Teleost HMGB1 paralogues mediate protective autophagy by interacting with NOD2 and ATG16L1 and activating ROS/Akt/mTOR pathway against Aeromonas hydrophila infection

Abstract

As a multifunctional regulator, high mobility group box 1 (HMGB1) plays an important role in DNA transcription, autophagy, infection and inflammation in mammals based on its cellular localization. Unlike in mammals, some teleost has two HMGB1 paralogues, and the DNA-binding and pro-inflammatory functions of extracellular HMGB1 have been characterized in various fish species, but the functional role of intracellular HMGB1 in bacterial infection remains elusive. In this study, the inducible effect of Aeromonas hydrophila (A. hydrophila) on autophagy was identified by using the autophagy related gene 7 (Atg7)-knockdown Epithelioma papulosum cyprini (EPC) cells, where fish HMGB1 paralogues have been defined as the autophagy regulator in our previous study. Consistently, the HMGB1 paralogues were proved to mediate A. hydrophila-induced autophagy by knockout HMGB1 paralogues and using HMGB1 inhibitors in the same cells, and suggested the defensive role of HMGB1 paralogues against A. hydrophila infection. Mechanistically, in accordance with the findings that nucleotide-binding oligomerization domain 2 (NOD2) but not NOD1 was involved in A. hydrophila-induced autophagy, HMGB1 mediated autophagy by interaction with NOD2 and ATG16L1. In addition, transcriptomic and western blotting assays further uncovered that HMGB1 regulated A. hydrophila-induced autophagy potentially by the ROS/AKT/mTOR signaling pathway. Furthermore, the physiological importance of HMGB1-mediated autophagic mechanism was strengthened in the primary neutrophils of grass carp, in which a complete autophagy flux mediated by HMGB1 facilitated A. hydrophila clearance. Notably, two HMGB1 paralogues played the same role in aforementioned events. Taken together, the intracellular role of fish HMGB1 paralogues in the autophagic response to A. hydrophila infection provided new insights into the immunological function of fish HMGB1, and offered clues as to how fish can defend themselves against bacterial infection.