Toby Lawrence

Transcriptional regulation of macrophage polarization: Enabling diversity with identity

In terms of both phenotype and function, macrophages have remarkable heterogeneity, which reflects the specialization of tissue-resident macrophages in microenvironments as different as liver, brain and bone. Also, marked changes in the activity and gene expression programmes of macrophages can occur when they come into contact with invading microorganisms or injured tissues. Therefore, the macrophage lineage includes a remarkable diversity of cells with different functions and functional states that are specified by a complex interplay between microenvironmental signals and a hardwired differentiation programme that determines macrophage identity. In this Review, we summarize the current knowledge of transcriptional and chromatin-mediated control of macrophage polarization in physiology and disease.

Nuclear Factor-κB and Tumor-Associated Macrophages

Tumor-associated macrophages (TAM) have been linked with the progression of cancer by favoring tumor angiogenesis, growth, and metastasis. The precise mechanisms that maintain the protumor phenotype of TAM are poorly understood, but recent research has highlighted a number of signaling pathways that are important in TAM phenotype and function. Nuclear factor-κB (NF-κB) is considered the master regulator of inflammatory and immune responses. Recently several genetic studies have indicated NF-κB is an important pathway in TAM for the integration of signals from the tumor microenvironment that promote carcinogenesis. This review will focus on the role of NF-κB in TAM and the potential of targeting this pathway as a novel therapeutic strategy against cancer. Clin Cancer Res; 16(3); 784–9

Homeostatic NF-κB Signaling in Steady-State Migratory Dendritic Cells Regulates Immune Homeostasis and Tolerance

Migratory non-lymphoid tissue dendritic cells (NLT-DCs) transport antigens to lymph nodes (LNs) and are required for protective immune responses in the context of inflammation and to promote tolerance to self-antigens in steady-state. However, the molecular mechanisms that elicit steady-state NLT-DC maturation and migration are unknown. By comparing the transcriptome of NLT-DCs in the skin with their migratory counterparts in draining LNs, we have identified a novel NF-κB-regulated gene network specific to migratory DCs. We show that targeted deletion of IKKβ in DCs, a major activator of NF-κB, prevents NLT-DC accumulation in LNs and compromises regulatory T cell conversion in vivo. This was associated with impaired tolerance and autoimmunity. NF-κB is generally considered the prototypical pro-inflammatory transcription factor, but this study describes a role for NF-κB signaling in DCs for immune homeostasis and tolerance that could have implications in autoimmune diseases and immunity.

Cigarette Smoke Induced Airway Inflammation Is Independent of NF-κB Signalling

Rationale COPD is an inflammatory lung disease largely associated with exposure to cigarette smoke (CS). The mechanism by which CS leads to the pathogenesis of COPD is currently unclear; it is known however that many of the inflammatory mediators present in the COPD lung can be produced via the actions of the transcription factor Nuclear Factor-kappaB (NF-κB) and its upstream signalling kinase, Inhibitor of κB kinase-2 (IKK-2). Therefore the NF-κB/IKK-2 signalling pathway may represent a therapeutic target to attenuate the inflammation associated with COPD. Aim To use a range of assays, genetically modified animals and pharmacological tools to determine the role of NF-κB in CS-induced airway inflammation. Methods NF-κB pathway activation was measured in pre-clinical models of CS-induced airway inflammation and in human lung tissue from COPD patients. This data was complemented by employing mice missing a functional NF-κB pathway in specific cell types (epithelial and myeloid cells) and with systemic inhibitors of IKK-2. Results We showed in an airway inflammation model known to be NF-κB-dependent that the NF-κB pathway activity assays and modulators were functional in the mouse lung. Then, using the same methods, we demonstrated that the NF-κB pathway appears not to play an important role in the inflammation observed after exposure to CS. Furthermore, assaying human lung tissue revealed that in the clinical samples there was also no increase in NF-κB pathway activation in the COPD lung, suggesting that our pre-clinical data is translational to human disease. Conclusions In this study we present compelling evidence that the IKK-2/NF-κB signalling pathway does not play a prominent role in the inflammatory response to CS exposure and that this pathway may not be important in COPD pathogenesis.

I kappa B kinase alpha (IKKα) activity is required for functional maturation of dendritic cells and acquired immunity to infection

Dendritic cells (DC) are required for priming antigen‐specific T cells and acquired immunity to many important human pathogens, including Mycobacteriuim tuberculosis (TB) and influenza. However, inappropriate priming of auto‐reactive T cells is linked with autoimmune disease. Understanding the molecular mechanisms that regulate the priming and activation of naïve T cells is critical for development of new improved vaccines and understanding the pathogenesis of autoimmune diseases. The serine/threonine kinase IKKα (CHUK) has previously been shown to have anti‐inflammatory activity and inhibit innate immunity. Here, we show that IKKα is required in DC for priming antigen‐specific T cells and acquired immunity to the human pathogen Listeria monocytogenes. We describe a new role for IKKα in regulation of IRF3 activity and the functional maturation of DC. This presents a unique role for IKKα in dampening inflammation while simultaneously promoting adaptive immunity that could have important implications for the development of new vaccine adjuvants and treatment of autoimmune diseases.

Bone Marrow-Specific Knock-In of a Non-Activatable Ikkα Kinase Mutant Influences Haematopoiesis but Not Atherosclerosis in Apoe-Deficient Mice

Background The Ikkα kinase, a subunit of the NF-κB-activating IKK complex, has emerged as an important regulator of inflammatory gene expression. However, the role of Ikkα-mediated phosphorylation in haematopoiesis and atherogenesis remains unexplored. In this study, we investigated the effect of a bone marrow (BM)-specific activation-resistant Ikkα mutant knock-in on haematopoiesis and atherosclerosis in mice. Methods and Results Apolipoprotein E (Apoe)-deficient mice were transplanted with BM carrying an activation-resistant Ikkα gene (IkkαAA/AAApoe−/−) or with Ikkα+/+Apoe−/− BM as control and were fed a high-cholesterol diet for 8 or 13 weeks. Interestingly, haematopoietic profiling by flow cytometry revealed a significant decrease in B-cells, regulatory T-cells and effector memory T-cells in IkkαAA/AAApoe−/− BM-chimeras, whereas the naive T-cell population was increased. Surprisingly, no differences were observed in the size, stage or cellular composition of atherosclerotic lesions in the aorta and aortic root of IkkαAA/AAApoe−/− vs Ikkα+/+Apoe−/− BM-transplanted mice, as shown by histological and immunofluorescent stainings. Necrotic core sizes, apoptosis, and intracellular lipid deposits in aortic root lesions were unaltered. In vitro, BM-derived macrophages from IkkαAA/AAApoe−/− vs Ikkα+/+Apoe−/− mice did not show significant differences in the uptake of oxidized low-density lipoproteins (oxLDL), and, with the exception of Il-12, the secretion of inflammatory proteins in conditions of Tnf-α or oxLDL stimulation was not significantly altered. Furthermore, serum levels of inflammatory proteins as measured with a cytokine bead array were comparable. Conclusion Our data reveal an important and previously unrecognized role of haematopoietic Ikkα kinase activation in the homeostasis of B-cells and regulatory T-cells. However, transplantation of IkkαAA mutant BM did not affect atherosclerosis in Apoe−/− mice. This suggests that the diverse functions of Ikkα in haematopoietic cells may counterbalance each other or may not be strong enough to influence atherogenesis, and reveals that targeting haematopoietic Ikkα kinase activity alone does not represent a therapeutic approach.

Receptor Activator of NF-κB Orchestrates Activation of Antiviral Memory CD8 T Cells in the Spleen Marginal Zone

Summary The spleen plays an important role in protective immunity to bloodborne pathogens. Macrophages and dendritic cells (DCs) in the spleen marginal zone capture microbial antigens to trigger adaptive immune responses. Marginal zone macrophages (MZMs) can also act as a replicative niche for intracellular pathogens, providing a platform for mounting the immune response. Here, we describe a role for RANK in the coordinated function of antigen-presenting cells in the spleen marginal zone and triggering anti-viral immunity. Targeted deletion of RANK results in the selective loss of CD169+ MZMs, which provide a niche for viral replication, while RANK signaling in DCs promotes the recruitment and activation of anti-viral memory CD8 T cells. These studies reveal a role for the RANKL/RANK signaling axis in the orchestration of protective immune responses in the spleen marginal zone that has important implications for the host response to viral infection and induction of acquired immunity.

Representing the Process of Inflammation as Key Events in Adverse Outcome Pathways

Inflammation is an important biological process involved in many target organ toxicities. However, there has been little consensus on how to represent inflammatory processes using the adverse outcome pathway (AOP) framework. In particular, there were concerns that inflammation was not being represented in a way that it would be recognized as a highly connected, central node within the global AOP network. The consideration of salient features common to the inflammatory process across tissues was used as a basis to propose 3 hub key events (KEs) for use in AOP network development. Each event, tissue resident cell activation, increased pro-inflammatory mediators, and leukocyte recruitment/activation, is viewed as a hallmark of inflammation, independent of tissue, and can be independently measured. Using these proposed hub KEs, it was possible to link together a series of AOPs that previously had no shared KEs. Significant challenges remain with regard to accurate prediction of inflammation-related toxicological outcomes even if a broader and more connected network of inflammation-centered AOPs is developed. Nonetheless, the current proposal addresses one of the major hurdles associated with representation of inflammation in AOPs and may aid fit-for-purpose evaluations of other AOPs operating in a network context.

Coordinated Regulation of Signaling Pathways during Macrophage Activation

The functional and phenotypic diversity of macrophages has long been appreciated, and it is now clear that it reflects a complex interplay between hard-wired differentiation pathways and instructive signals in specific tissues (Lawrence T, Natoli G. 2011, Nat Rev Immunol11:750-761). Recent studies have begun to unravel the molecular basis for the integration of these intrinsic developmental pathways with extracellular signals from the tissue microenvironment that confer the distinct phenotypes of tissue-resident macrophages (Lavin Y et al. 2014. Cell159:1312-1326; Gosselin D et al. 2014. Cell159:1327-1340). Macrophage phenotype and function is particularly dynamic during inflammation or infection, as blood monocytes are recruited into tissues and differentiate into macrophages, and depending on the nature of the inflammatory stimulus, they may acquire distinct functional phenotypes (Xue J et al. 2014. Immunity40:274-288; Murray PJ et al. 2014. Immunity41:14-20). Furthermore, these functional activation states can be rapidly modified in response to a changing microenvironment. Here we will discuss several key signaling pathways that drive macrophage activation during the inflammatory response and discuss how these pathways are integrated to fine-tune macrophage phenotype and function.

Role of NF-κB Activation in Macrophages

Nuclear factor-κB (NF-κB) is the prototypical pro-inflammatory transcription factor, this is primarily based on its activation by pro-inflammatory cytokines such as interleukin 1 (IL-1) and tumor necrosis factor α (TNFα), and pattern recognition receptors (PRRs) including Toll-like receptors (TLRs). In response to these pro-inflammatory signals NF-κB regulates the expression of other pro-inflammatory and cell survival genes that perpetuate the inflammatory response. It is this pivotal role in pro-inflammatory signaling pathways that has attracted a great deal of interest in NF-κB as a therapeutic target in inflammatory and autoimmune diseases. In light of their important role in orchestrating inflammation and immunity, NF-κB activation in macrophages has long been thought to be a major factor in the inflammatory response and consequently many inflammation-associated diseases. However, in recent years the development of sophisticated genetic tools to study the cell-specific role of the NF-κB in vivo has revealed previously unexpected anti-inflammatory and immunosuppressive roles for this pathway specifically in the macrophage lineage. In this chapter we will review these recent insights into the role of NF-κB activation in macrophages and related myeloid cells in the context of inflammation and immunity.