Dataset Information

Lipopolysaccharide-induced NF-?B nuclear translocation is primarily dependent on MyD88, but TNF? expression requires TRIF and MyD88.

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

SUBMITTER: Sakai J

PROVIDER: S-EPMC5431130 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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

Sakai Jiro J Cammarota Eugenia E Wright John A JA Cicuta Pietro P Gottschalk Rachel A RA Li Ning N Fraser Iain D C IDC Bryant Clare E CE

Scientific reports 20170503 1

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 ...[more]

PMID: 28469251

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Project description:The inflammatory response is a critical molecular defense mechanism of the innate immune system that mediates the elimination of disease-causing bacteria. Repair of the damaged tissue, and the reestablishment of homeostasis, must be accomplished after elimination of the pathogen. The innate defense regulators (IDRs) are short cationic peptides that mimic natural host defense peptides and are effective in eliminating pathogens by enhancing the activity of the immune system while controlling the inflammatory response. Although the role of different IDRs as modulators of inflammation has been reported, there have been only limited studies of the signaling molecules regulated by this type of peptide. The present study investigated the effect of IDR-1002 on nuclear factor κB (NF-κB) and cAMP-response element-binding protein (CREB) transcription factors that are responsible for triggering and controlling inflammation, respectively, in macrophages. We found that TNF-α and COX-2 expression, IκBα phosphorylation, and NF-κB nuclear translocation were strongly inhibited in macrophages pre-incubated with IDR-1002 and then stimulated with lipopolysaccharide (LPS). IDR-1002 also increased CREB phosphorylation at Ser133 via activation of the p38/ERK1/2-MSK1 signaling pathways without detectable expression of the cytokines IL-4, IL-10, and IL-13 involved is suppressing inflammation or alternative activation. Transcriptional activation of NF-κB and CREB is known to require interaction with the transcriptional coactivator CREB-binding protein (CBP). To test for CBP-NF-κB and CBP-CREB complex formation, we performed co-immunoprecipitation assays. These assays showed that IDR-1002 inhibited the interaction between CBP and NF-κB in macrophages stimulated with LPS, which might explain the inhibition of TNF-α and COX-2 expression. Furthermore, the complex between CBP and CREB in macrophages stimulated with IDR-1002 was also inhibited, which might explain why IDR-1002 did not lead to expression of IL-4, IL-10, and IL-13, even though it induced an increase in phospho-CREB relative abundance. In conclusion, our results indicated that IDR-1002 has a dual effect. On one hand, it inhibited NF-κB nuclear translocation through a mechanism that involved inhibition of IκBα phosphorylation, and on the other, it activated a protein kinase signaling cascade that phosphorylated CREB to selectively influence cytokine gene expression. Based on these results, we think IDR-1002 could be a potential good biopharmaceutical candidate to control inflammation.

Dataset Information

Lipopolysaccharide-induced NF-?B nuclear translocation is primarily dependent on MyD88, but TNF? expression requires TRIF and MyD88.

Publications

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

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