NF-κB/RelA signaling required for CD40-induced humoral immunity depends on specific NEMO lysine residues in mice

Abstract

Oxidative stress and genotoxic damage activate NF-κB signaling through intracellular pathways distinct from those initiated by membrane receptors. DNA damage selectively induces post-translational modifications at lysine residues 277 and 309 of the human ubiquitin-binding protein NEMO to promote NF-κB signaling, but the physiological importance of these modifications in vivo remains unclear. Here, we show that a newly developed mouse model (NEMODK) carrying germline arginine substitutions of the corresponding NEMO lysine residues exhibits B-cell-intrinsic defects in germinal center formation and anti-viral humoral responses. Mechanistically, we identify in NEMODK B-cells a CD40-specific NF-κB signaling defect that is not linked to the well-characterized canonical or noncanonical NF-κB pathways. These B-cells fail to secure sustained NEMO monoubiquitination following CD40-induced ROS generation, which specifically reduces downstream RelA (p65) signaling required for the transcriptomic and epigenetic remodeling underlying homotypic B-cell aggregation, cell proliferation, class-switch recombination, and antibody-secreting cell generation. Our results establish a physiological role of murine NEMO K270 and K302 in linking CD40 engagement to B-cell responses through enabling sustained NEMO modification and RelA transcriptional activity. Post-translational modifications of NEMO lysine residues following CD40 stimulation contribute to the regulation of NF-κB signaling and B-cell differentiation, but their function and physiological importance in vivo are incompletely understood. This work shows that murine NEMO residues K270 and K302 enable sustained activation of the canonical NF-κB pathway in response to CD40-induced ROS generation, enhancing RelA (p65) activity and promoting B-cell functions. Germline arginine substitutions of NEMO K270/K302 in mice result in B-cell-intrinsic defects in germinal center and antibody responses. NEMO K270/K302 are required for efficient homotypic aggregation, proliferation, class-switch recombination, and antibody-secreting cell differentiation regulated by CD40-induced NF-κB signaling. NEMO K270/K302-dependent CD40-activated NF-κB signaling is primarily mediated by the sustained activity of RelA in B-cells. NEMO K270/K302 contribute to class-switch recombination by enabling the transcription of AID and NBS1 genes. CD40-induced mitochondrial ROS, but not DNA damage, contributes to the monoubiquitination of NEMO K270/K302 and sustained NF-κB signaling for downstream B-cell functions. Germline arginine substitutions of NEMO K270/K302 in mice result in B-cell-intrinsic defects in germinal center and antibody responses. NEMO K270/K302 are required for efficient homotypic aggregation, proliferation, class-switch recombination, and antibody-secreting cell differentiation regulated by CD40-induced NF-κB signaling. NEMO K270/K302-dependent CD40-activated NF-κB signaling is primarily mediated by the sustained activity of RelA in B-cells. NEMO K270/K302 contribute to class-switch recombination by enabling the transcription of AID and NBS1 genes. CD40-induced mitochondrial ROS, but not DNA damage, contributes to the monoubiquitination of NEMO K270/K302 and sustained NF-κB signaling for downstream B-cell functions. Murine NEMO K270/K302 enable sustained NF-κB signaling and enhanced RelA activity to promote B-cell functions.