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Immunotherapeutic strategies for Alzheimer's and Parkinson's disease would be facilitated by better measures of inflammation. Here we established an ultra-sensitive single-molecule pull-down immunoassay combined with direct stochastic optical reconstruction microscopy (dSTORM) to measure the number, size and shape of individual extracellular inflammasome ASC specks. We assayed human post-mortem brain, serum and cerebrospinal fluid of patients with Parkinson's and Alzheimer's as well as healthy elderly. The number of ASC specks increased and showed altered morphology in the blood of early-stage Parkinson's and Alzheimer's patients compared to controls, mimicking those found in the brain and cerebrospinal fluid. In serum samples we also measured the number of Aβ, p-tau and α-syn aggregates and formed a composite biomarker of (ASC + p-tau)/Aβ and (ASC + α-syn)/Aβ ratios that distinguished age-matched healthy controls from patients with early-stage Alzheimer's with AUC of 92% and early-stage Parkinson's with AUC of 97%. Our findings confirm ASC specks as a fluid candidate biomarker of inflammation for neurodegenerative diseases with blood being the main focus for further development as convenient sample for diagnostics and clinical trials., Competing Interests: Competing interests The authors of the paper, E.L., Y.P.Z. and D.E., are inventors in the international patent application (application number: GB2317286.9, status: pending, applicant: Cambridge Enterprise Limited). The patent application has been filed on the methods of protein aggregate detection for diagnosis of neurodegenerative diseases including a method for detecting ASC specks in human biofluids, which is described in this manuscript. All other authors declare no competing interests., (© 2024. The Author(s).)

Some respiratory viruses, such as Human Rhinovirus, SARS-CoV-2, and Enterovirus D-68 (EV-D68), share the feature of hijacking host lipids in order to generate specialised replication organelles (ROs) with unique lipid compositions to enable viral replication. We have recently uncovered a novel non-canonical function of the stimulator of interferon genes (STING) pathway, as a critical factor in the formation of ROs in response to HRV infection. The STING pathway is the main DNA virus sensing system of the innate immune system controlling the type I IFN machinery. Although it is well-characterised as part of the DNA sensor machinery, the STING function in RNA viral infections is largely unexplored. In the current study, we investigated whether other RO-forming RNA viruses, such as EV-D68 and SARS-CoV-2, can also utilise STING for their replication. Using genetic and pharmacological inhibition, we demonstrate that STING is hijacked by these viruses and is utilised as part of the viral replication machinery. STING also co-localises with glycolytic enzymes needed to fuel the energy for replication. The inhibition of STING leads to the modulation of glucose metabolism in EV-D68-infected cells, suggesting that it might also manipulate immunometabolism. Therefore, for RO-generating RNA viruses, STING seems to have non-canonical functions in membrane lipid re-modelling, and the formation of replication vesicles, as well as immunometabolism.

The complement system is an ancient and critical part of innate immunity. Recent studies have highlighted novel roles of complement beyond lysis of invading pathogens with implications in regulating the innate immune response, as well as contributing to metabolic reprogramming of T-cells, synoviocytes as well as cells in the CNS. These findings hint that complement can be an immunometabolic regulator, but whether this is also the case for the terminal step of the complement pathway, the membrane attack complex (MAC) is not clear. In this study we focused on determining whether MAC is an immunometabolic regulator of the innate immune response in human monocyte-derived macrophages. Here, we uncover previously uncharacterized metabolic changes and mitochondrial dysfunction occurring downstream of MAC deposition. These alterations in glycolytic flux and mitochondrial morphology and function mediate NLRP3 inflammasome activation, pro-inflammatory cytokine release and gasdermin D formation. Together, these data elucidate a novel signalling cascade, with metabolic alterations at its center, in MAC-stimulated human macrophages that drives an inflammatory consequence in an immunologically relevant cell type., Competing Interests: Authors JK, RP-H, RA, ED, E-MN, CK, FZ, NG, LM, and LMB are employees and shareholders of GSK. All authors, except GJ-D, KT and MT, were employed by GSK at the time of the work. The Immunology Network at GSK funded GJ-Ds PhD studentship and research costs. The Immunology Network at GSK also funded secondments for KT and MT to undertake this research at GSK. The authors declare that the study was funded by GSK. The funder was involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication., (Copyright © 2022 Jimenez-Duran, Kozole, Peltier-Heap, Dickinson, Kwiatkowski, Zappacosta, Annan, Galwey, Nichols, Modis, Triantafilou, Triantafilou and Booty.)

Human rhinovirus (HRV), like coronavirus (HCoV), are positive-strand RNA viruses that cause both upper and lower respiratory tract illness, with their replication facilitated by concentrating RNA-synthesizing machinery in intracellular compartments made of modified host membranes, referred to as replication organelles (ROs). Here we report a non-canonical, essential function for stimulator of interferon genes (STING) during HRV infections. While the canonical function of STING is to detect cytosolic DNA and activate inflammatory responses, HRV infection triggers the release of STIM1-bound STING in the ER by lowering Ca 2+ , thereby allowing STING to interact with phosphatidylinositol 4-phosphate (PI4P) and traffic to ROs to facilitates viral replication and transmission via autophagy. Our results thus hint a critical function of STING in HRV viral replication and transmission, with possible implications for other RO-mediated RNA viruses., (© 2022. The Author(s).)

Abstract: As part of its role in the World Health Organization's (WHO) Global Influenza Surveillance and Response System (GISRS), the WHO Collaborating Centre for Reference and Research on Influenza in Melbourne received a record total of 9,266 human influenza positive samples during 2019. Viruses were analysed for their antigenic, genetic and antiviral susceptibility properties. Selected viruses were propagated in qualified cells or embryonated hen's eggs for potential use in seasonal influenza virus vaccines. In 2019, influenza A(H3N2) viruses predominated over influenza A(H1N1)pdm09 and B viruses, accounting for a total of 51% of all viruses analysed. The majority of A(H1N1)pdm09, A(H3N2) and influenza B viruses analysed at the Centre were found to be antigenically similar to the respective WHO recommended vaccine strains for the Southern Hemisphere in 2019. However, phylogenetic analysis indicated that a significant proportion of circulating A(H3N2) viruses had undergone genetic drift relative to the WHO recommended vaccine strain for 2019. Of 5,301 samples tested for susceptibility to the neuraminidase inhibitors oseltamivir and zanamivir, four A(H1N1)pdm09 viruses showed highly reduced inhibition with oseltamivir, one A(H1N1)pdm09 virus showed highly reduced inhibition with zanamivir and three B/Victoria viruses showed highly reduced inhibition with zanamivir., (© Commonwealth of Australia CC BY-NC-ND.)

Nucleotide-binding domain and leucine-rich repeat receptor (NLR)-mediated inflammasome activation is important in host response to microbes, danger-associated molecular patterns (DAMPs) and metabolic disease. Some NLRs have been shown to interact with distinct cell metabolic pathways and cause negative regulation, tumorigenesis and autoimmune disorders, interacting with multiple innate immune receptors to modulate disease. NLR activation is therefore crucial in host response and in the regulation of metabolic pathways that can trigger a wide range of immunometabolic diseases or syndromes. However, the exact mode by which some of the less well-studied NLR inflammasomes are activated, interact with other metabolites and immune receptors, and the role they play in the progression of metabolic diseases is still not fully elucidated. In this study, we review up-to-date evidence regarding NLR function in metabolic pathways and the interplay with other immune receptors involved in GPCR signalling, gut microbiota and the complement system, in order to gain a better understanding of its link to disease processes., (© 2021 The Authors. Immunology published by John Wiley & Sons Ltd.)

Macrophages are important drivers of pathogenesis and progression to AIDS in HIV infection. The virus in the later phases of the infection is often predominantly macrophage-tropic and this tropism contributes to a chronic inflammatory and immune activation state that is observed in HIV patients. Pattern recognition receptors of the innate immune system are the key molecules that recognise HIV and mount the inflammatory responses in macrophages. The innate immune response against HIV-1 is potent and elicits caspase-1-dependent pro-inflammatory cytokine production of IL-1β and IL-18. Although, NLRP3 has been reported as an inflammasome sensor dictating this response little is known about the pattern recognition receptors that trigger the "priming" signal for inflammasome activation, the NLRs involved or the HIV components that trigger the response. Using a combination of siRNA knockdowns in monocyte derived macrophages (MDMs) of different TLRs and NLRs as well as chemical inhibition, it was demonstrated that HIV Vpu could trigger inflammasome activation via TLR4/NLRP3 leading to IL-1β/IL-18 secretion. The priming signal is triggered via TLR4, whereas the activation signal is triggered by direct effects on Kv1.3 channels, causing K+ efflux. In contrast, HIV gp41 could trigger IL-18 production via NAIP/NLRC4, independently of priming, as a one-step inflammasome activation. NAIP binds directly to the cytoplasmic tail of HIV envelope protein gp41 and represents the first non-bacterial ligand for the NAIP/NLRC4 inflammasome. These divergent pathways represent novel targets to resolve specific inflammatory pathologies associated with HIV-1 infection in macrophages., Competing Interests: The authors have declared that no competing interests exist.

Different immune activation states require distinct metabolic features and activities in immune cells. For instance, inhibition of fatty acid synthase (FASN), which catalyzes the synthesis of long-chain fatty acids, prevents the proinflammatory response in macrophages; however, the precise role of this enzyme in this response remains poorly defined. Consistent with previous studies, we found here that FASN is essential for lipopolysaccharide-induced, Toll-like receptor (TLR)-mediated macrophage activation. Interestingly, only agents that block FASN upstream of acetoacetyl-CoA synthesis, including the well-characterized FASN inhibitor C75, inhibited TLR4 signaling, while those acting downstream had no effect. We found that acetoacetyl-CoA could overcome C75's inhibitory effect, whereas other FASN metabolites, including palmitate, did not prevent C75-mediated inhibition. This suggested an unexpected role for acetoacetyl-CoA in inflammation that is independent of its role in palmitate synthesis. Our evidence further suggested that acetoacetyl-CoA arising from FASN activity promotes cholesterol production, indicating a surprising link between fatty acid synthesis and cholesterol synthesis. We further demonstrate that this process is required for TLR4 to enter lipid rafts and facilitate TLR4 signaling. In conclusion, we have uncovered an unexpected link between FASN and cholesterol synthesis that appears to be required for TLR signal transduction and proinflammatory macrophage activation., (© 2018 Carroll et al.)

The inflammasome is a cytoplasmic protein complex that processes interleukins (IL)-1β and IL-18, and drives a form of cell death known as pyroptosis. Oligomerization of this complex is actually the second step of activation, and a priming step must occur first. This involves transcriptional upregulation of pro-IL-1β, inflammasome sensor NLRP3, or the non-canonical inflammasome sensor caspase-11. An additional aspect of priming is the post-translational modification of particular inflammasome constituents. Priming is typically accomplished in vitro using a microbial Toll-like receptor (TLR) ligand. However, it is now clear that inflammasomes are activated during the progression of sterile inflammatory diseases such as atherosclerosis, metabolic disease, and neuroinflammatory disorders. Therefore, it is time to consider the endogenous factors and mechanisms that may prime the inflammasome in these conditions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The innate immune system is an ancient surveillance system able to sense microbial invaders as well as aberrations in normal cell function. No longer viewed as a static and non-specific part of immunity, the innate immune system employs a plethora of specialized pattern recognition sensors to monitor and achieve homeostasis; these include the Toll-like receptors, the retinoic acid-inducible gene-like receptors, the nucleotide-binding oligomerization domain receptors (NLRs), the C-type lectins and the complement system. In order to increase specificity and diversity, innate immunity uses homotypic and heterotypic associations among these different components. Multi-molecular assemblies are formed both on the cell surface and in the cytosol to respond to pathogen and danger signals. Diverse, but tailored, responses to a changing environment are orchestrated depending on the the nature of the challenge and the repertoire of interacting receptors and components available in the sensing cell. It is now emerging that innate immunity operates a system of 'checks and balances' where interaction among the sensors is key in maintaining normal cell function. Complement sits at the heart of this alarm system and it is becoming apparent that it is capable of interacting with all the other pathways to effect a tailored immune response. In this review, we will focus on complement interactions with NLRs, the so-called 'inflammasomes', describing the molecular mechanisms that have been revealed so far and discussing the circumstantial evidence that exists for these interactions in disease states., (© 2015 John Wiley & Sons Ltd.)

Innate immunity has a primary role in lung antimicrobial defenses. The inflammasome has evolved for this purpose and is an important surveillance system that, when triggered, fights infection and eliminates pathogens. However, there is growing evidence that the inflammasome also plays a role in the pathogenesis of acute and chronic respiratory disease. Inflammasomes contribute to both the clearance of the pathogen as well as its pathogenesis - depending on the amount of inflammation triggered. How respiratory viruses trigger inflammasome activation remains unclear. Emerging evidence shows that ion flux is responsible for triggering inflammasome activation in the lung, causing lung pathology and disease exacerbations. Viroporins, encoded by all common respiratory viruses, are responsible for the changes in intracellular ion homeostasis that modulate inflammasome activation. This is a novel mechanism by which respiratory viral infection activates inflammasomes, and identifies sensing of disturbances in intracellular ionic concentrations as a novel pathogen-recognition pathway in the lung., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Certain low-abundance bacterial species, such as the periodontitis-associated oral bacterium Porphyromonas gingivalis, can subvert host immunity to remodel a normally symbiotic microbiota into a dysbiotic, disease-provoking state. However, such pathogens also exploit inflammation to thrive in dysbiotic conditions. How these bacteria evade immunity while maintaining inflammation is unclear. As previously reported, P. gingivalis remodels the oral microbiota into a dysbiotic state by exploiting complement. Now we show that in neutrophils P. gingivalis disarms a host-protective TLR2-MyD88 pathway via proteasomal degradation of MyD88, whereas it activates an alternate TLR2-Mal-PI3K pathway. This alternate TLR2-Mal-PI3K pathway blocks phagocytosis, provides "bystander" protection to otherwise susceptible bacteria, and promotes dysbiotic inflammation in vivo. This mechanism to disengage bacterial clearance from inflammation required an intimate crosstalk between TLR2 and the complement receptor C5aR and can contribute to the persistence of microbial communities that drive dysbiotic diseases., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Inflammation is mediated mainly by leukocytes that express both Toll-like receptor 4 (TLR4) and Fc γ receptors (FcγR). Dysregulated activation of leukocytes via exogenous and endogenous ligands of TLR4 results in a large number of inflammatory disorders that underlie a variety of human diseases. Thus, differentially blocking inflammatory cells while sparing structural cells, which are FcγR-negative, represents an elegant strategy when targeting the underlying causes of human diseases. Here, we report a novel tethering mechanism of the Fv and Fc portions of anti-TLR4 blocking antibodies that achieves increased potency on inflammatory cells. In the presence of ligand (e.g. lipopolysaccharide (LPS)), TLR4 traffics into glycolipoprotein microdomains, forming concentrated protein platforms that include FcγRs. This clustering produces a microenvironment allowing anti-TLR4 antibodies to co-engage TLR4 and FcγRs, increasing their avidity and thus substantially increasing their inhibitory potency. Tethering of antibodies to both TLR4 and FcγRs proves valuable in ameliorating inflammation in vivo. This novel mechanism of action therefore has the potential to enable selective intervention of relevant cell types in TLR4-driven diseases., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objective: The pathway by which herpes simplex virus 2 (HSV2) triggers the innate immune system in the urogenital system has not as yet been fully elucidated. In this study, we aimed to determine which pattern recognition receptors (PRRs) recognize HSV2 in primary vaginal epithelial cells. Once we deciphered the receptors involved, we aimed to target them to immunomodulate innate responses as a prophylactic or therapeutic intervention for early HSV2 infection., Study Design: To determine which PRRs are involved, receptor silencing as well as confocal microscopy was utilized. For immunomodulation, PRR agonists were utilized to induce a strong, local response to limit the infection, and we used 2 quantitative methods, flow cytometry and plaque assays, to determine their effect on HSV2 replication., Results: Our results show that HSV2 is detected by a plethora of PRRs: Toll-like receptors (TLR) 2 as well as deoxyribonucleic acid (DNA) sensors TLR9, DNA-dependent activator of interferon regulatory factors, and to a lesser extent interferon-inducible 16, which trigger cytokine secretion to protect the host. Using PRR agonists, such as lipoproteins, CpG DNA, and cyclic dinucleotides, we could significantly limit HSV2 replication., Conclusion: Different PRRs are strategically placed in different cell locations to detect virus invasion. Use of agonists that target and activate these PRRs appeared to be effective in preventing primary HSV2 infection in vaginal cells and could provide new insights in defense against HSV2 urogenital infections., (Copyright © 2014 Mosby, Inc. All rights reserved.)

Human rhinoviruses have been linked with underlying lung disorders, such as asthma and chronic obstructive pulmonary disease, in children and adults. However, the mechanism of virus-induced airway inflammation is poorly understood. In this study, using virus deletion mutants and silencing for nucleotide-binding oligomerization domain-like receptors (NLRs), we show that the rhinovirus ion channel protein 2B triggers NLRP3 and NLRC5 inflammasome activation and IL-1β secretion in bronchial cells. 2B protein targets the endoplasmic reticulum and Golgi and induces Ca(2+) reduction in these organelles, thereby disturbing the intracellular calcium homeostasis. NLRP3 and NLRC5 act in a cooperative manner during the inflammasome assembly by sensing intracellular Ca(2+) fluxes and trigger IL-1β secretion. These results reveal for the first time that human rhinovirus infection in primary bronchial cells triggers inflammasome activation.

The membrane attack complex of complement (MAC), apart from its classical role of lysing cells, can also trigger a range of non-lethal effects on cells, acting as a drive to inflammation. In the present study, we chose to investigate these non-lethal effects on inflammasome activation. We found that, following sublytic MAC attack, there is increased cytosolic Ca(2+) concentration, at least partly through Ca(2+) release from the endoplasmic reticulum lumen via the inositol 1,4,5-triphosphate receptor (IP3R) and ryanodine receptor (RyR) channels. This increase in intracellular Ca(2+) concentration leads to Ca(2+) accumulation in the mitochondrial matrix via the 'mitochondrial calcium uniporter' (MCU), and loss of mitochondrial transmembrane potential, triggering NLRP3 inflammasome activation and IL-1β release. NLRP3 co-localises with the mitochondria, probably sensing the increase in calcium and the resultant mitochondrial dysfunction, leading to caspase activation and apoptosis. This is the first study that links non-lethal effects of sublytic MAC attack with inflammasome activation and provides a mechanism by which sublytic MAC can drive inflammation and apoptosis.

Ureaplasma species are the most frequently isolated microorganisms inside the amniotic cavity and have been associated with spontaneous abortion, chorioamnionitis, premature rupture of the membranes (PROM), preterm labour (PL) pneumonia in neonates and bronchopulmonary dysplasia in neonates. The mechanisms by which Ureaplasmas cause such diseases remain unclear, but it is believed that inappropriate induction of inflammatory responses is involved, triggered by the innate immune system. As part of its mechanism of activation, the innate immune system employs germ-lined encoded receptors, called pattern recognition receptors (PRRs) in order to "sense" pathogens. One such family of PRRs are the Toll like receptor family (TLR). In the current study we aimed to elucidate the role of TLRs in Ureaplasma-induced inflammation in human amniotic epithelial cells. Using silencing, as well as human embryonic kidney (HEK) transfected cell lines, we demonstrate that TLR2, TLR6 and TLR9 are involved in the inflammatory responses against Ureaplasma parvum and urealyticum serovars. Ureaplasma lipoproteins, such as Multiple Banded antigen (MBA), trigger responses via TLR2/TLR6, whereas the whole bacterium is required for TLR9 activation. No major differences were observed between the different serovars. Cell activation by Ureaplasma parvum and urealyticum seem to require lipid raft function and formation of heterotypic receptor complexes comprising of TLR2 and TLR6 on the cell surface and TLR9 intracellularly.

Background: Respiratory syncytial virus (RSV) remains the leading cause of serious viral bronchiolitis and pneumonia in infants and young children throughout the world. The burden of disease is significant, with 70% of all infants being infected with RSV within the first year of their life. 40% of those children discharged from hospital have recurrent, repeated respiratory symptoms and wheezing for at least 10 years. The infection is also an important illness in the elderly and immunocompromised individuals. Ongoing symptoms relate to continued lung inflammation. One cytokine that is associated with RSV infection is IL-1β, but the mechanism of activation remain unclear., Objectives: In the current study, we set out to decipher the molecular mechanisms of RSV-induced inflammasome activation., Methods and Results: Using deletion mutants of the virus and measuring IL-1β secretion, as well as caspase 1 expression via western blotting, we demonstrate that the NLRP3 inflammasome is activated through the small hydrophobic (SH) RSV viroporin which induces membrane permeability to ions or small molecules. Confocal microscopy revealed that during virus infection, SH seems to accumulate within lipid rafts in the Golgi compartments., Conclusions: Upon RSV infection, SH gets localised in the cell membranes and intracellular organelle membranes, and then induces permeability by disrupting membrane architecture, thus leading us to believe that formation of viral ion channels in lipid bilayers of cells is a viral recognition pathway used by the host to signal inflammasome activation.

The innate immune system is a vital part of the body's defences against viral pathogens. The proteins retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation associated gene 5 (MDA5) function as cytoplasmic pattern recognition receptors that are involved in the elimination of actively replicating RNA viruses. Their location and their differential responses to RNA viruses emphasises the complexity of the innate detection system. Despite the wealth of information on the types of RNA that trigger RIG-I, much less is known about the nature of the RNAs that act as agonists for MDA5. In order to identify which RNA species triggers MDA5 activation during infection, we isolated viral ssRNA and replicative intermediates of RNA from positive sense ssRNA viruses. We reveal that MDA5 recognises not the genomic ssRNA but the dsRNA generated by the replication of these viruses. Furthermore, using fluorescent imaging we present the first report of the visualisation of dsRNA and MDA5, which provides unique evidence of the relationship between viral dsRNA and MDA5 and proves without a doubt that MDA5 is the key sensor for the dsRNA replicative intermediate form of positive sense ssRNA viruses.

Myristoylated alanine-rich C kinase substrate (MARCKS) is an intrinsically unfolded protein with a conserved cationic effector domain, which mediates the cross-talk between several signal transduction pathways. Transcription of MARCKS is increased by stimulation with bacterial LPS. We determined that MARCKS and MARCKS-related protein specifically bind to LPS and that the addition of the MARCKS effector peptide inhibited LPS-induced production of TNF-α in mononuclear cells. The LPS binding site within the effector domain of MARCKS was narrowed down to a heptapeptide that binds to LPS in an extended conformation as determined by nuclear magnetic resonance spectroscopy. After LPS stimulation, MARCKS moved from the plasma membrane to FYVE-positive endosomes, where it colocalized with LPS. MARCKS-deficient mouse embryonic fibroblasts (MEFs) responded to LPS with increased IL-6 production compared with the matched wild-type MEFs. Similarly, small interfering RNA knockdown of MARCKS also increased LPS signaling, whereas overexpression of MARCKS inhibited LPS signaling. TLR4 signaling was enhanced by the ablation of MARCKS, which had no effect on stimulation by TLR2, TLR3, and TLR5 agonists. These findings demonstrate that MARCKS contributes to the negative regulation of the cellular response to LPS.

Bacterial cell wall components, such LPS and LTA, are potent initiators of an inflammatory response that can lead to septic shock. The advances in the past were centered around membrane-bound receptors and intracellular events, but our understanding of the initial interactions of these bacterial components with serum proteins as they enter the bloodstream remain unclear. In this study we identified several serum proteins, which are involved in the innate recognition of bacterial products. Using affinity chromatography and mass spectrometry we performed proteomic analysis of LPS- and LTA-binding serum proteins. We isolated proteins from normal serum that can interact with LPS and LTA. Fluorescent binding experiments and cytokine assays revealed that serum proteins, such as apolipoprotein, LDL, transferrin and holotransferrin could neutralize LPS/LTA binding as well as the subsequent inflammatory response, suggesting that serum proteins modulate LPS/LTA-induced responses. When compared with the proteomic profile of serum from septic patients it was shown that these proteins were in lower abundance. Investigation of serum proteins in 25 critically ill patients with a mortality rate of 40% showed statistically higher levels of these proteins in survivors. Patients surviving sepsis had statistically significant higher levels of apolipoprotein, albumin, LDL, transferrin and holotransferrin than individuals that succumbed, suggesting that these proteins have an inhibitory effect on LPS/LTA-induced inflammatory responses and in their absence there might be an augmented inflammatory response in sepsis.

The innate immune system utilises a set of receptors, called pattern recognition receptors (PRRs), in order to recognise specific molecular patterns or motifs called pathogen-associated molecular patterns (PAMPs) on invading pathogens. The toll-like receptor (TLR) family of proteins is an integral part of the mammalian innate immune system. We are now beginning to decipher which TLRs are involved in the recognition of particular microbial patterns, but questions remain as to the homo- and heterotypic associations that TLRs form and how these associations affect their activation. Technical advances in fluorescence microscopy has enabled us to investigate the functional associations of TLRs and other PPRs in living cells in response to different pathogens using non-invasive fluorescence imaging methods. In this chapter, we will describe some of the fluorescent imaging techniques, such as FRET and FRAP, that we employ in order to study PAMP-PRR associations.

In the last twenty years, the general view of the plasma membrane has changed from a homogeneous arrangement of lipids to a mosaic of microdomains. It is currently thought that islands of highly ordered saturated lipids and cholesterol, which are laterally mobile, exist in the plane of the plasma membrane. Lipid rafts are thought to provide a means to explain the spatial segregation of certain signalling pathways emanating from the cell surface. They seem to provide the necessary microenvironment in order for certain specialised signalling events to take place, such as the innate immune recognition. The innate immune system seems to employ germ-lined encoded receptors, called pattern recognition receptors (PRRs), in order to detect pathogens. One family of such receptors are the Toll-like receptors (TLRs), which are the central "sensing" apparatus of the innate immune system. In recent years, it has become apparent that TLRs are recruited into membrane microdomains in response to ligands. These nanoscale assemblies of sphingolipid, cholesterol, and TLRs stabilize and coalesce, forming signalling platforms, which transduce signals that lead to innate immune activation. In the current paper, we will investigate all past and current literature concerning recruitment of extracellular and intracellular TLRs into lipid rafts and how this membrane organization modulates innate immune responses.

The early detection of invading viruses by the host depends on their identification by pathogen sensors. These include Toll-like receptors (TLRs) as well as cytoplasmic RNA helicases such as retinoic acid inducible protein I (RIG-I) and melanoma differentiation associated gene 5 (MDA-5). These pathogen sensors recognize specific molecular patterns found in viruses and trigger inflammatory and antiviral responses that result in the eradication of invading pathogens. In this study we investigated the specific recognition of Human rhinovirus 6 (HRV6) the common cold pathogen by the innate immune response in lung epithelial cells. Our experiments established that in the first stages on infection the TLRs play a crucial role in HRV recognition and that different constituents of HRV6 are recognized by different TLRs, while upon viral replication and generation of dsRNA the type I IFN inflammatory response is mediated by MDA-5. The HRV6 capsid is recognized via TLR2, whereas upon HRV6 ssRNA internalization the virus genome is recognized by TLR7 and TLR8. Upon generation of dsRNA the type I IFN response is mediated by MDA-5. The combined recognition by different TLRs and MDA5 and their upregulation concurs with the huge inflammatory response seen in the common cold caused by human rhinoviruses.

Lipoteichoic acid (LTA), a ubiquitous cell wall component of Gram-positive bacteria, represents a potent immunostimulatory molecule. Because LTA of a mutant Staphylococcus aureus strain lacking lipoproteins (Deltalgt-LTA) has been described to be immunobiologically inactive despite a lack of ascertained structural differences to wild-type LTA (wt-LTA), we investigated the functional requirements for the recognition of Deltalgt-LTA by human peripheral blood cells. In this study, we demonstrate that Deltalgt-LTA-induced immune activation critically depends on the immobilization of LTA and the presence of human serum components, which, to a lesser degree, was also observed for wt-LTA. Under experimental conditions allowing LTA-mediated stimulation, we found no differences between the immunostimulatory capacity of Deltalgt-LTA and wt-LTA in human blood cells, arguing for a limited contribution of possible lipoprotein contaminants to wt-LTA-mediated immune activation. In contrast to human blood cells, TLR2-transfected human embryonic kidney 293 cells could be activated only by wt-LTA, whereas activation of these cells by Deltalgt-LTA required the additional expression of TLR6 and CD14, suggesting that activation of human embryonic kidney 293 cells expressing solely TLR2 is probably mediated by residual lipoproteins in wt-LTA. Notably, in human peripheral blood, LTA-specific IgG Abs are essential for Deltalgt-LTA-mediated immune activation and appear to induce the phagocytic uptake of Deltalgt-LTA via engagement of FcgammaRII. In this study, we have elucidated a novel mechanism of LTA-induced cytokine induction in human peripheral blood cells that involves uptake of LTA and subsequent intracellular recognition driven by TLR2, TLR6, and CD14.

Crosstalk between complement and Toll-like receptors (TLRs) coordinates innate immunity. We report a previously unknown immune subversion mechanism involving microbial exploitation of communication between complement and TLRs. Porphyromonas gingivalis, a major oral and systemic pathogen with complement C5 convertase-like activity, synergizes with C5a (fragment of complement protein C5) to increase cyclic adenosine monophosphate (cAMP) concentrations, resulting in suppression of macrophage immune function and enhanced pathogen survival in vitro and in vivo. This synergy required TLR2 signaling, a pertussis toxin- and thapsigargin-sensitive C5a receptor pathway, with protein kinase A and glycogen synthase kinase-3beta as downstream effectors. Antagonistic blockade of the C5a receptor abrogated this evasive strategy and may thus have important therapeutic implications for periodontitis and atherosclerosis, diseases in which P. gingivalis is implicated. This first demonstration of complement-TLR crosstalk for immunosuppressive cAMP signaling indicates that pathogens may not simply undermine complement or TLRs (or both) as separate entities, but may also exploit their crosstalk pathways.