Antimycobacterial Role Of Human Eosinophil Cationic Protein By Inducing Autophagy Of Mouse Macrophage To Eliminate Intracellular M.aurum

Mycobacteria are the causative agent of serious health problems worldwide,such as tuberculosis which is still a big threat to human healthy and limitation to economic development.The emergence of multi-drug resistant TB strains and inherent incompatibilities with current anti-HIV treatments has worsened the clinical scenario,demanding the development of novel antimycobacterial agents.As one member of human RNase A superfamily,eosinophil cationic protein（ECP,also named as RNase3）not only catalyzes single strand RNA but also play an important role in host innate immune system which demonstrating antimicrobial activity against a variety of microbes.Objective:In this present study,we aimed at investigating the antimycobacterial activity of ECP and unveiling the underlying mechanism involved in.Method:Extracellular antimicrobial activity of ECP was evaluated by the 100%minimal inhibitory concentration(MIC₁₀₀)against mycobacteria specie that fast growing and non-pathogenic,M.aurum.Meanwhile,the performance of ECP toward M.aurum inside of mouse macrophage RAW264.7 cell line was also recorded and compared with the extracellular result.After that,we continued to explore if ECP is able to induce autophagy,using real-time quantitative（q）PCR for transcriptional detection of BECN1 and ATG5 of M.aurum infected mouse macrophages,moreover,western blot analysis and confocal microscopy for another autophagy marker,LC3.In the end,the antimicrobial performance of ECP against M.aurum was compared among groups that were co-treated with autophagy inducer（Rapamycin）or autophagy inhibitor（Bafilomycin A and 3methyladenine）.Result:Extracellularly,ECP is effective against M.aurum with MIC₁₀₀ of 5uM,while compared with in vitro,better performance of ECP was found ex vivo with a MIC₁₀₀ of2uM with which is not toxic to mouse macrophage.Compared with control group,0.75 uM ECP treatment(lower than MIC₁₀₀,2 uM)significantly up regulated the transcription of BECN1 and ATG5 gene by real time qPCR,and ECP improving transform of LC3Ⅰto LC3Ⅱwas confirmed by western blot analysis.What’s more,rapamycin improved the antimycobacterial activity of ECP while Bafilomycin A and 3 methyladenine inhibited ECP’s intracellular antimycobacterial activity.Conclusion:RNase3 is effective against M.aurum both in vitro and ex vivo.Despite the direct antimycobacterial activity by disrupting the membrane,RNase3 can also induce the autophagy of mouse macroohage which facilitates the eradication of intracellular mycobacterium.