Search

Age-related macular degeneration (AMD) is the leading cause of central vision loss in the over 50s worldwide. Activation of the immune system has been implicated in disease progression, but while polymorphisms in genes associated with the immune system have been identified as risk factors for disease, the underlying pathways and mechanisms involved in disease progression remain incompletely characterised. Typically inflammatory responses are mediated by microbial infection; however, in chronic conditions, a form of 'sterile' inflammation exists whereby immune responses occur in areas of the body, in the absence of microbes; 'sterile' inflammation is likely to be central to AMD. In this case the innate immune response is triggered when alarm signals released by stressed cells or damaged tissue are identified by pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are a family of membrane-spanning PRRs for which host-derived ligands have been identified; these include heat shock proteins, extracellular matrix breakdown products, mRNA from necrotic cells and modified lipids. Here we review the evidence for TLR involvement in the pathogenesis of AMD.

Toll-like receptors (TLR) survey the extracellular space, cytoplasm, and endosomal compartments for signs of infection or tissue injury. Over the past decade, it has become evident that TLR activation and signal transduction can be regulated by subcellular compartmentalization of both the receptors and their downstream signaling components. Immunofluorescence and/or overexpression of fluorescently "tagged"' proteins teamed with confocal microscopy presents a powerful technique for studying the spatial organization of TLRs, their signaling mediators, and the dynamic processes they activate. This chapter details the common methods for determining the subcellular location of TLRs in both live and fixed cells.

Since first being described in the fruit fly Drosophila melanogaster, Toll-like receptors (TLRs) have proven to be of great interest to immunologists and investigators interested in the molecular basis to inflammation. They recognize pathogen-derived factors and also products of inflamed tissue, and trigger signaling pathways that lead to activation of transcription factors such as nuclear factor-kappaB and the interferon regulatory factors. These in turn lead to induction of immune and inflammatory genes, including such important cytokines as tumor necrosis factor-alpha and type I interferon. Much evidence points to a role for TLRs in immune and inflammatory diseases and increasingly in cancer. Examples include clear roles for TLR4 in sepsis, rheumatoid arthritis, ischemia/reperfusion injury, and allergy. TLR2 has been implicated in similar pathologic conditions and also in systemic lupus erythematosus (SLE) and tumor metastasis. TLR7 has also been shown to be important in SLE. TLR5 has been shown to be radioprotective. Recent advances in our understanding of signaling pathways activated by TLRs, structural insights into TLRs bound to their ligands and antagonists, and approaches to inhibit TLRs (including antibodies, peptides, and small molecules) are providing possiblemeans by which to interfere with TLRs clinically. Here we review these recent advances and speculate about whether manipulating TLRs is likely to be successful in fighting off different diseases.

Toll-like receptors (TLRs) are key components of the innate immune system, functioning as pattern recognition receptors that recognise a wide range of microbial pathogens. TLRs represent a primary line of defence against invading pathogens in mammals, plants and insects. Recognition of microbial components by TLRs triggers a cascade of cellular signals that culminates in the activation of NFkappaB which leads to inflammatory gene expression and clearance of the infectious agent. The history of NFkappaB began with the TLR4 ligand lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, since this was the stimulus first used to activate NFkappaB in pre-B-cells. However, since those early days it has been a circuitous route, made possible by drawing on information provided by many different fields, that has led us not only to the discovery of TLRs but also to an understanding of the complex pathways that lead from TLR ligation to NFkappaB activation. In this review we will summarize the current knowledge of TLR-mediated NFkappaB activation, and also the recent discoveries that subtle differences in kappaB binding sequences and NFkappaB dimer formation result in specific gene expression profiles.

Toll-like receptor (TLR) signals that initiate innate immune responses to pathogens must be tightly regulated to prevent excessive inflammatory damage to the host. The adaptor protein Mal is specifically involved in signaling via TLR2 and TLR4. We demonstrate here that after TLR2 and TLR4 stimulation Mal becomes phosphorylated by Bruton's tyrosine kinase (Btk) and then interacts with SOCS-1, which results in Mal polyubiquitination and subsequent degradation. Removal of SOCS-1 regulation potentiates Mal-dependent p65 phosphorylation and transactivation of NF-kappaB, leading to amplified inflammatory responses. These data identify a target of SOCS-1 that regulates TLR signaling via a mechanism distinct from an autocrine cytokine response. The transient activation of Mal and subsequent SOCS-1-mediated degradation is a rapid and selective means of limiting primary innate immune response.

Fas (CD95) signalling is best known for its role in apoptosis, however, recent reports have shown it to be involved in other cellular responses as well, including inflammation. Fas and its adaptor protein FADD are known to negatively regulate LPS‐induced proinflammatory responses, but their role in LPS‐induced type I interferon production is unknown. Here, we demonstrate that Fas engagement on macrophages, using an agonistic Fas antibody CH11, augments LPS‐induced NF‐κB responses, causing increased production of TNFα, IL‐8, IL‐6 and IL‐12. Conversely, costimulation with both LPS and CH11 causes a significant reduction in the level of interferon‐beta (IFNβ) production. This differential effect involves the Fas adaptor FADD because while LPS‐induced IL‐6 production increased in FADD−/− murine embryonic fibroblasts, LPS‐induced IFNβ production was significantly reduced in these cells. Overexpression of a dominant negative form of FADD (FADD‐DD) inhibits LPS‐induced IFNβ luciferase but not LPS‐induced NF‐κB luciferase. In contrast, overexpression of full‐length FADD inhibited LPS‐induced NF‐κB luciferase activation but was seen to augment LPS‐induced IFNβ luciferase. Moreover, FADD‐DD inhibits TRIF‐, TRAM‐, IKKε‐, TBK‐1‐ and TRAF3‐induced IFNβ luciferase production, with coimmunoprecipitation experiments demonstrating an interaction between FADD and TRIF. These data identify FADD as a novel component of the noncanonical Toll‐like receptor 4/IFNβ signalling pathway and demonstrate that both Fas and its adaptor FADD can differentially regulate the production of LPS‐induced proinflammatory cytokines and type I interferons. We have identified that the death receptor Fas and its adaptor protein FADD are able to differentially regulate the MYD88‐dependent and ‐independent arms of the TLR4 signalling pathway. FADD is a negative regulator of the MyD88‐dependent arm (A) but is an essential component of the MyD88‐independent pathway (B), facilitating induction of LPS‐induced IFN‐beta through an interaction with TRIF. [ABSTRACT FROM AUTHOR]

Detection of microbes by TLRs on the plasma membrane leads to the induction of proinflammatory cytokines such as TNF-α, via activation of NF-κB. Alternatively, activation of endosomal TLRs leads to the induction of type I IFNs via IFN regulatory factors (IRFs). TLR4 signaling from the plasma membrane to NF-κB via the Toll/IL-1R (TIR) adaptor protein MyD88 requires the TIR sorting adaptor Mal, whereas endosomal TLR4 signaling to IRF3 via the TIR domain–containing adaptor-inducing IFN-β (TRIF) requires the TRIF-related adaptor molecule (TRAM). Similar to TLR4 homodimers, TLR2 heterodimers can also induce both proinflammatory cytokines and type I IFNs. TLR2 plasma membrane signaling to NF-κB is known to require MyD88 and Mal, whereas endosomal IRF activation by TLR2 requires MyD88. However, it was unclear whether TLR2 requires a sorting adaptor for endosomal signaling, like TLR4 does. In this study, we show that TLR2-dependent IRF7 activation at the endosome is both Mal- and TRAM-dependent, and that TRAM is required for the TLR2-dependent movement of MyD88 to endosomes following ligand engagement. TRAM interacted with both TLR2 and MyD88, suggesting that TRAM can act as a bridging adapter between these two molecules. Furthermore, infection of macrophages lacking TRAM with herpes viruses or the bacterium Staphylococcus aureus led to impaired induction of type I IFN, indicating a role for TRAM in TLR2-dependent responses to human pathogens. Our work reveals that TRAM acts as a sorting adaptor not only for TLR4, but also for TLR2, to facilitate signaling to IRF7 at the endosome, which explains how TLR2 is capable of causing type I IFN induction. [ABSTRACT FROM AUTHOR]

B cells signal through both the B cell receptor (BCR) which binds antigens and Toll-like receptors (TLRs) including TLR9 which recognises CpG DNA. Activation of TLR9 synergises with BCR signalling when the BCR and TLR9 co-localise within an auto-phagosome-like compartment. Here we report that Bruton’s tyrosine kinase (BTK) is required for synergistic IL6 production and up-regulation of surface expression of MHC-class-II, CD69 and CD86 in primary murine and human B cells. We show that BTK is essential for co-localisation of the BCR and TLR9 within a potential auto-phagosome-like compartment in the Namalwa human B cell line. Downstream of BTK we find that calcium acting via calmodulin is required for this process. These data provide new insights into the role of BTK, an important target for autoimmune diseases, in B cell activation. [ABSTRACT FROM AUTHOR]

Abstract: Polyunsaturated fatty acids (PUFA) can modulate the immune response, however the mechanism by which they exert this effect remains unclear. Previous studies have clearly demonstrated that the cis-9, trans-11 isomer of conjugated linoleic acid (c9,t11-CLA), found predominantly in beef and dairy products, can modulate the response of immune cells to the toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). This study aimed to investigate further the mechanism by which these effects are mediated. Treatment of macrophages with c9,t11-CLA significantly decreased CD14 expression and partially blocked its association with lipid rafts following stimulation with LPS. Furthermore the c9,t11-CLA isomer inhibited both nuclear factor-κB (NF-κB) and IRF3 activation following TLR4 ligation while eicosapentaenoic acid (EPA) only suppressed NF-κB activation. Given that the ability of LPS to activate IRF3 downstream of TLR4 depends on internalisation of the TLR4 complex and involves CD14, we examined TLR4 endocytosis. Indeed the internalisation of TLR4 to early endosomes following activation with LPS was markedly inhibited in c9,t11-CLA treated cells. These effects were not seen with the n-3 fatty acid, EPA, which was used as a comparison. Our data demonstrates that c9,t11-CLA inhibits IRF3 activation via its effects on CD14 expression and localisation. This results in a decrease in the endocytosis of TLR4 which is necessary for IRF3 activation, revealing a novel mechanism by which this PUFA exerts its anti-inflammatory effects. [Copyright &y& Elsevier]