TNFAIP8L2/TIPE2 impairs autolysosome reformation via modulating the RAC1-MTORC1 axis

Macroautophagy/Autophagy is an evolutionarily conserved process that involves the selective degradation of cytoplasmic components within lysosomes in response to starvation. Autophagy is an ancient defense mechanism that has been closely integrated with the immune system and has multiple effects on innate and adaptive immunity. The pro-inflammatory and anti-inflammatory cytokines can activate and inhibit Autophagy, respectively. TNFAIP8L2/TIPE2 (tumor necrosis factor, alpha-induced protein 8-like 2) is a newly identified immune negative regulator of innate and adaptive immunity that plays an important role in immune homeostasis. However, whether and how TNFAIP8L2 controls Autophagy is still unknown. Murine TNFAIP8L2 can directly bind to and block the RAC1 GTPase activity to regulate innate immunity. RAC1 can also bind to mTOR and regulate mTORC1 cellular localization and activity. Here, we find that TNFAIP8L2 can compete with mTOR for binding to the GTP-bound state of RAC1 and negatively regulate mTORC1 activity. Interestingly, TNFAIP8L2 overexpression fails to induce Autophagy flux by the suppression of the mTOR activity under glutamine and serum starvation. Instead, TNFAIP8L2 appears to impair autophagic lysosome reformation (ALR) during prolonged starvation. Finally, we demonstrate that TNFAIP8L2 overexpression leads to a defect in mTOR reactivation and disrupts Autophagy flux, thereby leading to cell death. Furthermore, TNFAIP8L2 deficiency can exacerbate the inflammatory response and lung injury by controlling the mTOR activity in an LPS-induced mouse endotoxemia model. Our study reveals a novel role of TNFAIP8L2 in Autophagy by regulating the RAC1-MTORC1 axis that supports its potential as a target for therapeutic treatment.Abbreviations: ALR: autophagic lysosome reformation; BafA₁: bafilomycin A₁; BMDMs: bone marrow-derived macrophages; Co-IP: Co-Immunoprecipitation; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTORC1: mechanistic target of rapamycin kinase complex 1; RAPA: rapamycin; RPS6: ribosomal protein S6; SQSTM1/p62: sequestosome 1; Starv: Starvation; TNFAIP8L2/TIPE2: tumor necrosis factor-alpha-induced protein-8 like-2.