Lipopolysaccharide-induced NF-κB nuclear translocation is primarily dependent on MyD88, but TNFα expression requires TRIF and MyD88

Abstract

TLR4 signalling through the MyD88 and TRIF-dependent pathways initiates translocation of the transcription factor NF-κB into the nucleus. In cell population studies using mathematical modeling and functional analyses, Cheng et al. suggested that LPS-driven activation of MyD88, in the absence of TRIF, impairs NF-κB translocation. We tested the model proposed by Cheng et al. using real-time single cell analysis in macrophages expressing EGFP-tagged p65 and a TNFα promoter-driven mCherry. Following LPS stimulation, cells lacking TRIF show a pattern of NF-κB dynamics that is unaltered from wild-type cells, but activation of the TNFα promoter is impaired. In macrophages lacking MyD88, there is minimal NF-κB translocation to the nucleus in response to LPS stimulation, and there is no activation of the TNFα promoter. These findings confirm that signalling through MyD88 is the primary driver for LPS-dependent NF-κB translocation to the nucleus. The pattern of NF-κB dynamics in TRIF-deficient cells does not, however, directly reflect the kinetics of TNFα promoter activation, supporting the concept that TRIF-dependent signalling plays an important role in the transcription of this cytokine.

Mathematical Modeling and Parameter Estimation of Intracellular Signaling Pathway: Application to LPS-induced NFκB Activation and TNFα Production in Macrophages

“…Profiles of de novo synthesized intracellular TNFα under the stimulation of LPS in the presence of Golgiplug ™ demonstrated that the TNFα production increased around one hour after the stimulation (Figure 2). At around the same time, the IκBα concentration reached its minimum, which is consistent with experimental observations in the literature [46][47][48]. Subsequently, the IκBα concentration increased due to the induction of IκB transcript (IκB t ) by nuclear translation of NFκB, while the TNFα production rate slowed down beyond 4 h of LPS stimulation (Figure 2).…”

Myeloid cell–targeted miR-146a mimic inhibits NF-κB–driven inflammation and leukemia progression in vivo

“…Activated NF-κB translocates to the nucleus, thus we examined the effect of C-miR146a on the localization of NF-κB in LPS-treated RAW264.7 cells expressing NF-κB/p65-eGFP fusion protein. 27 Confocal microscopy confirmed that C-miR146a, but not C-scrRNA, efficiently blocked the nuclear translocation of NF-κB ( Figure 2C). Correspondingly, the C-miR146a reduced the NF-κB DNA-binding activity in nuclear extracts from RAW264.7 and MDSL cells as measured using gelshift assays ( Figure 2D).…”

Indoxyl sulfate–induced TNF‐α is regulated by crosstalk between the aryl hydrocarbon receptor, NF‐κB, and SOCS2 in human macrophages

“…2C, D). NF-kB proteins are well-known to be key TFs involved in control of the immune response, for instance by inducing TNF-a expression in primary human macrophages in response to TLR agonists (34,35,53). The finding that IS-induced phosphorylation of NF-kB p65 on Ser536 (Fig.…”

“…Although multiple TFs are involved in inflammatory and immune responses, the NF-kB family plays a central role in inflammation, including in the induction of TNF-a gene transcription, especially in monocytes and macrophages (33)(34)(35). Therefore, to access whether IS activates Figure 1.…”