RESUMEN
Mankind's quest for a manned mission to Mars is placing increased emphasis on the development of innovative radio-protective countermeasures for long-term space travel. Hibernation confers radio-protective effects in hibernating animals, and this has led to the investigation of synthetic torpor to mitigate the deleterious effects of chronic low-dose-rate radiation exposure. Here we describe an induced torpor model we developed using the zebrafish. We explored the effects of radiation exposure on this model with a focus on the liver. Transcriptomic and behavioural analyses were performed. Radiation exposure resulted in transcriptomic perturbations in lipid metabolism and absorption, wound healing, immune response, and fibrogenic pathways. Induced torpor reduced metabolism and increased pro-survival, anti-apoptotic, and DNA repair pathways. Coupled with radiation exposure, induced torpor led to a stress response but also revealed maintenance of DNA repair mechanisms, pro-survival and anti-apoptotic signals. To further characterise our model of induced torpor, the zebrafish model was compared with hepatic transcriptomic data from hibernating grizzly bears (Ursus arctos horribilis) and active controls revealing conserved responses in gene expression associated with anti-apoptotic processes, DNA damage repair, cell survival, proliferation, and antioxidant response. Similarly, the radiation group was compared with space-flown mice revealing shared changes in lipid metabolism.
Asunto(s)
Hibernación , Exposición a la Radiación , Letargo , Animales , Ratones , Pez Cebra/genética , Hígado , Hibernación/fisiología , Letargo/fisiologíaRESUMEN
The development of the Artemis programme with the goal of returning to the moon is spurring technology advances that will eventually take humans to Mars and herald a new era of interplanetary space travel. However, long-term space travel poses unique challenges including exposure to ionising radiation from galactic cosmic rays and potential solar particle events, exposure to microgravity and specific nutritional challenges arising from earth independent exploration. Ionising radiation is one of the major obstacles facing future space travel as it can generate oxidative stress and directly damage cellular structures such as DNA, in turn causing genomic instability, telomere shortening, extracellular-matrix remodelling and persistent inflammation. In the gastrointestinal tract (GIT) this can lead to leaky gut syndrome, perforations and motility issues, which impact GIT functionality and affect nutritional status. While current countermeasures such as shielding from the spacecraft can attenuate harmful biological effects, they produce harmful secondary particles that contribute to radiation exposure. We hypothesised that induction of a torpor-like state would confer a radioprotective effect given the evidence that hibernation extends survival times in irradiated squirrels compared to active controls. To test this hypothesis, a torpor-like state was induced in zebrafish using melatonin treatment and reduced temperature, and radiation exposure was administered twice over the course of 10 days. The protective effects of induced-torpor were assessed via RNA sequencing and qPCR of mRNA extracted from the GIT. Pathway and network analysis were performed on the transcriptomic data to characterise the genomic signatures in radiation, torpor and torpor + radiation groups. Phenotypic analyses revealed that melatonin and reduced temperature successfully induced a torpor-like state in zebrafish as shown by decreased metabolism and activity levels. Genomic analyses indicated that low dose radiation caused DNA damage and oxidative stress triggering a stress response, including steroidal signalling and changes to metabolism, and cell cycle arrest. Torpor attenuated the stress response through an increase in pro-survival signals, reduced oxidative stress via the oxygen effect and detection and removal of misfolded proteins. This proof-of-concept model provides compelling initial evidence for utilizing an induced torpor-like state as a potential countermeasure for radiation exposure.
Asunto(s)
Exposición a la Radiación , Letargo/fisiología , Pez Cebra/fisiología , Animales , Ritmo Circadiano/genética , Ritmo Circadiano/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Degradación Asociada con el Retículo Endoplásmico/efectos de la radiación , Regulación de la Expresión Génica/efectos de la radiación , Redes Reguladoras de Genes/efectos de la radiación , Melatonina/farmacología , Modelos Animales , Fosforilación Oxidativa/efectos de la radiación , Reproducibilidad de los Resultados , Estrés Fisiológico/genética , Estrés Fisiológico/efectos de la radiación , Análisis de Supervivencia , Temperatura , Transcriptoma/genética , Transcriptoma/efectos de la radiación , Pez Cebra/genéticaRESUMEN
Chronic rhinosinusitis with nasal polyps (CRSwNP) is an inflammatory disease with an unknown etiology. Recent studies have implicated the complement system as a potential modulator of disease immunopathology. We performed proteomic pathway enrichment analysis of differentially increased proteins, and found an enrichment of complement cascade pathways in the nasal mucus of individuals with CRSwNP as compared to control subjects. Sinonasal mucus levels of complement 3 (C3) correlated with worse subjective disease severity, whereas no significant difference in systemic C3 levels could be determined in plasma samples. Given that human sinonasal epithelial cells were the predominate sinonasal source of C3 and complement anaphylatoxin 3a (C3a) staining, we focused on their role in in vitro studies. Baseline intracellular C3 levels were higher in CRSwNP cells, and following exposure to Aspergillus fumigatus (Af) extract, they released significantly more C3 and C3a. Inhibition of complement 3a receptor (C3aR) signaling led to a decrease in Af-induced C3 and C3a release, both in vitro and in vivo. Finally, we found in vivo that C3aR deficiency or inhibition significantly reduced inflammation and CRS development in a mouse model of Af-induced CRS. These findings demonstrate that local sinonasal complement activation correlates with subjective disease severity, and that local C3aR antagonism significantly ameliorates Af-induced CRS in a rodent model.
Asunto(s)
Receptores de Complemento/antagonistas & inhibidores , Rinosporidiosis/tratamiento farmacológico , Rinosporidiosis/inmunología , Animales , Aspergillus fumigatus/patogenicidad , Línea Celular , Enfermedad Crónica , Complemento C3/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Ratones , Ratones Endogámicos BALB C , Mucosa Nasal/efectos de los fármacos , Mucosa Nasal/inmunología , Pólipos Nasales/tratamiento farmacológico , Pólipos Nasales/inmunología , Proteoma/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunologíaRESUMEN
RATIONALE: Patients with chronic rhinosinusitis with nasal polyps (CRSwNP) have been shown to be vitamin D3 (VD3) deficient, which is associated with more severe disease and increased polyp size. To gain mechanistic insights into these observational studies, we examined the impact of VD3 deficiency on inflammation and VD3 metabolism in an Aspergillus fumigatus (Af) mouse model of chronic rhinosinusitis (Af-CRS). METHODS: Balb/c mice were fed control or VD3 deficient diet for 4 weeks. Mice were then sensitized with intraperitoneal Af, and one week later given Af intranasally every three days for four weeks while being maintained on control or VD3 deficient diet. Airway function, sinonasal immune cell infiltrate and sinonasal VD3 metabolism profiles were then examined. RESULTS: Mice with VD3 deficiency had increased Penh and sRaw values as compared to controls as well as exacerbated changes in sRaw when coupled with Af-CRS. As compared to controls, VD3 deficient and Af-CRS mice had reduced sinonasal 1α-hydroxylase and the active VD3 metabolite, 1,25(OH)2D3. Differential analysis of nasal lavage samples showed that VD3 deficiency alone and in combination with Af-CRS profoundly upregulated eosinophil, neutrophil and lymphocyte numbers. VD3 deficiency exacerbated increases in monocyte-derived dendritic cell (DC) associated with Af-CRS. Conversely, T-regulatory cells were decreased in both Af-CRS mice and VD3 deficient mice, though coupling VD3 deficiency with Af-CRS did not exacerbate CD4 or T-regulatory cells numbers. Lastly, VD3 deficiency had a modifying or exacerbating impact on nasal lavage levels of IFN-γ, IL-6, IL-10 and TNF-α, but had no impact on IL-17A. CONCLUSIONS: VD3 deficiency causes changes in sinonasal immunity, which in many ways mirrors the changes observed in Af-CRS mice, while selectively exacerbating inflammation. Furthermore, both VD3 deficiency and Af-CRS were associated with altered sinonasal VD3 metabolism causing reductions in local levels of the active VD3 metabolite, 1,25(OH)2D3, even with adequate circulating levels.
Asunto(s)
Colecalciferol/metabolismo , Inflamación/metabolismo , Pólipos Nasales/metabolismo , Rinitis/metabolismo , Sinusitis/metabolismo , Animales , Aspergillus fumigatus/patogenicidad , Recuento de Células Sanguíneas , Dieta , Suplementos Dietéticos , Modelos Animales de Enfermedad , Eosinófilos/patología , Humanos , Inflamación/dietoterapia , Inflamación/patología , Linfocitos/patología , Ratones , Lavado Nasal (Proceso) , Pólipos Nasales/dietoterapia , Pólipos Nasales/patología , Neutrófilos/patología , Rinitis/dietoterapia , Rinitis/patología , Sinusitis/dietoterapia , Sinusitis/patología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patología , Deficiencia de Vitamina D/metabolismo , Deficiencia de Vitamina D/patologíaRESUMEN
PURPOSE: Morphologic and genetic evidence exists that an overactive complement system driven by the complement alternative pathway (AP) is involved in pathogenesis of age-related macular degeneration (AMD). Smoking is the only modifiable risk factor for AMD. As we have shown that smoke-related ocular pathology can be prevented in mice that lack an essential activator of AP, we ask here whether this pathology can be reversed by increasing inhibition in AP. METHODS: Mice were exposed to either cigarette smoke (CS) or filtered air (6 hours/day, 5 days/week, 6 months). Smoke-exposed animals were then treated with the AP inhibitor (CR2-fH) or vehicle control (PBS) for the following 3 months. Spatial frequency and contrast sensitivity were assessed by optokinetic response paradigms at 6 and 9 months; additional readouts included assessment of retinal morphology by electron microscopy (EM) and gene expression analysis by quantitative RT-PCR. RESULTS: The CS mice treated with CR2-fH showed significant improvement in contrast threshold compared to PBS-treated mice, whereas spatial frequency was unaffected by CS or pharmacologic intervention. Treatment with CR2-fH in CS animals reversed thinning of the retina observed in PBS-treated mice as analyzed by spectral-domain optical coherence tomography, and reversed most morphologic changes in RPE and Bruch's membrane seen in CS animals by EM. CONCLUSIONS: Taken together, these findings suggest that AP inhibitors not only prevent, but have the potential to accelerate the clearance of complement-mediated ocular injury. Improving our understanding of the regulation of the AP is paramount to developing novel treatment approaches for AMD.
Asunto(s)
Vía Alternativa del Complemento/genética , Regulación de la Expresión Génica , Degeneración Macular/genética , Proteínas Recombinantes de Fusión/uso terapéutico , Recuperación de la Función , Retina/fisiopatología , Humo/efectos adversos , Animales , Vía Alternativa del Complemento/efectos de los fármacos , ADN/genética , Modelos Animales de Enfermedad , Electrorretinografía , Degeneración Macular/inducido químicamente , Degeneración Macular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Reacción en Cadena en Tiempo Real de la Polimerasa , Retina/metabolismo , Retina/ultraestructura , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/ultraestructura , Agudeza Visual/fisiologíaRESUMEN
The etiology of mitochondrial disease is poorly understood. Furthermore, treatment options are limited, and diagnostic methods often lack the sensitivity to detect disease in its early stages. Disrupted oxidative phosphorylation (OXPHOS) that inhibits ATP production is a common phenotype of mitochondrial disorders that can be induced in zebrafish by exposure to 2,4-dinitrophenol (DNP), a FDA-banned weight-loss agent and EPA-regulated environmental toxicant, traditionally used in research labs as an uncoupler of OXPHOS. Despite the DNP-induced OXPHOS inhibition we observed using in vivo respirometry, the development of the DNP-treated and control zebrafish were largely similar during the first half of embryogenesis. During this period, DNP-treated embryos induced gene expression of mitochondrial and nuclear genes that stimulated the production of new mitochondria and increased glycolysis to yield normal levels of ATP. DNP-treated embryos were incapable of sustaining this mitochondrial biogenic response past mid-embryogenesis, as shown by significantly lowered ATP production and ATP levels, decreased gene expression, and the onset of developmental defects. Examining neural tissues commonly affected by mitochondrial disease, we found that DNP exposure also inhibited motor neuron axon arbor outgrowth and the proper formation of the retina. We observed and quantified the molecular and physiological progression of mitochondrial dysfunction during development with this new model of OXPHOS dysfunction, which has great potential for use in diagnostics and therapies for mitochondrial disease.
Asunto(s)
Desarrollo Embrionario/genética , Metabolismo Energético/genética , Mitocondrias/genética , Enfermedades Mitocondriales/genética , 2,4-Dinitrofenol/toxicidad , Adenosina Trifosfato/biosíntesis , Animales , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Mitocondrias/metabolismo , Mitocondrias/patología , Enfermedades Mitocondriales/inducido químicamente , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Fosforilación Oxidativa/efectos de los fármacos , Retina/metabolismo , Retina/patología , Pez CebraRESUMEN
BACKGROUND: Age-related macular degeneration (AMD), a complex disease involving genetic variants and environmental insults, is among the leading causes of blindness in Western populations. Genetic and histologic evidence implicate the complement system in AMD pathogenesis; and smoking is the major environmental risk factor associated with increased disease risk. Although previous studies have demonstrated that cigarette smoke exposure (CE) causes retinal pigment epithelium (RPE) defects in mice, and smoking leads to complement activation in patients, it is unknown whether complement activation is causative in the development of CE pathology; and if so, which complement pathway is required. METHODS: Mice were exposed to cigarette smoke or clean, filtered air for 6 months. The effects of CE were analyzed in wildtype (WT) mice or mice without a functional complement alternative pathway (AP; CFB(-/-) ) using molecular, histological, electrophysiological, and behavioral outcomes. RESULTS: CE in WT mice exhibited a significant reduction in function of both rods and cones as determined by electroretinography and contrast sensitivity measurements, concomitant with a thinning of the nuclear layers as measured by SD-OCT imaging and histology. Gene expression analyses suggested that alterations in both photoreceptors and RPE/choroid might contribute to the observed loss of function, and visualization of complement C3d deposition implies the RPE/Bruch's membrane (BrM) complex as the target of AP activity. RPE/BrM alterations include an increase in mitochondrial size concomitant with an apical shift in mitochondrial distribution within the RPE and a thickening of BrM. CFB(-/-) mice were protected from developing these CE-mediated alterations. CONCLUSIONS: Taken together, these findings provide clear evidence that ocular pathology generated in CE mice is dependent on complement activation and requires the AP. Identifying animal models with RPE/BrM damage and verifying which aspects of pathology are dependent upon complement activation is essential for developing novel complement-based treatment approaches for the treatment of AMD.
Asunto(s)
Proteínas del Sistema Complemento/metabolismo , Oftalmopatías/patología , Oftalmopatías/fisiopatología , Transducción de Señal/efectos de los fármacos , Humo/efectos adversos , Animales , Complemento C3/metabolismo , Proteínas del Sistema Complemento/deficiencia , Proteínas del Sistema Complemento/genética , Oftalmopatías/inducido químicamente , Oftalmopatías/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Lectinas/metabolismo , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Factores de Tiempo , Nicotiana/efectos adversosRESUMEN
BACKGROUND: Brain death (BD) can immunologically prime the donor organ and is thought to lead to exacerbated ischemia/reperfusion injury after transplantation. Using a newly developed mouse model of BD, we investigated the effect of donor BD on posttransplantation cardiac ischemia/reperfusion injury. We further investigated the therapeutic effect of a targeted complement inhibitor in recipients of BD donor hearts and addressed the clinical relevance of these studies by analyzing human heart biopsies from BD and domino (living) donors. METHODS AND RESULTS: Hearts from living or BD donor C57BL/6 mice were transplanted into C57BL/6 or BALB/c recipients. Recipient mice were treated with the complement inhibitor CR2-Crry or vehicle control (n=6). Isografts were analyzed 48 hours after transplantation for injury, inflammation, and complement deposition, and allografts were monitored for graft survival. Human cardiac biopsies were analyzed for complement deposition and inflammatory cell infiltration. In the murine model, donor BD exacerbated ischemia/reperfusion injury and graft rejection, as demonstrated by increased myocardial injury, serum cardiac troponin, cellular infiltration, complement deposition, inflammatory chemokine and cytokine levels, and by decreased graft survival. CR2-Crry treatment of recipients significantly reduced all measured outcomes in grafts from both BD and living donors compared with controls. Analysis of human samples documented the relevance of our experimental findings and revealed exacerbated complement deposition and inflammation in grafts from BD donors compared with grafts from living donors. CONCLUSIONS: BD exacerbates posttransplantation cardiac ischemia/reperfusion injury in mice and humans and decreases survival of mouse allografts. Furthermore, targeted complement inhibition in recipient mice ameliorates BD-exacerbated ischemia/reperfusion injury.
Asunto(s)
Muerte Encefálica/fisiopatología , Proteínas del Sistema Complemento/fisiología , Trasplante de Corazón/fisiología , Daño por Reperfusión/fisiopatología , Donantes de Tejidos , Adolescente , Adulto , Animales , Biopsia , Citocinas/metabolismo , Femenino , Corazón/efectos de los fármacos , Trasplante de Corazón/mortalidad , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Persona de Mediana Edad , Modelos Animales , Miocardio/metabolismo , Miocardio/patología , Proteínas Recombinantes de Fusión/farmacología , Estudios Retrospectivos , Tasa de Supervivencia , Adulto JovenRESUMEN
Interactions between multiple predator species are frequent in natural communities and can have important implications for shared prey survival. Predator density may be an important component of these interactions between predator species, as the frequency of interactions between species is largely determined by species density. Here we experimentally examine the importance of predator density for interactions between predator species and subsequent impacts on prey. We show that aggressive interactions between the predatory shore crabs Carcinus maenas and Hemigrapsus sanguineus increased with predator density, yet did not increase as fast as negative interactions between conspecifics. At low density, interactions between conspecific and heterospecific predators had similar inhibitory impacts on predator function, whereas conspecific interference was greater than interference from heterospecifics at high predator density. Thus the impact of conspecific interference at high predator density was sufficient in itself that interactions with a second predator species had no additional impact on per capita predation. Spatial and temporal variability in predator density is a ubiquitous characteristic of natural systems that should be considered in studies of multiple predator species.