JNK Activation Correlates with Cognitive Impairment and Alteration of the Post-Synaptic Element in the 5xFAD AD Mouse Model.

The c-Jun N-terminal kinases (JNKs) are a family of proteins that, once activated by stress stimuli, can alter neuronal functions and survival. The JNK cascade plays a crucial role in the post-synaptic neuronal compartment by altering its structural organization and leading, at worst, to an overall impairment of neuronal communication. Increasing evidence suggests that synaptic impairment is the first neurodegenerative event in Alzheimer's disease (AD). To better elucidate this mechanism, we longitudinally studied 5xFAD mice at three selected time points representative of human AD symptom progression. We tested the mice cognitive performance by using the radial arm water maze (RAWM) in parallel with biochemical evaluations of post-synaptic enriched protein fraction and total cortical parenchyma. We found that 5xFAD mice presented a strong JNK activation at 3.5 months of age in the post-synaptic enriched protein fraction. This JNK activation correlates with a structural alteration of the post-synaptic density area and with memory impairment at this early stage of the disease that progressively declines to cause cell death. These findings pave the way for future studies on JNK as a key player in early neurodegeneration and as an important therapeutic target for the development of new compounds able to tackle synaptic impairment in the early phase of AD pathology.

Development of a rapid in vitro pre-screen for distinguishing effective liposome-adjuvant delivery systems.

Liposomes are a strong supporting tool in vaccine technology, as they are a versatile system that not only act as antigen delivery systems but also adjuvants that can be highly effective at stimulating both innate and adaptive immune responses. Their ability to induce cell-mediated immunity makes their use in vaccines a useful tool in the development of novel, more effective vaccines against intracellular infections (e.g. HIV, malaria and tuberculosis). Currently, screening of novel liposome formulations uses murine in vivo models which generate data that often correlates poorly with human data. In addition, these models are both high cost and low throughput, making them prohibitive for large scale screening of formulation libraries. This study uses the cationic liposome formulation DDA:TDB (known as cationic adjuvant formulation 01 (CAF01)), as a lead formulation, along with other liposome formulations of known in vivo efficacy to develop an in vitro screening tool for liposome formulation development. THP-1-derived macrophages were the model antigen presenting cell used to assess the ability of the liposome formulations to attract, associate with and activate antigen presenting cells in vitro, crucial steps necessary for an effective immune response to antigen. By using a combination of in vitro functions, the study highlights the potential use of an in vitro screening tool, to predict the in vivo efficacy of novel liposome formulations. CAF01 was predicted as the most effective liposome formulation when assessing all in vitro functions and a measure of in vitro activation was able to predict 80% of the liposome correctly for their ability to induce an in vivo IFN-Ò¯ response.

Emerging roles for AQP in mammalian extracellular vesicles.

Recent research in the aquaporin (AQP) field has identified a role for diverse AQPs in extracellular vesicles (EV). Though still in its infancy, there is a growing body of knowledge in the area; AQPs in EV have been suggested as biomarkers for disease, as drug targets and show potential as therapeutics. To advance further in this field, AQPs in EV must be better understood. Here we summarize current knowledge of the presence and function of AQPs in EV and hypothesise their roles in health and disease.

Macrophages: The Good, the Bad, and the Gluttony.

Macrophages are dynamic cells that play critical roles in the induction and resolution of sterile inflammation. In this review, we will compile and interpret recent findings on the plasticity of macrophages and how these cells contribute to the development of non-infectious inflammatory diseases, with a particular focus on allergic and autoimmune disorders. The critical roles of macrophages in the resolution of inflammation will then be examined, emphasizing the ability of macrophages to clear apoptotic immune cells. Rheumatoid arthritis (RA) is a chronic autoimmune-driven spectrum of diseases where persistent inflammation results in synovial hyperplasia and excessive immune cell accumulation, leading to remodeling and reduced function in affected joints. Macrophages are central to the pathophysiology of RA, driving episodic cycles of chronic inflammation and tissue destruction. RA patients have increased numbers of active M1 polarized pro-inflammatory macrophages and few or inactive M2 type cells. This imbalance in macrophage homeostasis is a main contributor to pro-inflammatory mediators in RA, resulting in continual activation of immune and stromal populations and accelerated tissue remodeling. Modulation of macrophage phenotype and function remains a key therapeutic goal for the treatment of this disease. Intriguingly, therapeutic intervention with glucocorticoids or other DMARDs promotes the re-polarization of M1 macrophages to an anti-inflammatory M2 phenotype; this reprogramming is dependent on metabolic changes to promote phenotypic switching. Allergic asthma is associated with Th2-polarised airway inflammation, structural remodeling of the large airways, and airway hyperresponsiveness. Macrophage polarization has a profound impact on asthma pathogenesis, as the response to allergen exposure is regulated by an intricate interplay between local immune factors including cytokines, chemokines and danger signals from neighboring cells. In the Th2-polarized environment characteristic of allergic asthma, high levels of IL-4 produced by locally infiltrating innate lymphoid cells and helper T cells promote the acquisition of an alternatively activated M2a phenotype in macrophages, with myriad effects on the local immune response and airway structure. Targeting regulators of macrophage plasticity is currently being pursued in the treatment of allergic asthma and other allergic diseases. Macrophages promote the re-balancing of pro-inflammatory responses towards pro-resolution responses and are thus central to the success of an inflammatory response. It has long been established that apoptosis supports monocyte and macrophage recruitment to sites of inflammation, facilitating subsequent corpse clearance. This drives resolution responses and mediates a phenotypic switch in the polarity of macrophages. However, the role of apoptotic cell-derived extracellular vesicles (ACdEV) in the recruitment and control of macrophage phenotype has received remarkably little attention. ACdEV are powerful mediators of intercellular communication, carrying a wealth of lipid and protein mediators that may modulate macrophage phenotype, including a cargo of active immune-modulating enzymes. The impact of such interactions may result in repair or disease in different contexts. In this review, we will discuss the origin, characterization, and activity of macrophages in sterile inflammatory diseases and the underlying mechanisms of macrophage polarization via ACdEV and apoptotic cell clearance, in order to provide new insights into therapeutic strategies that could exploit the capabilities of these agile and responsive cells.

Evidence of sequestration of triclabendazole and associated metabolites by extracellular vesicles of Fasciola hepatica.

Fascioliasis is a neglected zoonotic disease that infects humans and ruminant species worldwide. In the absence of vaccines, control of fascioliasis is primarily via anthelminthic treatment with triclabendazole (TCBZ). Parasitic flatworms, including Fasciola hepatica, are active secretors of extracellular vesicles (EVs), but research has not been undertaken investigating EV anthelmintic sequestration. Adult F. hepatica were cultured in lethal and sub-lethal doses of TCBZ and its active metabolites, in order to collect EVs and evaluate their morphological characteristics, production and anthelmintic metabolite content. Transmission electron microscopy demonstrated that F. hepatica exposed to TCBZ and its metabolites produced EVs of similar morphology, compared to non-TCBZ exposed controls, even though TCBZ dose and/or TCBZ metabolite led to measurable structural changes in the treated F. hepatica tegument. qNano particle analysis revealed that F. hepatica exposed to TCBZ and its metabolites produced at least five times greater EV concentrations than non-TCBZ controls. A combined mass spectrometry and qNano particle analysis confirmed the presence of TCBZ and the TCBZ-sulphoxide metabolite in anthelmintic exposed EVs, but limited TCBZ sulphone was detectable. This data suggests that EVs released from adult F. hepatica have a biological role in the sequestration of TCBZ and additional toxic xenobiotic metabolites.

CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups.

Tetraspanins exert a wide range of cellular functions of broad medical importance. Despite this, their biophysical characteristics are incompletely understood. Only two high-resolution structures of full-length tetraspanins have been solved. One is that of human CD81, which is involved in the infectivity of human pathogens including influenza, HIV, the malarial Plasmodium parasite and hepatitis C virus (HCV). The CD81 crystal structure identifies a cholesterol-binding pocket, which has been suggested to be important in the regulation of tetraspanin function. Here we investigate the use of styrene-maleic anhydride co-polymers (SMA) for the solubilisation and purification of CD81 within a lipid environment. When CD81 was expressed in the yeast Pichia pastoris, it could be solubilised and purified using SMA2000. This SMALP-encapsulated CD81 retained its native folded structure, as determined by the binding of two conformation-sensitive anti-CD81 antibodies. Analysis by size exclusion chromatography revealed two distinct populations of CD81, only one of which bound the HCV glycoprotein, E2. Optimization of expression and buffer conditions increased the proportion of E2-binding competent CD81 protein. Mass spectrometry analysis indicated that the lipid environment surrounding CD81 is enriched with negatively charged lipids. These results establish a platform to study the influence of protein-lipid interactions in tetraspanin biology.

Biophysical analysis of lipidic nanoparticles.

Biological nanoparticles include liposomes, extracellular vesicle and lipid-based discoidal systems. When studying such particles, there are several key parameters of interest, including particle size and concentration. Measuring these characteristics can be of particular importance in the research laboratory or when producing such particles as biotherapeutics. This article briefly describes the major types of lipid-containing nanoparticles and the techniques that can be used to study them. Such methodologies include electron microscopy, atomic force microscopy, dynamic light scattering, nanoparticle tracking analysis, flow cytometry, tunable resistive pulse sensing and microfluidic resistive pulse sensing. Whilst no technique is perfect for the analysis of all nanoparticles, this article provides advantages and disadvantages of each, highlighting the latest developments in the field. Finally, we demonstrate the use of microfluidic resistive pulse sensing for the analysis of biological nanoparticles.

Inflammation, Lipid (Per)oxidation, and Redox Regulation.

Significance: Inflammation increases during the aging process. It is linked to mitochondrial dysfunction and increased reactive oxygen species (ROS) production. Mitochondrial macromolecules are critical targets of oxidative damage; they contribute to respiratory uncoupling with increased ROS production, redox stress, and a cycle of senescence, cytokine production, and impaired oxidative phosphorylation. Targeting the formation or accumulation of oxidized biomolecules, particularly oxidized lipids, in immune cells and mitochondria could be beneficial for age-related inflammation and comorbidities. Recent Advances: Inflammation is central to age-related decline in health and exhibits a complex relationship with mitochondrial redox state and metabolic function. Improvements in mass spectrometric methods have led to the identification of families of oxidized phospholipids (OxPLs), cholesterols, and fatty acids that increase during inflammation and which modulate nuclear factor erythroid 2-related factor 2 (Nrf2), peroxisome proliferator-activated receptor gamma (PPARγ), activator protein 1 (AP1), and NF-κB redox-sensitive transcription factor activity. Critical Issues: The kinetic and spatial resolution of the modified lipidome has profound and sometimes opposing effects on inflammation, promoting initiation at high concentration and resolution at low concentration of OxPLs. Future Directions: There is an emerging opportunity to prevent or delay age-related inflammation and vascular comorbidity through a resolving (oxy)lipidome that is dependent on improving mitochondrial quality control and restoring redox homeostasis.

Formulation and manufacturing of lymphatic targeting liposomes using microfluidics.

The lymphatics are a target for a range of therapeutic purposes, including cancer therapy and vaccination, and both vesicle size and charge have been considered as factors controlling lymphatic targeting. Within this work, a range of liposomal formulations were investigated to develop a liposomal lymphatic targeting system. Initial screening of formulations considered the effect of charge, with neutral, cationic and anionic liposomes being investigated. Biodistribution studies demonstrated that after intramuscular injection, anionic liposomes offered the most rapid clearance to the draining lymphatics with cationic liposomes forming a depot at the injection site. Anionic liposomes containing phosphatidylserine showed higher clearance to the lymphatics and this may result form preferential uptake by macrophages. In terms of vesicle size, smaller unilamellar vesicles gave high lymphatic targeting and a 10-fold increase in concentration was achieved in dose escalation studies. Given that effective trafficking to the lymphatics was achieved, the next step was to enhance retention of the liposomes within the lymphatics, therefore the liposome formulation was combined with an avidin/biotin complex mechanism. The affinity of avidin for biotin allows biotinylated liposomes to complex in the presence of avidin. By pre-dosing with avidin, the biotin-avidin complex can be exploited to promote longer retention of the liposomes at the draining lymphatics. To load these small, biotinylated liposomes with protein, microfluidics manufacturing was used. Using microfluidics, protein could easily be incorporated in these small (~90nm) biotinylated liposomes. Both liposome and protein retention at the local draining lymph nodes was demonstrated with the liposome-biotin-avidin system. These results demonstrate that microfluidics can be used to prepare protein-loaded liposomes that offer enhanced lymphatic targeting and retention of both the liposomes and entrapped antigen.

Anaerobiosis influences virulence properties of Pseudomonas aeruginosa cystic fibrosis isolates and the interaction with Staphylococcus aureus.

The airways of individuals with cystic fibrosis (CF) are abundantly colonised by Staphylococcus aureus and Pseudomonas aeruginosa. Co-infecting hypoxic regions of static mucus within CF airways, together with decreases in pulmonary function, mucus plugging and oxygen consumption by host neutrophils gives rise to regions of anoxia. This study determined the impact of anaerobiosis upon S. aureus-P. aeruginosa interactions in planktonic co-culture and mixed species biofilms in vitro. Whilst anoxia reduced the ability for P. aeruginosa CF isolates to dominate over S. aureus, this occurred in an isolate dependent manner. Investigations into the underlying mechanisms suggest that the anti-staphylococcal compound facilitating P. aeruginosa dominance under normoxia and anoxia is greater than 3 kDa in size and is heat-stable. Not all interspecies interactions studied were antagonistic, as S. aureus exoproducts were shown to restore and enhance P. aeruginosa motility under normoxia and anoxia in an isolate dependent manner. Collectively, this study suggests changes in oxygen availability within regions of the CF lung is likely to influence interspecies interactions and in turn, potentially influence disease progression.

Apoptotic cell-derived extracellular vesicles: structure-function relationships.

Apoptosis is an essential process for normal physiology and plays a key role in the resolution of inflammation. Clearance of apoptotic cells (ACs) involves complex signalling between phagocytic cells, ACs, and the extracellular vesicles (EVs) they produce. Here, we discuss apoptotic cell-derived extracellular vesicles (ACdEVs) and how their structure relates to their function in AC clearance and the control of inflammation, focussing on the ACdEV proteome. We review the current knowledge, ongoing work and future directions for research in this field.

Communicating with the dead: lipids, lipid mediators and extracellular vesicles.

Apoptosis is a key event in the control of inflammation. However, for this to be successful, dying cells must efficiently and effectively communicate their presence to phagocytes to ensure timely removal of dying cells. Here, we consider apoptotic cell-derived extracellular vesicles and the role of contained lipids and lipid mediators in ensuring effective control of inflammation. We discuss key outstanding issues in the study of cell death and cell communication, and introduce the concept of the 'active extracellular vesicle' as a metabolically active and potentially changing intercellular communicator.

Simvastatin reduces circulating oxysterol levels in men with hypercholesterolaemia.

Oxysterols (OHC) are biologically active cholesterol metabolites circulating in plasma that may be formed enzymatically (e.g. 24S-OHC, 25-OHC and 27-OHC) or by autoxidative mechanisms (e.g. 7-ketocholesterol, 7ß-OHC and 25-OHC). Oxysterols are more soluble than cholesterol and are reported to exert inflammatory, cytoprotective and apoptotic effects according to concentration and species. Esterified oxysterols have been analysed in people with dementia and cardiovascular diseases although there is no consistent relationship between oxysterol esters and disease. However, oxysterol esters are held in lipoprotein core and may not relate to the concentration and activity of plasma free oxysterols. Methodological limitations have challenged the analysis of free oxysterols to date. We have developed a fast, sensitive and specific quantitative LC-MS/MS, multiple reaction monitoring (MRM) method to target five oxysterols in human plasma with analyte recoveries between 72% and 82% and sensitivities between 5 and 135 pg/ml. A novel method was used to investigate the hypothesis that simvastatin may reduce the concentrations of specific plasma free oxysterols in hypercholesterolaemia. Twenty healthy male volunteers were recruited (aged 41-63 years); ten were asymptomatic with high plasma cholesterol > 6.5 mM and ten were healthy with normal plasma cholesterol (< 6.5 mM). Simvastatin (40 mg/day) was prescribed to those with hypercholesterolaemia. Plasma samples were taken from both groups at baseline and after three months. Simvastatin reduced plasma cholesterol by ~35% (p < 0.05) at the end of three months. Oxysterols generated by autoxidation (but not enzymatically) were elevated up to 45 fold in hypercholesterolaemic midlife men. Plasma oxysterols were restored to those of healthy controls after simvastatin intervention suggesting that autoxidation is either prevented by simvastatin directly or that autoxidation is less prevalent when plasma cholesterol concentrations are within the normal range.

Technical challenges of working with extracellular vesicles.

Extracellular Vesicles (EVs) are gaining interest as central players in liquid biopsies, with potential applications in diagnosis, prognosis and therapeutic guidance in most pathological conditions. These nanosized particles transmit signals determined by their protein, lipid, nucleic acid and sugar content, and the unique molecular pattern of EVs dictates the type of signal to be transmitted to recipient cells. However, their small sizes and the limited quantities that can usually be obtained from patient-derived samples pose a number of challenges to their isolation, study and characterization. These challenges and some possible options to overcome them are discussed in this review.

Summary of the ISEV workshop on extracellular vesicles as disease biomarkers, held in Birmingham, UK, during December 2017.

This report summarises the presentations and activities of the ISEV Workshop on extracellular vesicle biomarkers held in Birmingham, UK during December 2017. Among the key messages was broad agreement about the importance of biospecimen science. Much greater attention needs to be paid towards the provenance of collected samples. The workshop also highlighted clear gaps in our knowledge about pre-analytical factors that alter extracellular vesicles (EVs). The future utility of certified standards for credentialing of instruments and software, to analyse EV and for tracking the influence of isolation steps on the structure and content of EVs were also discussed. Several example studies were presented, demonstrating the potential utility for EVs in disease diagnosis, prognosis, longitudinal serial testing and stratification of patients. The conclusion of the workshop was that more effort focused on pre-analytical issues and benchmarking of isolation methods is needed to strengthen collaborations and advance more effective biomarkers.

Porphyromonas gingivalis gingipains cause defective macrophage migration towards apoptotic cells and inhibit phagocytosis of primary apoptotic neutrophils.

Periodontal disease is a prevalent chronic inflammatory condition characterised by an aberrant host response to a pathogenic plaque biofilm resulting in local tissue damage and frustrated healing that can result in tooth loss. Cysteine proteases (gingipains) from the key periodontal pathogen Porphyromonas gingivalis have been implicated in periodontal disease pathogenesis by inhibiting inflammation resolution and are linked with systemic chronic inflammatory conditions such as rheumatoid arthritis. Efficient clearance of apoptotic cells is essential for the resolution of inflammation and tissue restoration. Here we sought to characterise the innate immune clearance of apoptotic cells and its modulation by gingipains. We examined the capacity of gingipain-treated macrophages to migrate towards and phagocytose apoptotic cells. Lysine gingipain treatment of macrophages impaired macrophage migration towards apoptotic neutrophils. Furthermore, lysine gingipain treatment reduced surface expression levels of CD14, a key macrophage receptor for apoptotic cells, which resulted in reduced macrophage interactions with apoptotic cells. Additionally, while apoptotic cells and their derived secretome were shown to inhibit TNF-α-induced expression by P. gingivalis lipopolysaccharide, we demonstrated that gingipain preparations induced a rapid inflammatory response in macrophages that was resistant to the anti-inflammatory effects of apoptotic cells or their secretome. Taken together, these data indicate that P. gingivalis may promote the chronic inflammation seen in periodontal disease patients by multiple mechanisms, including rapid, potent gingipain-mediated inflammation, coupled with receptor cleavage leading to defective clearance of apoptotic cells and reduced anti-inflammatory responses. Thus, gingipains represent a potential therapeutic target for intervention in the management of chronic periodontal disease.

Characterization of Microvesicles Released from Human Red Blood Cells.

BACKGROUND/AIMS: Extracellular vesicles (EVs) are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs) and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs) under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca2+ uptake and activation of protein kinase C. METHODS: Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. RESULTS: Treatment of RBCs with 4-bromo-A23187 (positive control), lysophosphatidic acid (LPA), or phorbol-12 myristate-13 acetate (PMA) in the presence of 2 mM extracellular Ca2+ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS) and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca2+, especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potential of MVs was determined in the range from - 40 mV to - 10 mV depended on the solutions and buffers used. CONCLUSION: An increase of intracellular Ca2+ or an activation of protein kinase C leads to the formation and release of MVs in human RBCs.

Age-associated changes in long-chain fatty acid profile during healthy aging promote pro-inflammatory monocyte polarization via PPARγ.

Differences in lipid metabolism associate with age-related disease development and lifespan. Inflammation is a common link between metabolic dysregulation and aging. Saturated fatty acids (FAs) initiate pro-inflammatory signalling from many cells including monocytes; however, no existing studies have quantified age-associated changes in individual FAs in relation to inflammatory phenotype. Therefore, we have determined the plasma concentrations of distinct FAs by gas chromatography in 26 healthy younger individuals (age < 30 years) and 21 healthy FA individuals (age > 50 years). Linear mixed models were used to explore the association between circulating FAs, age and cytokines. We showed that plasma saturated, poly- and mono-unsaturated FAs increase with age. Circulating TNF-α and IL-6 concentrations increased with age, whereas IL-10 and TGF-ß1 concentrations decreased. Oxidation of MitoSOX Red was higher in leucocytes from FA adults, and plasma oxidized glutathione concentrations were higher. There was significant colinearity between plasma saturated FAs, indicative of their metabolic relationships. Higher levels of the saturated FAs C18:0 and C24:0 were associated with lower TGF-ß1 concentrations, and higher C16:0 were associated with higher TNF-α concentrations. We further examined effects of the aging FA profile on monocyte polarization and metabolism in THP1 monocytes. Monocytes preincubated with C16:0 increased secretion of pro-inflammatory cytokines in response to phorbol myristate acetate-induced differentiation through ceramide-dependent inhibition of PPARγ activity. Conversely, C18:1 primed a pro-resolving macrophage which was PPARγ dependent and ceramide dependent and which required oxidative phosphorylation. These data suggest that a midlife adult FA profile impairs the switch from proinflammatory to lower energy, requiring anti-inflammatory macrophages through metabolic reprogramming.

Designing liposomal adjuvants for the next generation of vaccines.

Liposomes not only offer the ability to enhance drug delivery, but can effectively act as vaccine delivery systems and adjuvants. Their flexibility in size, charge, bilayer rigidity and composition allow for targeted antigen delivery via a range of administration routes. In the development of liposomal adjuvants, the type of immune response promoted has been linked to their physico-chemical characteristics, with the size and charge of the liposomal particles impacting on liposome biodistribution, exposure in the lymph nodes and recruitment of the innate immune system. The addition of immunostimulatory agents can further potentiate their immunogenic properties. Here, we outline the attributes that should be considered in the design and manufacture of liposomal adjuvants for the delivery of sub-unit and nucleic acid based vaccines.