Toll-like receptors drive specific patterns of tolerance and training on restimulation of macrophages

Tolerance is a long-recognized property of macrophages that leads to an altered response to repeated or chronic exposure to endotoxin. The physiological role of tolerance is to limit the potential damage to host tissue that may otherwise result from prolonged production of pro-inflammatory cytokines. Tolerance is induced by all toll-like receptor (TLR) ligands tested to date, however, tolerance induced by the TLR4 ligand lipopolysaccharide (LPS) is by far the best studied. LPS tolerance involves a global transcriptional shift from a pro-inflammatory response toward one characterized by the expression of anti-inflammatory and pro-resolution factors. Although largely reversible, LPS-tolerance leads to a hybrid macrophage activation state that is pro-inflammatory in nature, but possesses distinct regulatory anti-inflammatory features. Remarkably, a comparative transcriptomic analysis of tolerance induced by different TLR ligands has not previously been reported. Here, we describe the transcriptomic profiles of mouse macrophages tolerized with ligands for TLR2, TLR3, TLR4 and TLR 9. While we identified TLR-specific transcriptional profiles in macrophages tolerized with each ligand, tolerance induced by TLR4 represented an archetype pattern, such that each gene tolerized by any of the TLRs tested was also found to be tolerized by TLR4. Pro-inflammatory cytokines are not universally suppressed in all tolerant cells, but distinct patterns of cytokine expression distinguished TLR-specific tolerance. Analysis of gene regulatory regions revealed specific DNA sequence motifs associated with distinct states of TLR tolerance, implicating previously identified as well as novel transcriptional regulators of tolerance in macrophages. These data provide a basis for the future exploitation of TLR-specific tolerant states to achieve therapeutic re-programming of the innate immune response

Defining the structure of the NF-ĸB pathway in human immune cells using quantitative proteomic data

The NF-ĸB transcription factor is a critical regulator of immune homeostasis and inflammatory responses and is a critical factor in the pathogenesis of inflammatory disease. The pathways to NF-ĸB activation are paradigms for signal-induced ubiquitination and proteasomal degradation, control of transcription factor function by subcellular localisation, and the control of gene transcription and physiological processes by signal transduction mechanisms. Despite the importance of NF-ĸB in disease, the NF-ĸB pathway remains unexploited for the treatment of inflammatory disease. Our understanding of NF-ĸB comes mostly from studies of transgenic mice and cell lines where components of the pathway have been deleted or over expressed. Recent advances in quantitative proteomics offer new opportunities to understand the NF-ĸB pathway using the absolute abundance of individual pathway components. We have analysed available quantitative proteomic datasets to establish the structure of the NF-ĸB pathway in human immune cells under both steady state and activated conditions. This reveals a conserved NF-κB pathway structure across different immune cell lineages and identifies important differences to the current model of the NF-ĸB pathway. These include the findings that the IKK complex in most cells is likely to consist predominantly of IKKβ homodimers, that the relative abundancies of IκB proteins show strong cell type variation, and that the components of the non-canonical NF-ĸB pathway are significantly increased in activated immune cells. These findings challenge aspects of our current view of the NF-κB pathway and identify outstanding questions important for defining the role of key components in regulating inflammation and immunity

The regulation of sequence specific NF-kB DNA binding and transcription by IKKβ phosphorylation of NF-kB p50 at serine 80

peer-reviewedPhosphorylation of the NF-kB transcription factor is an important regulatory mechanism for the control of transcription. Here we identify serine 80 (S80) as a phosphorylation site on the p50 subunit of NF-kB, and IKKβ as a p50 kinase. Transcriptomic analysis of cells expressing a p50 S80A mutant reveals a critical role for S80 in selectively regulating the TNF inducible expression of a subset of NF-kB target genes including pro-inflammatory cytokines and chemokines. S80 phosphorylation regulates the binding of p50 to NF-kB binding ( B) sites in a sequence specific manner. Specifically, phosphorylation of S80 reduces the binding of p50 at B sites with an adenine at the −1 position. Our analyses demonstrate that p50 S80 phosphorylation predominantly regulates transcription through the p50:p65 heterodimer, where S80 phosphorylation acts in trans to limit the NF- kB mediated transcription of pro-inflammatory genes. The regulation of a functional class of pro-inflammatory genes by the interaction of S80 phosphorylated p50 with a specific kB sequence describes a novel mechanism for the control of cytokine-induced transcriptional responses

Nuclear factor-kappaB: activation and regulation during toll-like receptor signaling

Toll-like receptors (TLRs) recognize distinct microbial components to initiate the innate and adaptive immune responses. TLR activation culminates in the expression of appropriate pro-inflammatory and immunomodulatory factors to meet pathogenic challenges. The transcription factor NF-kappaB is the master regulator of all TLR-induced responses and its activation is the pivotal event in TLR-mediated activation of the innate immune response. Many of the key molecular events required for TLR-induced NF-kappaB activation have been elucidated. However, much remains to be learned about the ability of TLRs to generate pathogen-specific responses using a limited number of transcription factors. This review will focus on our current understanding of NF-kappaB activation by TLRs and potential mechanisms for achieving a signal-specific response through NF-kappaB

NF-κB and the Transcriptional Control of Inflammation

The NF-κB transcription factor was discovered 30 years ago and has since emerged as the master regulator of inflammation and immune homeostasis. It achieves this status by means of the large number of important pro- and antiinflammatory factors under its transcriptional control. NF-κB has a central role in inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, and autoimmunity, as well as diseases comprising a significant inflammatory component such as cancer and atherosclerosis. Here, we provide an overview of the studies that form the basis of our understanding of the role of NF-κB subunits and their regulators in controlling inflammation. We also describe the emerging importance of posttranslational modifications of NF-κB in the regulation of inflammation, and highlight the future challenges faced by researchers who aim to target NF-κB transcriptional activity for therapeutic benefit in treating chronic inflammatory diseases

The Ubiquitination of NF-κB Subunits in the Control of Transcription

Nuclear factor (NF)-κB has evolved as a latent, inducible family of transcription factors fundamental in the control of the inflammatory response. The transcription of hundreds of genes involved in inflammation and immune homeostasis require NF-κB, necessitating the need for its strict control. The inducible ubiquitination and proteasomal degradation of the cytoplasmic inhibitor of κB (IκB) proteins promotes the nuclear translocation and transcriptional activity of NF-κB. More recently, an additional role for ubiquitination in the regulation of NF-κB activity has been identified. In this case, the ubiquitination and degradation of the NF-κB subunits themselves plays a critical role in the termination of NF-κB activity and the associated transcriptional response. While there is still much to discover, a number of NF-κB ubiquitin ligases and deubiquitinases have now been identified which coordinate to regulate the NF-κB transcriptional response. This review will focus the regulation of NF-κB subunits by ubiquitination, the key regulatory components and their impact on NF-κB directed transcription

A novel mechanism of nuclear factor-kappaB regulation by adenoviral protein 14.7K

Viruses have evolved many different ways to evade immune attacks. The adenoviral E3 protein 14.7K effectively inhibits antiviral immunity and inflammation. However, the underlying mechanism for this effect is unclear. Here we show that 14.7K is a potent inhibitor of nuclear factor (NF)-kappaB transcriptional activity following Toll-like receptor (TLR) or tumour necrosis factor (TNF) receptor signalling. The inhibition of the NF-kappaB activity occurs downstream of IkappaBalpha degradation and NF-kappaB translocation into the nucleus. Analysis of NF-kappaB DNA binding reveals that 14.7K specifically inhibits p50 homodimer DNA binding and that this inhibition is mediated through the interaction of 14.7K with p50. We propose that 14.7K inhibits NF-kappaB activity through directly blocking p50 binding to DNA and that this is the basis for its anti-inflammatory properties. Our data also indicate a role for p50 homodimer-dependent transcription in inflammation

The Regulation of NF-κB Subunits by Phosphorylation

The NF-κB transcription factor is the master regulator of the inflammatory response and is essential for the homeostasis of the immune system. NF-κB regulates the transcription of genes that control inflammation, immune cell development, cell cycle, proliferation, and cell death. The fundamental role that NF-κB plays in key physiological processes makes it an important factor in determining health and disease. The importance of NF-κB in tissue homeostasis and immunity has frustrated therapeutic approaches aimed at inhibiting NF-κB activation. However, significant research efforts have revealed the crucial contribution of NF-κB phosphorylation to controlling NF-κB directed transactivation. Importantly, NF-κB phosphorylation controls transcription in a gene-specific manner, offering new opportunities to selectively target NF-κB for therapeutic benefit. This review will focus on the phosphorylation of the NF-κB subunits and the impact on NF-κB function

Control of NF-κB subunits by ubiquitination

NF-κB is an essential regulator of inflammation and is also required for normal immune development and homeostasis. The inducible activation of NF-κB by a wide range of immuno-receptors such as the toll-like receptors (TLR), Tumour Necrosis Factor receptor (TNFR), and antigen T cell and B cell receptors requires the ubiquitin-triggered proteasomal degradation of IκBα to promote the nuclear translocation and transcriptional activity of NF-κB dimers. More recently, an additional role for ubiquitination and proteasomal degradation in the control of NF-κB activity has been uncovered. In this case, it is the ubiquitination and proteasomal degradation of the NF-κB subunits that play a critical role in the termination of the NF-κB-dependent transcriptional response induced by receptor activation. The primary trigger of NF-κB ubiquitination is DNA binding by NF-κB dimers and is further controlled by specific phosphorylation events which regulate the interaction of NF-κB with the E3 ligase complex and the deubiquitinase enzyme USP7. It is the balance between ubiquitination and deubiquitination that shapes the NF-κB-mediated transcriptional response. This chapter describes methods for the analysis of NF-κB ubiquitination