Dept. of Earth, Environment and Life Sciences (DISTAV), University of Genoa, Italy.
Dept. of Experimental Medicine (DIMES), University of Genoa, Italy.
Fish Shellfish Immunol. 2018 Feb;73:66-74. doi: 10.1016/j.fsi.2017.12.003. Epub 2017 Dec 5.
Autophagy is a highly conserved and regulated catabolic process involved in maintaining cell homeostasis in response to different stressors. The autophagic machinery is also used as an innate immune mechanism against microbial infection. In invertebrates, that lack acquired immunity, autophagy may thus play a key role in the protection against potential pathogens. In aquatic molluscs, evidence has been provided for induction of autophagy by starvation and different environmental stressors; however, no information is available on autophagic pathways in the immune cells, the hemocytes. In this work, the autophagic processes were investigated in the hemocytes of the marine bivalve, the mussel Mytilus galloprovincialis. The effects of classical inducers/inhibitors of mammalian autophagy were first tested. Rapamycin induced a decrease in lysosomal membrane stability-LMS that was prevented by the autophagy inhibitor Wortmannin. Increased MDC fluorescence and expression of LC3-II were also observed. Moreover, responses to in vitro challenge with the bivalve pathogen Vibrio tapetis were evaluated. Mussel hemocytes were unable to activate the immune response towards V. tapetis; however, bacterial challenge induced a moderate decrease in LMS, corresponding to lysosomal activation but no cytotoxicity; the effect was prevented by Wortmannin. TEM observations showed that V. tapetis resulted in rapid formation of autophagosomes and autolysosomes. Accordingly, increased LC3-II expression, decreased levels of phosphorylated mTor and of p62 were observed. The results represent the first evidence for autophagic processes in bivalve hemocytes in response to bacterial challenge, and underline the protective role of autophagy towards potential pathogenic vibrios.
自噬是一种高度保守和受调控的分解代谢过程，参与细胞在不同应激条件下的维持细胞内稳态。自噬机制也被用作抵抗微生物感染的先天免疫机制。在缺乏获得性免疫的无脊椎动物中，自噬可能在对抗潜在病原体方面发挥关键作用。在水生软体动物中，已经有证据表明自噬可被饥饿和不同的环境应激诱导；然而，关于免疫细胞（血细胞）中的自噬途径尚无信息。在这项工作中，研究了海洋双壳贝类贻贝 Mytilus galloprovincialis 血细胞中的自噬过程。首先测试了经典哺乳动物自噬诱导剂/抑制剂的作用。雷帕霉素诱导溶酶体膜稳定性（LMS）降低，该作用被自噬抑制剂 Wortmannin 所阻止。还观察到 MDC 荧光增加和 LC3-II 表达增加。此外，还评估了体外挑战双壳贝类病原体鳗弧菌时的反应。贻贝血细胞无法激活针对 V. tapetis 的免疫反应；然而，细菌挑战导致 LMS 适度降低，对应于溶酶体激活但无细胞毒性；Wortmannin 可阻止该作用。TEM 观察表明，V. tapetis 导致自噬体和自溶体的快速形成。因此，观察到 LC3-II 表达增加，磷酸化 mTor 和 p62 的水平降低。这些结果代表了贝类血细胞在细菌挑战下发生自噬过程的首次证据，并强调了自噬对潜在致病性弧菌的保护作用。
