TIPE2, a negative regulator of innate and adaptive immunity that maintains immune homeostasis.

Immune homeostasis is essential for the normal functioning of the immune system, and its breakdown leads to fatal inflammatory diseases. We report here the identification of a member of the tumor necrosis factor-alpha-induced protein-8 (TNFAIP8) family, designated TIPE2, that is required for maintaining immune homeostasis. TIPE2 is preferentially expressed in lymphoid tissues, and its deletion in mice leads to multiorgan inflammation, splenomegaly, and premature death. TIPE2-deficient animals are hypersensitive to septic shock, and TIPE2-deficient cells are hyper-responsive to Toll-like receptor (TLR) and T cell receptor (TCR) activation. Importantly, TIPE2 binds to caspase-8 and inhibits activating protein-1 and nuclear factor-kappaB activation while promoting Fas-induced apoptosis. Inhibiting caspase-8 significantly blocks the hyper-responsiveness of TIPE2-deficient cells. These results establish that TIPE2 is an essential negative regulator of TLR and TCR function, and its selective expression in the immune system prevents hyperresponsiveness and maintains immune homeostasis.

The IκB-protein BCL-3 controls Toll-like receptor-induced MAPK activity by promoting TPL-2 degradation in the nucleus.

Proinflammatory responses induced by Toll-like receptors (TLRs) are dependent on the activation of the NF-ĸB and mitogen-activated protein kinase (MAPK) pathways, which coordinate the transcription and synthesis of proinflammatory cytokines. We demonstrate that BCL-3, a nuclear IĸB protein that regulates NF-ĸB, also controls TLR-induced MAPK activity by regulating the stability of the TPL-2 kinase. TPL-2 is essential for MAPK activation by TLR ligands, and the rapid proteasomal degradation of active TPL-2 is a critical mechanism limiting TLR-induced MAPK activity. We reveal that TPL-2 is a nucleocytoplasmic shuttling protein and identify the nucleus as the primary site for TPL-2 degradation. BCL-3 interacts with TPL-2 and promotes its degradation by promoting its nuclear localization. As a consequence, Bcl3-/- macrophages have increased TPL-2 stability following TLR stimulation, leading to increased MAPK activity and MAPK-dependent responses. Moreover, BCL-3-mediated regulation of TPL-2 stability sets the MAPK activation threshold and determines the amount of TLR ligand required to initiate the production of inflammatory cytokines. Thus, the nucleus is a key site in the regulation of TLR-induced MAPK activity. BCL-3 links control of the MAPK and NF-ĸB pathways in the nucleus, and BCL-3-mediated TPL-2 regulation impacts on the cellular decision to initiate proinflammatory cytokine production in response to TLR activation.

The deubiquitinase USP7 uses a distinct ubiquitin-like domain to deubiquitinate NF-ĸB subunits.

The transcription factor NF-ĸB is a master regulator of the innate immune response and plays a central role in inflammatory diseases by mediating the expression of pro-inflammatory cytokines. Ubiquitination-triggered proteasomal degradation of DNA-bound NF-ĸB strongly limits the expression of its target genes. Conversely, USP7 (deubiquitinase ubiquitin-specific peptidase 7) opposes the activities of E3 ligases, stabilizes DNA-bound NF-ĸB, and thereby promotes NF-ĸB-mediated transcription. Using gene expression and synthetic peptide arrays on membrane support and overlay analyses, we found here that inhibiting USP7 increases NF-ĸB ubiquitination and degradation, prevents Toll-like receptor-induced pro-inflammatory cytokine expression, and represents an effective strategy for controlling inflammation. However, the broad regulatory roles of USP7 in cell death pathways, chromatin, and DNA damage responses limit the use of catalytic inhibitors of USP7 as anti-inflammatory agents. To this end, we identified an NF-ĸB-binding site in USP7, ubiquitin-like domain 2, that selectively mediates interactions of USP7 with NF-ĸB subunits but is dispensable for interactions with other proteins. Moreover, we found that the amino acids 757LDEL760 in USP7 critically contribute to the interaction with the p65 subunit of NF-ĸB. Our findings support the notion that USP7 activity could be potentially targeted in a substrate-selective manner through the development of noncatalytic inhibitors of this deubiquitinase to abrogate NF-ĸB activity.

The regulation of sequence specific NF-κB DNA binding and transcription by IKKß phosphorylation of NF-κB p50 at serine 80.

Phosphorylation of the NF-κB 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-κB, and IKKß as a p50 kinase. Transcriptomic analysis of cells expressing a p50 S80A mutant reveals a critical role for S80 in selectively regulating the TNFα inducible expression of a subset of NF-κB target genes including pro-inflammatory cytokines and chemokines. S80 phosphorylation regulates the binding of p50 to NF-κB binding (κB) sites in a sequence specific manner. Specifically, phosphorylation of S80 reduces the binding of p50 at κB 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-κB 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 κB sequence describes a novel mechanism for the control of cytokine-induced transcriptional responses.

CRISPR Gene Editing of Murine Blood Stem and Progenitor Cells Induces MLL-AF9 Chromosomal Translocation and MLL-AF9 Leukaemogenesis.

Chromosomal rearrangements of the mixed lineage leukaemia (MLL, also known as KMT2A) gene on chromosome 11q23 are amongst the most common genetic abnormalities observed in human acute leukaemias. MLL rearrangements (MLLr) are the most common cytogenetic abnormalities in infant and childhood acute myeloid leukaemia (AML) and acute lymphocytic leukaemia (ALL) and do not normally acquire secondary mutations compared to other leukaemias. To model these leukaemias, we have used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce MLL-AF9 (MA9) chromosomal rearrangements in murine hematopoietic stem and progenitor cell lines and primary cells. By utilizing a dual-single guide RNA (sgRNA) approach targeting the breakpoint cluster region of murine Mll and Af9 equivalent to that in human MA9 rearrangements, we show efficient de novo generation of MA9 fusion product at the DNA and RNA levels in the bulk population. The leukaemic features of MA9-induced disease were observed including increased clonogenicity, enrichment of c-Kit-positive leukaemic stem cells and increased MA9 target gene expression. This approach provided a rapid and reliable means of de novo generation of Mll-Af9 genetic rearrangements in murine haematopoietic stem and progenitor cells (HSPCs), using CRISPR/Cas9 technology to produce a cellular model of MA9 leukaemias which faithfully reproduces many features of the human disease in vitro.

Expression of STAT3-regulated genes in circulating CD4+ T cells discriminates rheumatoid arthritis independently of clinical parameters in early arthritis.

OBJECTIVES: Dysregulated signal transduction and activator of transcription-3 (STAT3) signalling in CD4+ T cells has been proposed as an early pathophysiological event in RA. We sought further evidence for this observation, and to determine its clinical relevance. METHODS: Microarray technology was used to measure gene expression in purified peripheral blood CD4+ T cells from treatment-naïve RA patients and disease controls newly recruited from an early arthritis clinic. Analysis focused on 12 previously proposed transcripts, and concurrent STAT3 pathway activation was determined in the same cells by flow cytometry. A pooled analysis of previous and current gene expression findings incorporated detailed clinical parameters and employed multivariate analysis. RESULTS: In an independent cohort of 161 patients, expression of 11 of 12 proposed signature genes differed significantly between RA patients and controls, robustly validating the earlier findings. Differential regulation was most pronounced for the STAT3 target genes PIM1, BCL3 and SOCS3 (>1.3-fold difference; P < 0.005), each of whose expression correlated strongly with paired intracellular phospho-STAT3. In a meta-analysis of 279 patients the same three genes accounted for the majority of the signature's ability to discriminate RA patients, which was found to be independent of age, joint involvement or acute phase response. CONCLUSION: The STAT3-mediated dysregulation of BCL3, SOCS3 and PIM1 in circulating CD4+ T cells is a discriminatory feature of early RA that occurs independently of acute phase response. The mechanistic and functional implications of this observation at a cellular level warrant clarification.

Mapping the Interaction of B Cell Leukemia 3 (BCL-3) and Nuclear Factor κB (NF-κB) p50 Identifies a BCL-3-mimetic Anti-inflammatory Peptide.

The NF-κB transcriptional response is tightly regulated by a number of processes including the phosphorylation, ubiquitination, and subsequent proteasomal degradation of NF-κB subunits. The IκB family protein BCL-3 stabilizes a NF-κB p50 homodimer·DNA complex through inhibition of p50 ubiquitination. This complex inhibits the binding of the transcriptionally active NF-κB subunits p65 and c-Rel on the promoters of NF-κB target genes and functions to suppress inflammatory gene expression. We have previously shown that the direct interaction between p50 and BCL-3 is required for BCL-3-mediated inhibition of pro-inflammatory gene expression. In this study we have used immobilized peptide array technology to define regions of BCl-3 that mediate interaction with p50 homodimers. Our data show that BCL-3 makes extensive contacts with p50 homodimers and in particular with ankyrin repeats (ANK) 1, 6, and 7, and the N-terminal region of Bcl-3. Using these data we have designed a BCL-3 mimetic peptide based on a region of the ANK1 of BCL-3 that interacts with p50 and shares low sequence similarity with other IκB proteins. When fused to a cargo carrying peptide sequence this BCL-3-derived peptide, but not a mutated peptide, inhibited Toll-like receptor-induced cytokine expression in vitro. The BCL-3 mimetic peptide was also effective in preventing inflammation in vivo in the carrageenan-induced paw edema mouse model. This study demonstrates that therapeutic strategies aimed at mimicking the functional activity of BCL-3 may be effective in the treatment of inflammatory disease.

Glycolysis and pyrimidine biosynthesis are required for replication of adherent-invasive Escherichia coli in macrophages.

Adherent-invasive Escherichia coli (AIEC) have been implicated in the aetiology of Crohn's disease (CD), a chronic inflammatory bowel condition. It has been proposed that AIEC-infected macrophages produce high levels of pro-inflammatory cytokines thus contributing to the inflammation observed in CD. AIEC can replicate in macrophages and we wanted to determine if bacterial replication was linked to the high level of cytokine production associated with AIEC-infected macrophages. Therefore, we undertook a genetic analysis of the metabolic requirements for AIEC replication in the macrophage and we show that AIEC replication in this niche is dependent on bacterial glycolysis. In addition, our analyses indicate that AIEC have access to a wide range of nutrients in the macrophage, although the levels of purines and pyrimidines do appear to be limiting. Finally, we show that the macrophage response to AIEC infection is indistinguishable from the response to the non-replicating glycolysis mutant (ΔpfkAB) and a non-pathogenic strain of E. coli, MG1655. Therefore, AIEC does not appear to subvert the normal macrophage response to E. coli during infection.

The Regulation of Endotoxin Tolerance and its Impact on Macrophage Activation.

Endotoxin tolerance in macrophages is a key regulatory mechanism to limit the innate immune response to infection or injury. Long considered a state of unresponsiveness to Toll-like receptor activation, tolerance is now recognized as a state of altered responsiveness to infection or injury. Endotoxin tolerance leads to a shift away from a pro-inflammatory response toward a response with key anti-inflammatory and pro-resolution features. Advances in our understanding of Toll-like receptor function have identified a number of molecular mechanisms that promote tolerance, but how these are integrated to achieve gene-specific regulation is an important outstanding question. The potential to harness the mechanisms of endotoxin tolerance to promote the resolution of chronic inflammation warrants the continued investigation of this fundamental feature of innate immunity. This review focuses on the endotoxin tolerant state, our understanding of the underlying molecular mechanisms, and the clinical significance of endotoxin tolerance.

Identification of a unique hybrid macrophage-polarization state following recovery from lipopolysaccharide tolerance.

LPS tolerance is an essential immune-homeostatic response to repeated exposure to LPS that prevents excessive inflammatory responses. LPS tolerance induces a state of altered responsiveness in macrophages, resulting in repression of proinflammatory gene expression and increased expression of factors that mediate the resolution of inflammation. In this study, we analyzed the transcriptional plasticity of macrophages following LPS tolerance using genome-wide transcriptional profiling. We demonstrate that LPS tolerance is a transient state and that the expression of proinflammatory genes is restored to levels comparable to the acute response to LPS. However, following recovery from LPS tolerance a number of genes remained locked in a tolerizable state, including IL-33, CD86, IL-10, and NFIL3. Furthermore, we identified of a number of genes uniquely induced following recovery from LPS tolerance. Thus, macrophages adopt a unique transcriptional profile following recovery from LPS tolerance and have a distinct expression pattern of regulators of Ag presentation, antiviral responses, and transcription factors. Our data suggest that recovery from LPS tolerance leads to a hybrid macrophage activation state that is proinflammatory and microbicidal in nature but that possesses a regulatory anti-inflammatory profile distinct from that of LPS-tolerant and LPS-activated macrophages.

Deubiquitination of NF-κB by Ubiquitin-Specific Protease-7 promotes transcription.

NF-κB is the master regulator of the immune response and is responsible for the transcription of hundreds of genes controlling inflammation and immunity. Activation of NF-κB occurs in the cytoplasm through the kinase activity of the IκB kinase complex, which leads to translocation of NF-κB to the nucleus. Once in the nucleus, NF-κB transcriptional activity is regulated by DNA binding-dependent ubiquitin-mediated proteasomal degradation. We have identified the deubiquitinase Ubiquitin Specific Protease-7 (USP7) as a regulator of NF-κB transcriptional activity. USP7 deubiquitination of NF-κB leads to increased transcription. Loss of USP7 activity results in increased ubiquitination of NF-κB, leading to reduced promoter occupancy and reduced expression of target genes in response to Toll-like- and TNF-receptor activation. These findings reveal a unique mechanism controlling NF-κB activity and demonstrate that the deubiquitination of NF-κB by USP7 is critical for target gene transcription.

Inhibition of transcription by B cell Leukemia 3 (Bcl-3) protein requires interaction with nuclear factor κB (NF-κB) p50.

B cell leukemia 3 (Bcl-3) is an essential negative regulator of NF-κB during Toll-like receptor and TNF receptor signaling. Bcl-3 also interacts with a number of transcriptional regulators, including homodimers of the NF-κB p50 subunit. Deletion of Bcl-3 results in increased NF-κB p50 ubiquitination and proteasomal degradation and increased inflammatory gene expression. We employed immobilized peptide array technology to define a region of p50 required for the formation of a Bcl-3·p50 homodimer immunosuppressor complex. Our data demonstrate that amino acids 359-361 and 363 of p50 are critical for interaction with Bcl-3 and essential for Bcl-3-mediated inhibition of inflammatory gene expression. Bcl-3 is unable to interact with p50 when these amino acids are mutated, rendering it incapable of inhibiting the transcriptional activity of NF-κB. Bcl-3 interaction-defective p50 is hyperubiquitinated and has a significantly reduced half-life relative to wild-type p50. Nfkb1(-/-) cells reconstituted with mutated p50 precursor p105 are hyperresponsive to TNFα stimulation relative to wild-type p105, as measured by inflammatory gene expression. Mutant p105 recapitulates a Bcl3(-/-) phenotype. This study demonstrates that interaction with p50 is necessary and sufficient for the anti-inflammatory properties of Bcl-3 and further highlights the importance of p50 homodimer stability in the control of NF-κB target gene expression.

Nuclear factor-κB binding motifs specify Toll-like receptor-induced gene repression through an inducible repressosome.

Sustained Toll-like receptor (TLR) stimulation continuously activates antimicrobial genes but paradoxically represses inflammatory genes. This phenomenon, termed TLR tolerance, is essential for preventing fatal inflammatory conditions such as sepsis, but its underlying mechanisms are unclear. We report here that NF-κB binding nucleic acids of gene promoters are tolerogenic motifs, which selectively recruit an NcoR-Hdac3-deacetylated-p50 repressosome to inflammatory genes. Genome-wide analyses of TLR4-induced genes revealed that NF-κB motifs were the only regulatory elements significantly enriched in tolerizable genes. Mutating the NF-κB motifs of tolerizable genes converted them into nontolerizable ones, whereas inserting NF-κB binding motifs into nontolerizable genes conferred the tolerance. Although NF-κB p50 was essential for assembling the repressosome, genetic disruption of the NcoR-Hdac3 interaction alone was sufficient to completely abolish TLR4 tolerance and to render mice vulnerable to sepsis. Thus, the specificity of TLR tolerance is dictated by evolutionally conserved nucleic acid motifs that bound by NF-κB and the NcoR repressosome.

IĸB Protein BCL3 as a Controller of Osteogenesis and Bone Health.

OBJECTIVE: IĸB protein B cell lymphoma 3-encoded protein (BCL3) is a regulator of the NF-κB family of transcription factors. NF-κB signaling fundamentally influences the fate of bone-forming osteoblasts and bone-resorbing osteoclasts, but the role of BCL3 in bone biology has not been investigated. The objective of this study was to evaluate BCL3 in skeletal development, maintenance, and osteoarthritic pathology. METHODS: To assess the contribution of BCL3 to skeletal homeostasis, neonatal mice (n = 6-14) lacking BCL3 (Bcl3-/- ) and wild-type (WT) controls were characterized for bone phenotype and density. To reveal the contribution to bone phenotype by the osteoblast compartment in Bcl3-/- mice, transcriptomic analysis of early osteogenic differentiation and cellular function (n = 3-7) were assessed. Osteoclast differentiation and function in Bcl3-/- mice (n = 3-5) was assessed. Adult 20-week Bcl3-/- and WT mice bone phenotype, strength, and turnover were assessed. A destabilization of the medial meniscus model of osteoarthritic osteophytogenesis was used to understand adult bone formation in Bcl3-/- mice (n = 11-13). RESULTS: Evaluation of Bcl3-/- mice revealed congenitally increased bone density, long bone dwarfism, increased bone biomechanical strength, and altered bone turnover. Molecular and cellular characterization of mesenchymal precursors showed that Bcl3-/- cells displayed an accelerated osteogenic transcriptional profile that led to enhanced differentiation into osteoblasts with increased functional activity, which could be reversed with a mimetic peptide. In a model of osteoarthritis-induced osteophytogenesis, Bcl3-/- mice exhibited decreased pathological osteophyte formation (P < 0.05). CONCLUSION: Cumulatively, these findings demonstrate that BCL3 controls developmental mineralization to enable appropriate bone formation, whereas in a pathological setting, it contributes to skeletal pathology.

Evidence that marine-derived, multi-mineral, Aquamin inhibits the NF-κB signaling pathway in vitro.

It is well established that nuclear factor kappa B (NF-κB) is a central regulator of the immune response and that dysregulation of NF-κB contributes to the pathogenesis of many autoimmune and inflammatory diseases. The food supplement Aquamin is a natural multi-mineral derived from the red algae Lithothamnion corallioides, rich in calcium, magnesium and 72 other trace minerals. This study describes an anti-inflammatory role for Aquamin in inhibiting NF-κB activation through reducing the phosphorylation and degradation of its upstream inhibitor IκBα. Aquamin inhibition of NF-κB activation results in significantly reduced cyclo-oxygenase-2 gene expression following treatment of macrophage cells with lipopolysaccharide. These data suggest that nutritional supplements such as Aquamin may play an important role in regulating the inflammatory response by modulating the nuclear factor kappa B signalling pathway.

Defining the Role of Nuclear Factor (NF)-κB p105 Subunit in Human Macrophage by Transcriptomic Analysis of NFKB1 Knockout THP1 Cells.

Since its discovery over 30 years ago the NF-ĸB family of transcription factors has gained the status of master regulator of the immune response. Much of what we understand of the role of NF-ĸB in immune development, homeostasis and inflammation comes from studies of mice null for specific NF-ĸB subunit encoding genes. The role of inflammation in diseases that affect a majority of individuals with health problems globally further establishes NF-ĸB as an important pathogenic factor. More recently, genomic sequencing has revealed loss of function mutations in the NFKB1 gene as the most common monogenic cause of common variable immunodeficiencies in Europeans. NFKB1 encodes the p105 subunit of NF-ĸB which is processed to generate the NF-ĸB p50 subunit. NFKB1 is the most highly expressed transcription factor in macrophages, key cellular drivers of inflammation and immunity. Although a key role for NFKB1 in the control of the immune system is apparent from Nfkb1-/- mouse studies, we know relatively little of the role of NFKB1 in regulating human macrophage responses. In this study we use the THP1 monocyte cell line and CRISPR/Cas9 gene editing to generate a model of NFKB1-/- human macrophages. Transcriptomic analysis reveals that activated NFKB1-/- macrophages are more pro-inflammatory than wild type controls and express elevated levels of TNF, IL6, and IL1B, but also have reduced expression of co-stimulatory factors important for the activation of T cells and adaptive immune responses such as CD70, CD83 and CD209. NFKB1-/- THP1 macrophages recapitulate key observations in individuals with NFKB1 haploinsufficiency including decreased IL10 expression. These data supporting their utility as an in vitro model for understanding the role of NFKB1 in human monocytes and macrophages and indicate that of loss of function NFKB1 mutations in these cells is an important component in the associated pathology.

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 IKKß 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.

IL-6 Mediated Transcriptional Programming of Naïve CD4+ T Cells in Early Rheumatoid Arthritis Drives Dysregulated Effector Function.

Objective: We have previously shown that increased circulating interleukin-6 (IL-6) results in enhanced CD4+ T cell signaling via signal transduction and activator of transcription-3 (STAT3) in early rheumatoid arthritis (RA). We tested the hypothesis that transcriptional "imprinting" of T-cells by this mechanism skews downstream effector responses, reinforcing immune dysregulation at a critical, but targetable, disease phase. Methods: We modeled naïve CD4+ T cell exposure to pathophysiological concentrations of IL-6 in vitro, assessing the dynamic transcriptional and functional consequences for downstream effector cells utilizing microarray and flow cytometry. Fresh blood from treatment-naïve early arthritis patients was phenotyped in parallel for comparison. Results: T cell sensitivity to IL-6 was most marked in the naïve subset, and related to gp130 rather than IL-6R expression. Exposure of healthy naïve CD4+ T cells to IL-6 induced the same STAT3 target genes as previously seen to discriminate RA patients from disease controls. After TCR stimulation IL-6 pre-exposed cells exhibited enhanced proliferative capacity, activation, and a propensity toward Th1 differentiation, compared to non-exposed cells. An entirely analogous phenotype was observed in early RA compared to control CD4+ T cells. Conclusions: Sustained IL-6 exposure at a critical point in the natural history of RA "primes" the adaptive immune system to respond aberrantly to TCR stimulation, potentiating disease induction with implications for the optimal timing of targeted therapy.

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.

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.