Assessing the scale and ecological impact of derelict and discarded fishing gear across Thailand via the MARsCI citizen science protocol.

South-East Asia is among the least studied regions for the growing issue of marine debris pollution, despite being a major contributor towards global marine debris. In the present study, we provide the preliminary results from the MARsCI project, a survey protocol designed to utilise citizen science to facilitate data collection on the ecological impact of discarded fishing gear (DFG) in Thailand. Over a three-year period, 103 surveys were carried out across Thailand, resulting in impact assessment of 606 pieces of DFG. Our findings indicate corals are regularly impacted by DFG in Thai waters and that isolated marine habitats may be more severely impacted than near-shore sites. We further identify crabs, muricid snails, and demersal fish to be among the most regularly entangled animals. We discuss our findings in the context of earlier work from Thailand, and conduct a critical review of the protocol itself, identifying improvements for future efforts.

Neurological disorders after severe pneumonia are associated with translocation of endogenous bacteria from the lung to the brain.

Neurological disorders are a common feature in patients who recover from severe acute pneumonia. However, the underlying mechanisms remain poorly understood. Here, we show that the neurological syndromes after severe acute pneumonia are partly attributed to the translocation of endogenous bacteria from the lung to the brain during pneumonia. Using principal components analysis, similarities were found between the brain's flora species and those of the lungs, indicating that the bacteria detected in the brain may originate from the lungs. We also observed impairment of both the lung-blood and brain-blood barriers, allowing endogenous lung bacteria to invade the brain during pneumonia. An elevated microglia and astrocyte activation signature via bacterial infection-related pathways was observed, indicating a bacterial-induced disruption of brain homeostasis. Collectively, we identify endogenous lung bacteria that play a role in altering brain homeostasis, which provides insight into the mechanism of neurological syndromes after severe pneumonia.

Inhibition of pro-inflammatory signaling in human primary macrophages by enhancing arginase-2 via target site blockers.

The modulation of macrophage phenotype from a pro-inflammatory to an anti-inflammatory state holds therapeutic potential in the treatment of inflammatory disease. We have previously shown that arginase-2 (Arg2), a mitochondrial enzyme, is a key regulator of the macrophage anti-inflammatory response. Here, we investigate the therapeutic potential of Arg2 enhancement via target site blockers (TSBs) in human macrophages. TSBs are locked nucleic acid antisense oligonucleotides that were specifically designed to protect specific microRNA recognition elements (MREs) in human ARG2 3' UTR mRNA. TSBs targeting miR-155 (TSB-155) and miR-3202 (TSB-3202) MREs increased ARG2 expression in human monocyte-derived macrophages. This resulted in decreased gene expression and cytokine production of TNF-α and CCL2 and, for TSB-3202, in an increase in the anti-inflammatory macrophage marker, CD206. Proteomic analysis demonstrated that a network of pro-inflammatory responsive proteins was modulated by TSBs. In silico bioinformatic analysis predicted that TSB-3202 suppressed upstream pro-inflammatory regulators including STAT-1 while enhancing anti-inflammatory associated proteins. Proteomic data were validated by confirming increased levels of sequestosome-1 and decreased levels of phosphorylated STAT-1 and STAT-1 upon TSB treatment. In conclusion, upregulation of Arg2 by TSBs inhibits pro-inflammatory signaling and is a promising novel therapeutic strategy to modulate inflammatory signaling in human macrophages.

Enhancing arginase 2 expression using target site blockers as a strategy to modulate macrophage phenotype.

Macrophages are plastic cells playing a crucial role in innate immunity. While fundamental in responding to infections, when persistently maintained in a pro-inflammatory state they can initiate and sustain inflammatory diseases. Therefore, a strategy that reprograms pro-inflammatory macrophages toward an anti-inflammatory phenotype could hold therapeutic potential in that context. We have recently shown that arginase 2 (Arg2), a mitochondrial enzyme involved in arginine metabolism, promotes the resolution of inflammation in macrophages and it is targeted by miR-155. Here, we designed and tested a target site blocker (TSB) that specifically interferes and blocks the interaction between miR-155 and Arg2 mRNA, leading to Arg2 increased expression and activity. In bone marrow-derived macrophages transfected with Arg2 TSB (in the presence or absence of the pro-inflammatory stimulus LPS), we observed an overall shift of the polarization status of macrophages toward an anti-inflammatory phenotype, as shown by significant changes in surface markers (CD80 and CD71), metabolic parameters (mitochondrial oxidative phosphorylation) and cytokines secretion (IL-1ß, IL-6, and TNF). Moreover, in an in vivo model of LPS-induced acute inflammation, intraperitoneal administration of Arg2 TSB led to an overall decrease in systemic levels of pro-inflammatory cytokines. Overall, this proof-of-concept strategy represent a promising approach to modulating macrophage phenotype.

Multi-factorial nerve guidance conduit engineering improves outcomes in inflammation, angiogenesis and large defect nerve repair.

Nerve guidance conduits (NGCs) are sub-optimal for long-distance injuries with inflammation and poor vascularization related to poor axonal repair. This study used a multi-factorial approach to create an optimized biomaterial NGC to address each of these issues. Through stepwise optimization, a collagen-chondroitin-6-sulfate (Coll-CS) biomaterial was functionalized with extracellular matrix (ECM) components; fibronectin, laminin 1 and laminin 2 (FibL1L2) in specific ratios. A snap-cooled freeze-drying process was then developed with optimal pore architecture and alignment to guide axonal bridging. Culture of adult rat dorsal root ganglia on NGCs demonstrated significant improvements in inflammation, neurogenesis and angiogenesis in the specific Fib:L1:L2 ratio of 1:4:1. In clinically relevant, large 15 mm rat sciatic nerve defects, FibL1L2-NGCs demonstrated significant improvements in axonal density and angiogenesis compared to unmodified NGCs with functional equivalence to autografts. Therefore, a multiparameter ECM-driven strategy can significantly improve axonal repair across large defects, without exogenous cells or growth factors.

Biomaterial and Therapeutic Approaches for the Manipulation of Macrophage Phenotype in Peripheral and Central Nerve Repair.

Injury to the peripheral or central nervous systems often results in extensive loss of motor and sensory function that can greatly diminish quality of life. In both cases, macrophage infiltration into the injury site plays an integral role in the host tissue inflammatory response. In particular, the temporally related transition of macrophage phenotype between the M1/M2 inflammatory/repair states is critical for successful tissue repair. In recent years, biomaterial implants have emerged as a novel approach to bridge lesion sites and provide a growth-inductive environment for regenerating axons. This has more recently seen these two areas of research increasingly intersecting in the creation of 'immune-modulatory' biomaterials. These synthetic or naturally derived materials are fabricated to drive macrophages towards a pro-repair phenotype. This review considers the macrophage-mediated inflammatory events that occur following nervous tissue injury and outlines the latest developments in biomaterial-based strategies to influence macrophage phenotype and enhance repair.

Mitochondrial arginase-2 is essential for IL-10 metabolic reprogramming of inflammatory macrophages.

Mitochondria are important regulators of macrophage polarisation. Here, we show that arginase-2 (Arg2) is a microRNA-155 (miR-155) and interleukin-10 (IL-10) regulated protein localized at the mitochondria in inflammatory macrophages, and is critical for IL-10-induced modulation of mitochondrial dynamics and oxidative respiration. Mechanistically, the catalytic activity and presence of Arg2 at the mitochondria is crucial for oxidative phosphorylation. We further show that Arg2 mediates this process by increasing the activity of complex II (succinate dehydrogenase). Moreover, Arg2 is essential for IL-10-mediated downregulation of the inflammatory mediators succinate, hypoxia inducible factor 1α (HIF-1α) and IL-1ß in vitro. Accordingly, HIF-1α and IL-1ß are highly expressed in an LPS-induced in vivo model of acute inflammation using Arg2-/- mice. These findings shed light on a new arm of IL-10-mediated metabolic regulation, working to resolve the inflammatory status of the cell.

Impact of Exercise on Immunometabolism in Multiple Sclerosis.

Multiple Sclerosis (MS) is a chronic, autoimmune condition characterized by demyelinating lesions and axonal degradation. Even though the cause of MS is heterogeneous, it is known that peripheral immune invasion in the central nervous system (CNS) drives pathology at least in the most common form of MS, relapse-remitting MS (RRMS). The more progressive forms' mechanisms of action remain more elusive yet an innate immune dysfunction combined with neurodegeneration are likely drivers. Recently, increasing studies have focused on the influence of metabolism in regulating immune cell function. In this regard, exercise has long been known to regulate metabolism, and has emerged as a promising therapy for management of autoimmune disorders. Hence, in this review, we inspect the role of key immunometabolic pathways specifically dysregulated in MS and highlight potential therapeutic benefits of exercise in modulating those pathways to harness an anti-inflammatory state. Finally, we touch upon current challenges and future directions for the field of exercise and immunometabolism in MS.

Novel mineralocorticoid receptor mechanisms regulate cardiac tissue inflammation in male mice.

MR activation in macrophages is critical for the development of cardiac inflammation and fibrosis. We previously showed that MR activation modifies macrophage pro-inflammatory signalling, changing the cardiac tissue response to injury via both direct gene transcription and JNK/AP-1 second messenger pathways. In contrast, MR-mediated renal electrolyte homeostasis is critically determined by DNA-binding-dependent processes. Hence, ascertaining the relative contribution of MR actions via DNA binding or alternative pathways on macrophage behaviour and cardiac inflammation may provide therapeutic opportunities which separate the cardioprotective effects of MR antagonists from their undesirable renal potassium-conserving effects. We developed new macrophage cell lines either lacking MR or harbouring a mutant MR incapable of DNA binding. Western blot analysis demonstrated that MR DNA binding is required for lipopolysaccharide (LPS), but not phorbol 12-myristate-13-acetate (PMA), induction of the MAPK/pJNK pathway in macrophages. Quantitative RTPCR for pro-inflammatory and pro-fibrotic targets revealed subsets of LPS- and PMA-induced genes that were either enhanced or repressed by the MR via actions that do not always require direct MR-DNA binding. Analysis of the MR target gene and profibrotic factor MMP12 identified promoter elements that are regulated by combined MR/MAPK/JNK signalling. Evaluation of cardiac tissue responses to an 8-day DOC/salt challenge in mice selectively lacking MR DNA-binding in macrophages demonstrated levels of inflammatory markers equivalent to WT, indicating non-DNA binding-dependent MR signalling in macrophages is sufficient for DOC/salt-induced tissue inflammation. Our data demonstrate that the MR regulates a macrophage pro-inflammatory phenotype and cardiac tissue inflammation, partially via pathways that do not require DNA binding.

Parsing the IL-37-Mediated Suppression of Inflammasome Function.

Interleukin (IL)-37 is a member of the IL-1 family of cytokines. Although its broad anti-inflammatory properties are well described, the effects of IL-37 on inflammasome function remain poorly understood. Performing gene expression analyses, ASC oligomerization/speck assays and caspase-1 assays in bone marrow-derived macrophages (BMDM), and employing an in vivo endotoxemia model, we studied how IL-37 affects the expression and maturation of IL-1ß and IL-18, inflammasome activation, and pyroptosis in detail. IL-37 inhibited IL-1ß production by NLRP3 and AIM2 inflammasomes, and IL-18 production by the NLRP3 inflammasome. This inhibition was partially attributable to effects on gene expression: whereas IL-37 did not affect lipopolysaccharide (LPS)-induced mRNA expression of Il18 or inflammasome components, IL-37-transgenic BMDM displayed an up to 83% inhibition of baseline and LPS-stimulated Il1b compared to their wild-type counterparts. Importantly, we observed that IL-37 suppresses nigericin- and silica-induced ASC oligomerization/speck formation (a step in inflammasome activation and subsequent caspase-1 activation), and pyroptosis (-50%). In mice subjected to endotoxemia, IL-37 inhibited plasma IL-1ß (-78% compared to wild-type animals) and IL-18 (-61%). Thus, our study adds suppression of inflammasome activity to the portfolio of anti-inflammatory pathways employed by IL-37, highlighting this cytokine as a potential tool for treating inflammasome-driven diseases.

The Single Nucleotide Polymorphism Mal-D96N Mice Provide New Insights into Functionality of Mal in TLR Immune Responses.

MyD88 adaptor-like (Mal) protein is the most polymorphic of the four key adaptor proteins involved in TLR signaling. TLRs play a critical role in the recognition and immune response to pathogens through activation of the prototypic inflammatory transcription factor NF-κB. The study of single nucleotide polymorphisms in TLRs, adaptors, and signaling mediators has provided key insights into the function of the corresponding genes but also into the susceptibility to infectious diseases in humans. In this study, we have analyzed the immune response of mice carrying the human Mal-D96N genetic variation that has previously been proposed to confer protection against septic shock. We have found that Mal-D96N macrophages display reduced cytokine expression in response to TLR4 and TLR2 ligand challenge. Mal-D96N macrophages also display reduced MAPK activation, NF-κB transactivation, and delayed NF-κB nuclear translocation, presumably via delayed kinetics of Mal interaction with MyD88 following LPS stimulation. Importantly, Mal-D96N genetic variation confers a physiological protective phenotype to in vivo models of LPS-, Escherichia coli-, and influenza A virus-induced hyperinflammatory disease in a gene dosage-dependent manner. Together, these results highlight the critical role Mal plays in regulating optimal TLR-induced inflammatory signaling pathways and suggest the potential therapeutic advantages of targeting the Mal D96 signaling nexus.

Membrane vesicles from Pseudomonas aeruginosa activate the noncanonical inflammasome through caspase-5 in human monocytes.

Outer membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria both in vivo and in vitro. These lipid-bound structures carry a range of immunogenic components derived from the parent cell, which are transported into host target cells and activate the innate immune system. Recent advances in the field have shed light on some of the multifaceted roles of OMVs in host-pathogen interactions. In this study, we investigated the ability of OMVs from two clinically important pathogens, Pseudomonas aeruginosa and Helicobacter pylori, to activate canonical and noncanonical inflammasomes. P. aeruginosa OMVs induced inflammasome activation in mouse macrophages, as evidenced by "speck" formation, as well as the cleavage and secretion of interleukin-1ß and caspase-1. These responses were independent of AIM2 and NLRC4 canonical inflammasomes, but dependent on the noncanonical caspase-11 pathway. Moreover, P. aeruginosa OMVs alone were able to activate the inflammasome in a TLR-dependent manner, without requiring an exogenous priming signal. In contrast, H. pylori OMVs were not able to induce inflammasome activation in macrophages. Using CRISPR/Cas9 knockout THP-1 cells lacking the human caspase-11 homologs, caspase-4 and -5,we demonstrated that caspase-5 but not caspase-4 is required for inflammasome activation by P. aeruginosa OMVs in human monocytes. In contrast, free P. aeruginosa lipopolysaccharide (LPS) transfected into cells induced inflammasome responses via caspase-4. This suggests that caspase-4 and caspase-5 differentially recognize LPS depending on its physical form or route of delivery into the cell. These findings have relevance to Gram-negative infections in humans and the use of OMVs as novel vaccines.

PB1-F2 Peptide Derived from Avian Influenza A Virus H7N9 Induces Inflammation via Activation of the NLRP3 Inflammasome.

The emergence of avian H7N9 influenza A virus in humans with associated high mortality has highlighted the threat of a potential pandemic. Fatal H7N9 infections are characterized by hyperinflammation and increased cellular infiltrates in the lung. Currently there are limited therapies to address the pathologies associated with H7N9 infection and the virulence factors that contribute to these pathologies. We have found that PB1-F2 derived from H7N9 activates the NLRP3 inflammasome and induces lung inflammation and cellular recruitment that is NLRP3-dependent. We have also shown that H7N9 and A/Puerto Rico/H1N1 (PR8)PB1-F2 peptide treatment induces significant mitochondrial reactive oxygen production, which contributes to NLRP3 activation. Importantly, treatment of cells or mice with the specific NLRP3 inhibitor MCC950 significantly reduces IL-1ß maturation, lung cellular recruitment, and cytokine production. Together, these results suggest that PB1-F2 from H7N9 avian influenza A virus may be a major contributory factor to disease pathophysiology and excessive inflammation characteristic of clinical infections and that targeting the NLRP3 inflammasome may be an effective means to reduce the inflammatory burden associated with H7N9 infections.

Reassessing the role of the NLRP3 inflammasome during pathogenic influenza A virus infection via temporal inhibition.

The inflammasome NLRP3 is activated by pathogen associated molecular patterns (PAMPs) during infection, including RNA and proteins from influenza A virus (IAV). However, chronic activation by danger associated molecular patterns (DAMPs) can be deleterious to the host. We show that blocking NLRP3 activation can be either protective or detrimental at different stages of lethal influenza A virus (IAV). Administration of the specific NLRP3 inhibitor MCC950 to mice from one day following IAV challenge resulted in hypersusceptibility to lethality. In contrast, delaying treatment with MCC950 until the height of disease (a more likely clinical scenario) significantly protected mice from severe and highly virulent IAV-induced disease. These findings identify for the first time that NLRP3 plays a detrimental role later in infection, contributing to IAV pathogenesis through increased cytokine production and lung cellular infiltrates. These studies also provide the first evidence identifying NLRP3 inhibition as a novel therapeutic target to reduce IAV disease severity.

Toll-like receptors: the swiss army knife of immunity and vaccine development.

Innate immune cells have a critical role in defense against infection and disease. Central to this is the broad specificity with which they can detect pathogen-associated patterns and danger-associated patterns via the pattern recognition receptors (PRRs) they express. Several families of PRRs have been identified including: Toll-like receptors (TLRs), C-type lectin-like receptors, retinoic acid-inducible gene-like receptors and nucleotide-binding oligomerization domain-like receptors. TLRs are one of the most largely studied families of PRRs. The binding of ligands to TLRs on antigen presenting cells (APCs), mainly dendritic cells, leads to APC maturation, induction of inflammatory cytokines and the priming of naive T cells to drive acquired immunity. Therefore, activation of TLRs promotes both innate inflammatory responses and the induction of adaptive immunity. Consequently, in the last two decades mounting evidence has inextricably linked TLR activation with the pathogenesis of immune diseases and cancer. It has become advantageous to harness these aspects of TLR signaling therapeutically to accelerate and enhance the induction of vaccine-specific responses and also target TLRs with the use of biologics and small molecule inhibitors for the treatment of disease. In these respects, TLRs may be considered a 'Swiss Army' knife of the immune system, ready to respond in a multitude of infectious and disease states. Here we describe the latest advances in TLR-targeted therapeutics and the use of TLR ligands as vaccine adjuvants.

Toll-Like Receptors: Ligands, Cell-Based Models, and Readouts for Receptor Action.

This chapter details Toll-like receptors (TLRs) and the tools available to study their biology in vitro. Key parameters to consider before exploring TLR action such as receptor localization, signaling pathways, nature of ligands and cellular expression are introduced. Cellular models (i.e., host cells and readouts) based on the use of cell lines, primary cells, or whole blood are presented. The use of modified TLRs to circumvent some technical problems is also discussed.

Promyelocytic leukemia protein interacts with the apoptosis-associated speck-like protein to limit inflammasome activation.

The apoptosis-associated speck-like protein containing a caspase-activating recruitment domain (ASC) is an essential component of several inflammasomes, multiprotein complexes that regulate caspase-1 activation and inflammation. We report here an interaction between promyelocytic leukemia protein (PML) and ASC. We observed enhanced formation of ASC dimers in PML-deficient macrophages. These macrophages also display enhanced levels of ASC in the cytosol. Furthermore, IL-1ß production was markedly enhanced in these macrophages in response to both NLRP3 and AIM2 inflammasome activation and following bone marrow-derived macrophage infection with herpes simplex virus-1 (HSV-1) and Salmonella typhimurium. Collectively, our data indicate that PML limits ASC function, retaining ASC in the nucleus.

Activation of liver X receptor suppresses the production of the IL-12 family of cytokines by blocking nuclear translocation of NF-κBp50.

There is now convincing evidence that liver X receptor (LXR) is an important modulator of the inflammatory response; however, its mechanism of action remains unclear. This study aimed to examine the effect of LXR on the IL-12 family of cytokines and examined the mechanism by which LXR exerted this effect. We first demonstrated that activation of murine-derived dendritic cells (DC) with a specific agonist to LXR enhanced expression of LXR following activation with LPS, suggesting a role in inflammation. Furthermore, we showed LXR expression to be increased in vivo in dextrane sulphate sodium-induced colitis. LXR activation also suppressed production of IL-12p40, IL-12p70, IL-27 and IL-23 in murine-derived DC following stimulation with LPS, and specifically targeted the p35, p40 and EBI3 subunits of the IL-12 cytokine family, which are under the control of the NF-κB subunit p50 (NF-κBp50). Finally, we demonstrated that LXR can associate with NF-κBp50 in DC and that LXR activation prevents translocation of the p50 subunit into the nucleus. In summary, our study indicates that LXR can specifically suppress the IL-12 family of cytokines though its association with NF-κBp50 and highlights its potential as a therapeutic target for chronic inflammatory diseases.

Conjugated linoleic acid suppresses IRF3 activation via modulation of CD14.

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.

Biochemical regulation of the inflammasome.

The extensively studied cytokine IL-1ß is an important mediator of the inflammatory response. However, dysregulated release of IL-1ß can be detrimental and is attributed to the progression and pathogenesis of multiple inflammatory diseases including, rhuematoid arthritis (RA), atherosclerosis, type 2 diabetes (T2D), Alzheimers disease and gout. IL-1ß is encoded as a pro-protein. A multi-protein molecular scaffold termed the "Inflammasome" is responsible for the tightly controlled and coordinated processing of pro-IL-1ß. The activation of several NLR (nucleotide-binding oligomerization domain (NOD)-like receptor) family members and PYHIN (pyrin and HIN domain) proteins can drive the formation of inflammasomes. However, the exact biochemical mechanisms governing their activation have been the subject of much research. Different inflammasomes have been demonstrated to respond to the same pathogen inducing a cooperative immune response accountable for the clearance of infection. Here, we review current knowledge surrounding the biochemical regulation of the NLRP1, NLRP3, NLRC4, AIM2 and IFI16 inflammasomes.