Abstract: 【Abstract】 Objective To explore the effect of Aspergillus fumigatus （A. fumigatus）on the autophagic flux in murine bone marrow-derived macrophages （BMDM）. Methods Murine BMDM were in vitro cultured with heat-killed A. fumigatus for 0, 0.5, 4, and 12 hours. Then, cellular proteins were extracted, and Western blot analysis was performed to detect the conversion of the key autophagy protein microtubule-associated protein 1 light chain 3 （LC3）-Ⅰto LC3-Ⅱ, and to determine the protein expression of phosphorylated mammalian target of rapamycin （p-mTOR） Ser2481. Additionally, murine BMDM were in vitro cultured with A. fumigatus alone or in combination with different lysosomal inhibitors, including the cysteine cathepsin inhibitor E-64d + pepstatin, bafilomycin-A1 （BAF-A1）, ammonium chloride （NH4Cl）, and chloroquine, for 4 or 12 hours. Then, Western blot analysis was performed to investigate the effect of A. fumigatus on newly formed LC3-Ⅱ and basal autophagic flux, and confocal laser scanning fluorescence microscopy to analyze the colocalization of A. fumigatus with LC3 and Rubicon （a RUN domain Beclin-1-interacting and cysteine-rich-domain-containing protein）. Experimental results at different treatment time points were analyzed by using unpaired t test, and results of experiments evaluating the effect of two factors （A. fumigatus spores and autophagosome inhibitors） were analyzed by 2 × 2 factorial analysis. Results After in vitro co-culture with A. fumigatus for 0.5, 4, 12 hours, Western blot analysis showed that the conversion of LC3-Ⅰ to LC3-Ⅱ increased over time in murine BMDM compared with the control (0 hour) group （t = 6.58, 3.28, 3.02, respectively, all P < 0.05）, but the protein expression level of p-mTOR （Ser2481） did not significantly differ at different treatment time points（t = 0.441, 0.477, 0.382, P = 0.682, 0.660, 0.722, respectively）. After 4- and 12-hour in vitro treatment, the accumulation levels of LC3-Ⅱ in BMDM significantly increased in the A. fumigatus + chloroquine group compared with the chloroquine-alone group （t = 2.13, 2.78, respectively, both P < 0.05）, in the A. fumigatus + NH4Cl group compared with the NH4Cl-alone group （t = 2.92, 2.92, respectively, both P < 0.05）, in the A. fumigatus + BAF-A1 group compared with the BAF-A1-alone group （t = 2.13, 2.13, respectively, both P < 0.05）, and in the A. fumigatus + E-64d + pepstatin group compared with the E-64d + pepstatin group （t = 2.13, 2.92, respectively, both P < 0.05）. After 8-hour treatment with calcofluor white-labeled A. fumigatus spores, confocal laser scanning fluorescence microscopy showed that LC3 and Rubicon mainly surrounded A. fumigatus, suggesting their colocalization with A. fumigatus. Conclusion A. fumigatus can in vitro increase the basal autophagic flux in murine BMDM.

Key words: Aspergillus fumigatus, Macrophages, Autophagy, Microtubule-associated proteins, Microtubule-associated protein 1 light chain 3, LC3-associated phagocytosis