Aspergillus Fumigatus-derived Extracellular Vesicles Modulate Immune Cell Function And Play A Protective Role In Fungal Keratitis

Objective:To investigate the modulatory effect of extracellular vesicles（EVs）extracted from Aspergillus fumigatus（AF）on immune cell function and its protective effect in murine AF keratitis.Methods:1.The liquid culturemedium of AF was used to extract the AF-derived EVs produced by AF through filtration,ultrafiltration and ultracentrifugation.And the EVs were analyzed by transmission electronic microscopy（TEM）and nanoparticle tracking analysis（NTA）,to observe the morphology,size and particle concentration.The approximate molecular weight and origin of AF-derived EVs were identified by SDS polyacrylamide gel electrophoresis（SDS-PAGE）and protein immunoblotting（Western blot）.2.The cell proliferation toxicity assay（CCK-8）was used to detect the effects of different levels of AF-derived EVs on mouse monocyte macrophage leukemia cells（RAW264.7 cells）,mouse peritoneal neutrophils（PMNs）,and human corneal epithelial cells（HCECs）.3.AF-derived EVs and RAW264.7 cells was observed by fluorescence microscopy with different fluorescent labels.EVs and RAW264.7 cells by fluorescence microscopy to investigate the membrane fusion of RAW264.7 cells with AF-derived EVs and the active phagocytosis of AF-derived EVs.4.q RT-PCR（quantificational real time-Polymerase chain reaction）was used to detect the expression level of inflammatory factors（both RAW264.7 cells and PMNs）and polarization markers（RAW264.7 cells）;the effect of AF-derived EVs on the ability of immune cells to phagocytose and kill conidia was measured by colony forming unit（CFU）counting.5.The toxicity of AF-derived EVs on the ocular surface of mice was examined by subconjunctival injection of AF-derived EVs.6.The production of secretory Ig A（s Ig A）in mouse tears was examined after subconjunctival injection of AF-derived EVs.7.After pretreatment of mice with AF-derived EVs,mice models of AF keratitis were established,and a slit lamp and clinical scores were used to assess the severity of fungal keratitis.8.CFU counting method,HE staining,q RT-PCR and ELISA were used to detect the fungal load and the degree of inflammatory response in mouse corneal tissue after AF infection.Results:1.A typical bilayer membrane,spherical EVs structure was observed by TEM.NTA observed that the diameter distribution of AF-derived EVs was about 100-200 nm,and the particle concentration was 5.7×10¹⁰Particle/m L.SDS-PAGE determined the protein molecular weight of AF-derived EVs.Western blot experiments identified the EVs as the source of AF.2.It was concluded from CCK-8 experiments that AF-derived EVs did not affect RAW264.7 cells,HCEC cells,and PMNs cells at particle concentrations below10⁷Particle/m L,10⁶Particle/m L,and 10⁷Particle/m L,respectively;3.RAW264.7 cells could undergo membrane fusion with AF-derived EVs as observed by fluorescence microscopy,and RAW264.7 cells could actively phagocytose AF-derived EVs;4.AF-derived EVs could induce RAW264.7 cells to polarize in the M1 direction and upregulate RAW264.7 cells and PMNs cells expressed inflammatory factors and promoted cellular phagocytosis and conidia killing ability;5.No significant clouding and ulceration of mouse corneal tissue after subconjunctival injection of AF-derived EVs;6.ELISA experiments suggested that a large amount of s Ig A was produced in tears after the treatment of AF-derived EVs;7.Pre-treated mice with AF-derived EVs had significantly lower severity and clinical scores than the non-pre-treated group;8.AF-derived EVs pre-treated mice with AF keratitis model had significantly lower AF load compared to the non-pre-treated group;9.The expression of corneal inflammatory factors was significantly decreased in the mouse AF keratitis model after subconjunctival injection of AF-derived EVs detected by q RT-PCR and ELISA.Conclusions:AF produced EVs and expressed immunogenic proteins on them;AF-derived EVs modulated the inflammatory response and conidia-clearing capacity of immune cells;AF-derived EVs enhanced s Ig A expression in tear fluid and their pretreatment on mice attenuated the severity of AF keratitis,reduced corneal fungal load,and inhibited the corneal inflammatory response.