively regulating the IFN-γ signaling pathway through γδT cells, thereby suppressing macrophage recruitment and fungal clearance. This study elucidates the functional role of the γδT cell-IFN-γ axis in fungal immune evasion and provides a novel theoretical foundation for immunotargeted therapies against invasive pulmonary aspergillosis.