Autophagy limits inflammatory gene expression through targeting of nuclear p65/RelA by LC3 and p62 for lysosomal degradation

The interplay between NF-κB signaling and autophagy regulates inflammatory signaling in different cellular contexts and in response to different stimuli. The impairment of this crosstalk may play a role in chronic inflammation and in tumorigenesis. However, the molecular mechanism by which these two pathways interact to regulate the inflammatory response remains elusive. By using biochemical analysis and imaging techniques, we characterized the interaction of the endogenous autophagic marker LC3 and NF-κB/p65 in response to different stress conditions. Following irradiation or TNFα stimulation, nuclear accumulation of LC3 strongly co-localized with p65, suggesting that nuclear p65 is targeted for autophagic degradation. Mechanistically, we showed that the nuclear p65-LC3 interaction is mediated by ubiquitination of the same p65, which is recognized by the cargo receptor p62, resulting in its cytoplasmic export and lysosomal proteolysis. Accordingly, autophagy inhibition by depletion of the essential autophagy gene ATG16L1 selectively stabilizes nuclear p65, in turn enhancing NF-κB gene expression of pro-inflammatory cytokine. Our results revealed a novel molecular mechanism that modulates the NF-κB inflammatory response through nuclear sequestration of the NF-κB/p65 subunit by autophagy proteins. These findings are of importance for developing novel therapeutic strategies against chronic inflammatory diseases displaying defective autophagy and constitutive NF-κB activity. ### Competing Interest Statement The authors have declared no competing interest. * Ang II : Angiotensin II ATG : Autophagy-related genes ATG16L1 : Autophagy related 16 like 1 CHX : Cycloheximide CQ : Chloroquine CTCF : Corrected total cell fluorescence DSS : Dextran sodium sulfate DUBs : Deubiquitinases EBSS : Earle’s Balanced Salt Solution GFP : Green fluorescent protein IBD : Inflammatory bowel disease IEC : Intestinal epithelial cell IKK : IκB kinase iNOS : Nitric oxide IR : Irradiation IκBα : NF-kappa-B inhibitor alpha KO : Knock-out LAMP1 : Lysosome-associated membrane glycoprotein LC3 : Microtubule-associated protein light chain 3 LIR : LC3-interacting region LMB : Leptomycin B LPS : Lipopolysaccharide LSD : LIR docking site NEM : N-ethylmaleimide NF-κB : Nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells NIK : NF kappa B inducing kinase PLA : Proximity ligation assay RelA : REL-associated protein RHD : Rel homology domain SQSTM1 : Sequestosome 1 TLR4 : Toll-like receptor 4 TNFα : Tumor necrosis factor-alpha UBA : Ubiquitin-associated UUO : Unilateral ureteric obstruction WT : Wild-type