Understanding the dynamics of Toll-like Receptor 5 response to flagellin and its regulation by estradiol

© 2017 The Author(s). Toll-like receptors (TLRs) are major players of the innate immune system. Once activated, they trigger a signalling cascade that leads to NF-ΰ B translocation from the cytoplasm to the nucleus. Single cell analysis shows that NF-ΰ B signalling dynamics are a critical determinant of transcriptional regulation. Moreover, the outcome of innate immune response is also affected by the cross-talk between TLRs and estrogen signalling. Here, we characterized the dynamics of TLR5 signalling, responsible for the recognition of flagellated bacteria, and those changes induced by estradiol in its signalling at the single cell level. TLR5 activation in MCF7 cells induced a single and sustained NF-k B translocation into the nucleus that resulted in high NF-k B transcription activity. The overall magnitude of NF-k B transcription activity was not influenced by the duration of the stimulus. No significant changes are observed in the dynamics of NF-k B translocation to the nucleus when MCF7 cells are incubated with estradiol. However, estradiol significantly decreased NF-k B transcriptional activity while increasing TLR5-mediated AP-1 transcription. The effect of estradiol on transcriptional activity was dependent on the estrogen receptor activated. This fine tuning seems to occur mainly in the nucleus at the transcription level rather than affecting the translocation of the NF-k B transcription factor

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

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 TNF$\alpha$ 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 TNF$\alpha$ 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 TNF$\alpha$ 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 TNF$\alpha$ promoter activation, supporting the concept that TRIF-dependent signalling plays an important role in the transcription of this cytokine.J.S. is supported by the Cambridge Commonwealth, European and International Trust. CEB was supported by a BBSRC fellowship (BB/H021930/1) and a Wellcome Trust Investigator award (WT108045AIA). E.C. and P.C. acknowledge EU-ITN Transpol and EU-ERC Hydrosync. I.D.C.F. is supported by the intramural Research Program of the National Institute of Allergy and Infectious Diseases