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Interleukin-1α is a suggested dual-function cytokine that diverged from interleukin-1ß in mammals potentially by acquiring additional biological roles that relate to highly conserved regions in the pro-domain of interleukin-1α, including a nuclear localisation sequence and histone acetyltransferase-binding domains. Why evolution modified pro-interleukin-1α's subcellular location and protein interactome, and how this shaped interleukin-1α's intracellular role, is unknown. Here we show that TurboID proximity labelling with pro-interleukin-1α suggests a nuclear role for pro-interleukin-1α that involves interaction with histone acetyltransferases, including EP300. We also identify and validate inactivating mutations in the pro-interleukin-1α nuclear localisation sequence of multiple mammalian species, including toothed whales, castorimorpha and marsupials. However, histone acetyltransferase-binding domains are conserved in those species that have lost pro-interleukin-1α nuclear localisation. Together, these data suggest that histone acetyltransferase binding and nuclear localisation occurred together, and that while some species lost the nuclear localisation sequence in their pro-interleukin-1α, histone acetyltransferase binding ability was maintained. The nuclear localisation sequence was lost from several distinct species at different evolutionary times, suggesting convergent evolution, and that the loss of the nuclear localisation sequence confers some important biological outcome.
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Núcleo Celular , Evolución Molecular , Interleucina-1alfa , Interleucina-1alfa/metabolismo , Interleucina-1alfa/genética , Animales , Núcleo Celular/metabolismo , Humanos , Proteína p300 Asociada a E1A/metabolismo , Proteína p300 Asociada a E1A/genética , Histona Acetiltransferasas/metabolismo , Histona Acetiltransferasas/genética , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , Unión Proteica , Secuencia de AminoácidosRESUMEN
Microplastic and nanoplastic research has proliferated in recent years in response to the escalating plastic pollution crisis. However, a lack of optimised methods for sampling and sample processing has potential implications for contaminating samples resulting in an overestimation of the quantity of microplastics and nanoplastics present in environmental samples. In response, a series of recommendations have been made, but most have not been quantified or validated sources of contamination. In the present study, we investigated sources of plastic contamination in common laboratory procedures including water sources (e.g., Milli-Q), consumables (e.g., unburnt glassware), airflow (e.g., fume hood) and dust. Using flow cytometry, we identified water, air flow and dust as sources of significant contamination. Milli-Q and reverse osmosis were the least contaminated sources when compared with tap water. Interestingly, current recommendations are to use glass consumables in replacement of plastic consumables, however, we have identified glassware and glass consumables as a significant source of contamination. Current best practice is to cover the glass tube with aluminium foil to reduce airborne contamination, but we found fresh aluminium foil to be a significant source of contamination, bringing light to the limitations foil has as a contamination control measure. Lastly, we identified significant quantities of microplastics and nanoplastics present in dust collected within the laboratory, suggesting this is a widespread and underestimated source of contamination. We have provided validated sources of contamination for both consumables and common laboratory procedures and provided mitigation strategies based on these. Additional recommendations include the appropriate design of experimental controls to quantify levels of introduced contamination based on methods and the detection techniques utilised. The application of these mitigation strategies and appropriate experimental design will allow for more accurate estimations on the level of microplastic and nanoplastic contamination within environmental samples.
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The global impact of pollution on human and wildlife health is a growing concern. The health impacts of pollution are significant and far-reaching yet poorly understood as no one field of research has the practices and methodologies required to encapsulate the diversity of these consequences. This paper advocates that interdisciplinary research is essential to comprehend the full extent of the impact of pollution. Medical and ecological research play a key role in investigating the health consequences of the pollution crisis, yet the wildlife experience is often neglected. This paper outlines how applying advanced techniques and expertise adapted in medical research to wildlife exposed to pollutants offers a unique perspective to understanding the full diversity of impacts to health. The challenges that impede the progress of this research include the lack of support for interdisciplinary research among funding streams, limitations in field-specific techniques, and a lack of communication between researchers from different disciplines. Of awarded funding from major national research councils across Australia, Europe, and the United States of America, only 0.5% is dedicated to pollution focused research. This is inclusive of laboratory equipment, mitigation strategies, quantification of environmental samples and health consequences research. Of that, 0.03% of funding is awarded to explaining the wildlife experience and documenting the health consequences observed despite being model organisms to environmentally and biologically relevant models for pollution exposure. This calls for a coordinated effort to overcome these hurdles and to promote interdisciplinary research in order to fully comprehend the consequences of pollution exposure and protect the health of humans, wildlife, and the environment. An interdisciplinary approach to this problem is timely given the magnitude of negative health consequences associated with exposure, the number of pollutants already present within the environment and the continual development of new compounds.
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As biota are increasingly exposed to plastic pollution, there is a need to closely examine the sub-lethal 'hidden' impacts of plastic ingestion. This emerging field of study has been limited to model species in controlled laboratory settings, with little data available for wild, free-living organisms. Highly impacted by plastic ingestion, Flesh-footed Shearwaters (Ardenna carneipes) are thus an apt species to examine these impacts in an environmentally relevant manner. A Masson's Trichrome stain was used to document any evidence of plastic-induced fibrosis, using collagen as a marker for scar tissue formation in the proventriculus (stomach) of 30 Flesh-footed Shearwater fledglings from Lord Howe Island, Australia. Plastic presence was highly associated with widespread scar tissue formation and extensive changes to, and even loss of, tissue structure within the mucosa and submucosa. Additionally, despite naturally occurring indigestible items, such as pumice, also being found in the gastrointestinal tract, this did not cause similar scarring. This highlights the unique pathological properties of plastics and raises concerns for other species impacted by plastic ingestion. Further, the extent and severity of fibrosis documented in this study gives support for a novel, plastic-induced fibrotic disease, which we define as 'Plasticosis,'.
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Microplásticos , Contaminantes Químicos del Agua , Animales , Plásticos , Monitoreo del Ambiente , Cicatriz , Aves , Contaminantes Químicos del Agua/análisisRESUMEN
Coastlines, including estuaries, mudflats, and beaches, are particularly susceptible to plastic pollution, which can accumulate from both marine and terrestrial sources. While numerous studies have confirmed the presence of microplastics (1-5 mm) along coastlines, few have focused on very small particles (<1 µm) or quantified exposure within the organisms that inhabit these areas, such as shorebirds. Here, we quantified small plastics (200 nm-70 µm) in two resident shorebird species in Tasmania, and compared this to quantities found in the surrounding sediments in order to investigate the potential exposure and transfer of particles within these ecosystems. Analysis was performed using a combination of flow cytometry for quantification of micro- and nanoplastics (200 nm-70 µm), and µm-FT-IR for validation and polymer identification of particles >5.5 × 5.5 µm. Micro- and nano-plastics were detected in 100% of guano samples from surface-feeding Eastern Hooded Plovers (Thinornis cucullatus) and 90% of Australian Pied Oystercatcher (Haematopus longirostris) guano, a species that forages for coastal invertebrates at 60-90 mm depth, and 100% of beach sediments. Hooded Plover guano contained 32 × more plastics, on average, than Pied Oystercatcher guano. Interestingly, the abundance of plastic particles within sediments collected from shorebird foraging sites did not appear to have a significant effect on the number of plastics the birds had ingested, suggesting the difference between species is likely a result of other variables, such as prey selection. The results of this study highlight the importance of including techniques that provide quantitative data on the abundance and size of the smallest possible particle sizes, and demonstrate the significant proportion of small plastics that are 'missed' using standard analysis tools.
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Charadriiformes , Contaminantes Químicos del Agua , Animales , Ecosistema , Microplásticos , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Plásticos , Australia , Monitoreo del Ambiente/métodos , Contaminantes Químicos del Agua/análisisRESUMEN
Plastic ingestion has been documented in a plethora of taxa. However, there is a significant gap in the detection of nano- and ultrafine particles due to size limitations of commonly used techniques. Using two Australian seabird species as case studies, the flesh-footed shearwater (FFSH) Ardenna carneipes and short-tailed shearwater (STSH) A. tenuirostris, we tested a novel approach of flow cytometry to quantify ingested particles <70 µm in the fecal precursor (guano; colon and cloacal contents) of both species. This method provided the first baseline data set for these species for plastics in the 200 nm-70 µm particle size ranges and detected a mean of 553.50 ± 91.21 and 350.70 ± 52.08 plastics (count/mg fecal precursor, wet mass) in STSH and FFSH, respectively, whereas Fourier transform infrared spectroscopy (FT-IR) provided accurate measurements of polymer compositions and quantities in the size range above 5.5 × 5.5 µm2. The abundance of nano- and ultrafine particles in the guano (count/mg) was not significantly different between species (p-value = 0.051), suggesting that foraging distribution or prey items, but not species, may contribute to the consumption of small plastics. In addition, there was no correlation between macroplastics in the stomach compared to the fecal precursor, indicating that small particles are likely bioaccumulating (e.g., through shedding and digestive fragmentation) and/or being directly ingested. Combining flow cytometry with FT-IR provides a powerful quantitative and qualitative analysis tool for detecting particles orders of magnitude smaller than that are currently explored with wider applications across taxa and marine environments.
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Plásticos , Contaminantes Químicos del Agua , Animales , Plásticos/análisis , Australia , Espectroscopía Infrarroja por Transformada de Fourier , Residuos/análisis , Monitoreo del Ambiente/métodos , Aves , Contaminantes Químicos del Agua/análisisRESUMEN
Plastic pollution in the world's oceans is ubiquitous and increasing. The environment is inundated with microplastics (< 1 mm), and the health effects of these less conspicuous pollutants is poorly known. In addition, there is now evidence that macroplastics can release microplastics in the form of shedding or digestive fragmentation, meaning there is potential for macroplastic exposure to induce direct and indirect pathology through microplastics. Therefore, there is an urgent need for data from wild populations on the relationship between macro- and microplastic exposure and the potential compounding pathological effects of these forms of plastics. We investigated the presence and impact of microplastics in multiple tissues from Flesh-footed Shearwaters Ardenna carneipes, a species that ingests considerable quantities of plastics, and used histopathological techniques to measure physiological responses and inflammation from the plastics. All organs examined (kidney, spleen, proventriculus) had embedded microplastic particles and this correlated with macroplastic exposure. Considerable tissue damage was recorded, including a significant reduction in tubular glands and rugae in the proventriculus, and evidence of inflammation, fibrosis, and loss of organ structures in the kidney and spleen. This indicates macroplastics can induce damage directly at the site of exposure, while microplastics can be mobilised throughout the body causing widespread pathology. Collectively, these results indicate the scope and severity of the health impacts of plastic pollution may be grossly underestimated.
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Plásticos , Contaminantes Químicos del Agua , Animales , Plásticos/toxicidad , Microplásticos/toxicidad , Monitoreo del Ambiente/métodos , Residuos/análisis , Contaminantes Químicos del Agua/toxicidad , Contaminantes Químicos del Agua/análisis , Aves , InflamaciónRESUMEN
BACKGROUND: Gasdermins are ancient (>500million-years-ago) proteins, constituting a family of pore-forming proteins that allow the release of intracellular content including proinflammatory cytokines. Despite their importance in the immune response, and although gasdermin and gasdermin-like genes have been identified across a wide range of animal and non-animal species, there is limited information about the evolutionary history of the gasdermin family, and their functional roles after infection. In this study, we assess the lytic functions of different gasdermins across Metazoa species, and use a mouse model of sepsis to evaluate the expression of the different gasdermins during infection. RESULTS: We show that the majority of gasdermin family members from distantly related animal clades are pore-forming, in line with the function of the ancestral proto-gasdermin and gasdermin-like proteins of Bacteria. We demonstrate the first expansion of this family occurred through a duplication of the ancestral gasdermin gene which formed gasdermin E and pejvakin prior to the divergence of cartilaginous fish and bony fish ~475 mya. We show that pejvakin from cartilaginous fish and mammals lost the pore-forming functionality and thus its role in cell lysis. We describe that the pore-forming gasdermin A formed ~320 mya as a duplication of gasdermin E prior to the divergence of the Sauropsida clade (the ancestral lineage of reptiles, turtles, and birds) and the Synapsid clade (the ancestral lineage of mammals). We then demonstrate that the gasdermin A gene duplicated to form the rest of the gasdermin family including gasdermins B, C, and D: pore-forming proteins that present a high variation of the exons in the linker sequence, which in turn allows for diverse activation pathways. Finally, we describe expression of murine gasdermin family members in different tissues in a mouse sepsis model, indicating function during infection response. CONCLUSIONS: In this study we explored the evolutionary history of the gasdermin proteins in animals and demonstrated that the pore-formation functionality has been conserved from the ancient proto-gasdermin protein. We also showed that one gasdermin family member, pejvakin, lost its pore-forming functionality, but that all gasdermin family members, including pejvakin, likely retained a role in inflammation and the physiological response to infection.
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Piroptosis , Sepsis , Animales , Muerte Celular , Citocinas , Inflamación/genética , Inflamación/metabolismo , Mamíferos , Ratones , Proteínas , Piroptosis/fisiologíaRESUMEN
Plastic pollution is the focus of substantial scientific and public interest, leading many to believe the issue is well documented and managed, with effective mitigation in place. However, many aspects are poorly understood, including fundamental questions relating to the scope and severity of impacts (e.g., demographic consequences at the population level). Plastics accumulate in significant quantities on beaches globally, yet the consequences for these terrestrial environments are largely unknown. Using real world, in situ measurements of circadian thermal fluctuations of beach sediment on Henderson Island and Cocos (Keeling) Islands, we demonstrate that plastics increase circadian temperature extremes. Particular plastic levels were associated with increases in daily maximum temperatures of 2.45°C and decreases of daily minimum by - 1.50°C at 5 cm depth below the accumulated plastic. Mass of surface plastic was high on both islands (Henderson: 571 ± 197 g/m2; Cocos: 3164 ± 1989 g/m2), but did not affect thermal conductivity, specific heat capacity, thermal diffusivity, or moisture content of beach sediments. Therefore, we suggest plastic effects sediment temperatures by altering thermal inputs and outputs (e.g., infrared radiation absorption). The resulting circadian temperature fluctuations have potentially significant implications for terrestrial ectotherms, many of which have narrow thermal tolerance limits and are functionally important in beach habitats.
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Plásticos , Residuos , Monitoreo del Ambiente , Calor , Temperatura , Residuos/análisisRESUMEN
Blood-circulating biomarkers have the potential to detect Alzheimer's disease (AD) pathology before clinical symptoms emerge and to improve the outcomes of clinical trials for disease-modifying therapies. Despite recent advances in understanding concomitant systemic abnormalities, there are currently no validated or clinically used blood-based biomarkers for AD. The extremely low concentration of neurodegeneration-associated proteins in blood necessitates the development of analytical platforms to address the "signal-to-noise" issue and to allow an in-depth analysis of the plasma proteome. Here, we aimed to discover and longitudinally track alterations of the blood proteome in a transgenic mouse model of AD, using a nanoparticle-based proteomics enrichment approach. We employed blood-circulating, lipid-based nanoparticles to extract, analyze and monitor AD-specific protein signatures and to systemically uncover molecular pathways associated with AD progression. Our data revealed the existence of multiple proteomic signals in blood, indicative of the asymptomatic stages of AD. Comprehensive analysis of the nanoparticle-recovered blood proteome by label-free liquid chromatography-tandem mass spectrometry resulted in the discovery of AD-monitoring signatures that could discriminate the asymptomatic phase from amyloidopathy and cognitive deterioration. While the majority of differentially abundant plasma proteins were found to be upregulated at the initial asymptomatic stages, the abundance of these molecules was significantly reduced as a result of amyloidosis, suggesting a disease-stage-dependent fluctuation of the AD-specific blood proteome. The potential use of the proposed nano-omics approach to uncover information in the blood that is directly associated with brain neurodegeneration was further exemplified by the recovery of focal adhesion cascade proteins. We herein propose the integration of nanotechnology with already existing proteomic analytical tools in order to enrich the identification of blood-circulating signals of neurodegeneration, reinvigorating the potential clinical utility of the blood proteome at predicting the onset and kinetics of the AD progression trajectory.
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Enfermedad de Alzheimer , Nanopartículas , Enfermedad de Alzheimer/diagnóstico , Animales , Biomarcadores , Proteínas Sanguíneas , Ratones , Proteoma , ProteómicaRESUMEN
Alzheimer's disease is a devastating neurodegenerative disease with a dramatically increasing prevalence and no disease-modifying treatment. Inflammatory lifestyle factors increase the risk of developing Alzheimer's disease. Zinc deficiency is the most prevalent malnutrition in the world and may be a risk factor for Alzheimer's disease potentially through enhanced inflammation, although evidence for this is limited. Here we provide epidemiological evidence suggesting that zinc supplementation was associated with reduced risk and slower cognitive decline, in people with Alzheimer's disease and mild cognitive impairment. Using the APP/PS1 mouse model of Alzheimer's disease fed a control (35 mg/kg zinc) or diet deficient in zinc (3 mg/kg zinc), we determined that zinc deficiency accelerated Alzheimer's-like memory deficits without modifying amyloid ß plaque burden in the brains of male mice. The NLRP3-inflammasome complex is one of the most important regulators of inflammation, and we show here that zinc deficiency in immune cells, including microglia, potentiated NLRP3 responses to inflammatory stimuli in vitro, including amyloid oligomers, while zinc supplementation inhibited NLRP3 activation. APP/PS1 mice deficient in NLRP3 were protected against the accelerated cognitive decline with zinc deficiency. Collectively, this research suggests that zinc status is linked to inflammatory reactivity and may be modified in people to reduce the risk and slow the progression of Alzheimer's disease.SIGNIFICANCE STATEMENT Alzheimer's disease is a common condition mostly affecting the elderly. Zinc deficiency is also a global problem, especially in the elderly and also in people with Alzheimer's disease. Zinc deficiency contributes to many clinical disorders, including immune dysfunction. Inflammation is known to contribute to the risk and progression of Alzheimer's disease; thus, we hypothesized that zinc status would affect Alzheimer's disease progression. Here we show that zinc supplementation reduced the prevalence and symptomatic decline in people with Alzheimer's disease. In an animal model of Alzheimer's disease, zinc deficiency worsened cognitive decline because of an enhancement in NLRP3-driven inflammation. Overall, our data suggest that zinc status affects Alzheimer's disease progression, and that zinc supplementation could slow the rate of cognitive decline.
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Enfermedad de Alzheimer/sangre , Disfunción Cognitiva/sangre , Progresión de la Enfermedad , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Zinc/sangre , Adulto , Anciano , Enfermedad de Alzheimer/diagnóstico por imagen , Enfermedad de Alzheimer/dietoterapia , Animales , Células Cultivadas , Disfunción Cognitiva/diagnóstico por imagen , Disfunción Cognitiva/dietoterapia , Suplementos Dietéticos , Femenino , Estudios de Seguimiento , Humanos , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Zinc/administración & dosificación , Zinc/deficienciaRESUMEN
Graphene oxide (GO) holds great potential for biomedical applications, however fundamental understanding of the way it interacts with biological systems is still lacking even though it is essential for successful clinical translation. In this study, we exploit intrinsic fluorescent properties of thin GO sheets to establish the relationship between lateral dimensions of the material, its cellular uptake mechanisms and intracellular fate over time. Label-free GO with distinct lateral dimensions, small (s-GO) and ultra-small (us-GO) were thoroughly characterised both in water and in biologically relevant cell culture medium. Interactions of the material with a range of non-phagocytic mammalian cell lines (BEAS-2B, NIH/3T3, HaCaT, 293T) were studied using a combination of complementary analytical techniques (confocal microscopy, flow cytometry and TEM). The uptake mechanism was initially interrogated using a range of pharmaceutical inhibitors and validated using polystyrene beads of different diameters (0.1 and 1 µm). Subsequently, RNA-Seq was used to follow the changes in the uptake mechanism used to internalize s-GO flakes over time. Regardless of lateral dimensions, both types of GO were found to interact with the plasma membrane and to be internalized by a panel of cell lines studied. However, s-GO was internalized mainly via macropinocytosis while us-GO was mainly internalized via clathrin- and caveolae-mediated endocytosis. Importantly, we report the shift from macropinocytosis to clathrin-dependent endocytosis in the uptake of s-GO at 24 h, mediated by upregulation of mTORC1/2 pathway. Finally, we show that both s-GO and us-GO terminate in lysosomal compartments for up to 48 h. Our results offer an insight into the mechanism of interaction of GO with non-phagocytic cell lines over time that can be exploited for the design of biomedically-applicable 2D transport systems.
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Epidemiological evidence suggests non-steroidal anti-inflammatory drugs reduce the risk of Alzheimer's disease. However, clinical trials have found no evidence of non-steroidal anti-inflammatory drug efficacy. This incongruence may be due to the wrong non-steroidal anti-inflammatory drugs being tested in robust clinical trials or the epidemiological findings being caused by confounding factors. Therefore, this study used logistic regression and the innovative approach of negative binomial generalized linear mixed modelling to investigate both prevalence and cognitive decline, respectively, in the Alzheimer's Disease Neuroimaging dataset for each commonly used non-steroidal anti-inflammatory drug and paracetamol. Use of most non-steroidal anti-inflammatories was associated with reduced Alzheimer's disease prevalence yet no effect on cognitive decline was observed. Paracetamol had a similar effect on prevalence to these non-steroidal anti-inflammatory drugs suggesting this association is independent of the anti-inflammatory effects and that previous results may be due to spurious associations. Interestingly, diclofenac use was significantly associated with both reduce incidence and slower cognitive decline warranting further research into the potential therapeutic effects of diclofenac in Alzheimer's disease.
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During recovery, stroke patients are at risk of developing long-term complications that impact quality of life, including changes in body weight and composition, depression and anxiety, as well as an increased risk of subsequent vascular events. The aetiologies and time-course of these post-stroke complications have not been extensively studied and are poorly understood. Therefore, we assessed long-term changes in body composition, metabolic markers and behaviour after middle cerebral artery occlusion in mice. These outcomes were also studied in the context of obesity, a common stroke co-morbidity proposed to protect against post-stroke weight loss in patients. We found that stroke induced long-term changes in body composition, characterised by a sustained loss of fat mass with a recovery of lean weight loss. These global changes in response to stroke were accompanied by an altered lipid profile (increased plasma free fatty acids and triglycerides) and increased adipokine release at 60 days. After stroke, the liver also showed histological changes indicative of liver damage and a decrease in plasma alanine aminotransferase (ALT) was observed. Stroke induced depression and anxiety-like behaviours in mice, illustrated by deficits in exploration, nest building and burrowing behaviours. When initial infarct volumes were matched between mice with and without comorbid obesity, these outcomes were not drastically altered. Overall, we found that stroke induced long-term changes in depressive/anxiety-like behaviours, and changes in plasma lipids, adipokines and the liver that may impact negatively on future vascular health.
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Composición Corporal , Metabolismo de los Lípidos , Hígado/metabolismo , Accidente Cerebrovascular/metabolismo , Animales , Modelos Animales de Enfermedad , Infarto de la Arteria Cerebral Media/metabolismo , Masculino , Ratones Endogámicos C57BLRESUMEN
Interleukin (IL)-1 family cytokines potently regulate inflammation, with the majority of the IL-1 family proteins being secreted from immune cells via unconventional pathways. In many cases, secretion of IL-1 cytokines appears to be closely coupled to cell death, yet the secretory mechanisms involved remain poorly understood. Here, we studied the secretion of the three best-characterized members of the IL-1 superfamily, IL-1α, IL-1ß, and IL-18, in a range of conditions and cell types, including murine bone marrow-derived and peritoneal macrophages, human monocyte-derived macrophages, HeLa cells, and mouse embryonic fibroblasts. We discovered that IL-1ß and IL-18 share a common secretory pathway that depends upon membrane permeability and can operate in the absence of complete cell lysis and cell death. We also found that the pathway regulating the trafficking of IL-1α is distinct from the pathway regulating IL-1ß and IL-18. Although the release of IL-1α could also be dissociated from cell death, it was independent of the effects of the membrane-stabilizing agent punicalagin, which inhibited both IL-1ß and IL-18 release. These results reveal that in addition to their role as danger signals released from dead cells, IL-1 family cytokines can be secreted in the absence of cell death. We propose that models used in the study of IL-1 release should be considered context-dependently.
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Células de la Médula Ósea/metabolismo , Interleucina-18/metabolismo , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Macrófagos Peritoneales/metabolismo , Animales , Células de la Médula Ósea/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Células HeLa , Humanos , Taninos Hidrolizables/farmacología , Macrófagos Peritoneales/citología , Ratones , Transporte de Proteínas/efectos de los fármacosRESUMEN
Graphene oxide (GO), an oxidized form of graphene, has potential applications in biomedical research. However, how GO interacts with biological systems, including the innate immune system, is poorly understood. Here, we elucidate the effects of GO sheets on macrophages, identifying distinctive effects of GO on the inflammatory phenotype. Small, thin (s)-GO dose-dependently inhibited release of interleukin (IL)-1ß and IL-6 but not tumor necrosis factor α. NLRP3 inflammasome and caspase-1 activation was not affected. The effect of s-GO was pretranslational, as s-GO blocked Toll-like receptor 4-dependent expression of Il1b and Il6 but not Nlrp3 or Tnf mRNA transcripts. s-GO was internalized by immortalized bone-marrow-derived macrophages, suggesting a potential intracellular action. Uptake of polystyrene beads with similar lateral dimensions and surface charge did not phenocopy the effects of s-GO, suggesting that s-GO-mediated inhibition of interleukin expression was not simply due to particle phagocytosis. RNA-Seq analysis established that s-GO had profound effects on the immunometabolism of the cells, leading to activation of the transcription factor nuclear factor erythroid 2-related factor 2, which inhibited expression of cytokines such as IL-1ß and IL-6. Thus, we have identified immunometabolic effects of GO that reveal another dimension to its effects on cells. These findings suggest that s-GO may be used as a valuable tool to generate further insights into inflammatory mechanisms and indicate its potential applications in biomedicine.
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Citocinas/metabolismo , Grafito/química , Factor 2 Relacionado con NF-E2/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Inflamasomas/inmunología , Inflamasomas/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Neuroglía/metabolismo , ARN Mensajero/metabolismo , Receptor Toll-Like 4/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Intracerebral haemorrhage (ICH) is a devastating condition with limited treatment options, and current understanding of pathophysiology is incomplete. Spontaneous cerebral bleeding is a characteristic of the human condition that has proven difficult to recapitulate in existing pre-clinical rodent models. Zebrafish larvae are frequently used as vertebrate disease models and are associated with several advantages, including high fecundity, optical translucency and non-protected status prior to 5 days post-fertilisation. Furthermore, other groups have shown that zebrafish larvae can exhibit spontaneous ICH. The aim of this study was to investigate whether such models can be utilised to study the pathological consequences of bleeding in the brain, in the context of pre-clinical ICH research. Here, we compared existing genetic (bubblehead) and chemically inducible (atorvastatin) zebrafish larval models of spontaneous ICH and studied the subsequent disease processes. Through live, non-invasive imaging of transgenic fluorescent reporter lines and behavioural assessment we quantified brain injury, locomotor function and neuroinflammation following ICH. We show that ICH in both zebrafish larval models is comparable in timing, frequency and location. ICH results in increased brain cell death and a persistent locomotor deficit. Additionally, in haemorrhaged larvae we observed a significant increase in macrophage recruitment to the site of injury. Live in vivo imaging allowed us to track active macrophage-based phagocytosis of dying brain cells 24 hours after haemorrhage. Morphological analyses and quantification indicated that an increase in overall macrophage activation occurs in the haemorrhaged brain. Our study shows that in zebrafish larvae, bleeding in the brain induces quantifiable phenotypic outcomes that mimic key features of human ICH. We hope that this methodology will enable the pre-clinical ICH community to adopt the zebrafish larval model as an alternative to rodents, supporting future high throughput drug screening and as a complementary approach to elucidating crucial mechanisms associated with ICH pathophysiology.
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Restoration of cerebral blood flow (CBF) and upregulation of angiogenesis are crucial for brain repair and functional recovery after cerebral ischaemia. Pentraxin 3 (PTX3) is a key regulator of angiogenesis and is emerging as a promising target for cerebrovascular repair after stroke. Here, we investigated for the first time the role of PTX3 in long-term CBF, angiogenesis, and neuronal viability after ischaemic stroke induced by transient middle cerebral artery occlusion (MCAo). Lack of PTX3 had no effect on early brain damage, but significantly impaired restoration of CBF, 14 and 28 days after MCAo, compared to wild-type (WT) mice. Immunohistochemical analysis revealed that PTX3 KO mice have significantly greater neuronal loss, significantly decreased vessel diameter, vessel proliferation, vascular density, and reactive astrocytes and decreased expression of vascular endothelial growth factor receptor 2 (VEGR2), vascular extracellular matrix (ECM)-proteins (collagen IV, laminin), and integrin-ß, in the ipsilateral (stroke) hemisphere compared to WT mice, 28 days after MCAo. Therefore, PTX3 promotes sustained long-term recovery of CBF, angiogenesis, and neuronal viability after cerebral ischaemia. Collectively, these findings demonstrate the potential and clinical relevance of PTX3 as a promising therapeutic target, providing sustained long-term post-stroke neurovascular repair and reducing the loss of neurons. KEY MESSAGES: Pentraxin 3 (PTX3) is a key regulator of angiogenesis and is emerging as a promising target for cerebrovascular repair after stroke. Restoration of cerebral blood flow (CBF) and angiogenesis are crucial for brain repair and functional recovery after cerebral ischaemia. PTX3 promotes sustained long-term recovery of CBF, angiogenesis, and neuronal viability after cerebral ischaemia.
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Proteína C-Reactiva/fisiología , Circulación Cerebrovascular , Infarto de la Arteria Cerebral Media/fisiopatología , Neovascularización Fisiológica , Componente Amiloide P Sérico/fisiología , Animales , Encéfalo/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/fisiologíaRESUMEN
The interleukin-1 (IL-1) receptor and ligand families are components of the immune system. Knowledge of their evolutionary history is essential to understand their function. Using chromosomal anatomy and sequence similarity, we show that IL-1 receptor family members are related and nine members are likely formed from duplication and modification of a proto-IL-1R1 receptor. The IL-1 ligands have a different evolutionary history. The first proto-IL-1ß gene coincided with proto-IL-1R1 and duplication events resulted in the majority of IL-1 ligand family members. However, large evolutionary distances are observed for IL-1α, IL-18 and IL-33 proteins. Further analysis show that IL-33 and IL-18 have poor sequence similarity and no chromosomal evidence of common ancestry with the IL-1ß cluster and therefore should not be included in the IL-1 ligand ancestral family. IL-1α formed from the duplication of IL-1ß, and moonlighting functions of pro-IL-1α acted as divergent selection pressures for the observed sequence dissimilarity.
Asunto(s)
Evolución Molecular , Interleucina-1beta/genética , Animales , Humanos , Interleucina-18/genética , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Interleucina-33/genética , Interleucina-33/metabolismo , Familia de Multigenes , Filogenia , Receptores Tipo I de Interleucina-1/genética , Receptores Tipo I de Interleucina-1/metabolismo , Vertebrados/clasificación , Vertebrados/genéticaRESUMEN
NLRP3 is a receptor important for host responses to infection, yet is also known to contribute to devastating diseases such as Alzheimer's disease, diabetes, atherosclerosis, and others, making inhibitors for NLRP3 sought after. One of the inhibitors currently in use is 2-aminoethoxy diphenylborinate (2APB). Unfortunately, in addition to inhibiting NLRP3, 2APB also displays non-selective effects on cellular Ca2+ homeostasis. Here, we use 2APB as a chemical scaffold to build a series of inhibitors, the NBC series, which inhibit the NLRP3 inflammasome in vitro and in vivo without affecting Ca2+ homeostasis. The core chemical insight of this work is that the oxazaborine ring is a critical feature of the NBC series, and the main biological insight the use of NBC inhibitors led to was that NLRP3 inflammasome activation was independent of Ca2+. The NBC compounds represent useful tools to dissect NLRP3 function, and may lead to oxazaborine ring-containing therapeutics.