Pellino-1 and Pellino-3b in endotoxin tolerance and TLR signaling (INM9P.458)

Abstract Endotoxin tolerance (ET) reprograms Toll-like receptor (TLR) 4-inducible responses in monocytes and macrophages by attenuating expression of pro-inflammatory cytokines without suppressing anti-inflammatory cytokines and antimicrobial effectors. We previously demonstrated deficient LPS-mediated TLR4-driven activation of IL-1 receptor-associated kinase (IRAK) 4 and IRAK1 as critical hallmarks of endotoxin tolerance. E3 ubiquitin ligases Pellino regulate TLR signaling pathways via modifications of IRAK kinases. We examined the impact of ET on Pellino-1/3b expression and utilized overexpression and gene ablation approaches to establish their functions in TLR2/4 signaling. LPS stimulation increased Pellino-1 mRNA and protein expression in control THP-1 cells and human monocytes. Endotoxin-tolerant cells showed attenuated expression of Pellino-1 but maintained increased levels of Pellino-3b proteins. Overexpression of Pellino-1 in 293/TLR4/MD2 and 293/TLR2 cells enhanced Pam3Cys- and LPS-mediated NF-κB activation and cytokine gene expression, while Pellino-1 ablation reduced these responses demonstrating Pellino-1 as a positive regulator of TLR4 signaling. Pellino-3b overexpression reduced, whereas Pellino-3b gene knockdown increased TLR2/4-driven NF-kB activation and expression of inflammatory cytokines, establishing it as a negative regulator. These data suggest non-redundant functions for Pellino-1 and Pellino-3b within the TLR4 signaling pathways and implicate their role in ET.