RESUMO
Autophagy is an evolutionarily conserved process that plays a key role in the maintenance of overall cellular health. While it has been suggested that autophagy may elicit cardioprotective and pro-survival modulating functions, excessive activation of autophagy can also be detrimental. In this regard, the zebrafish is considered a hallmark model for vertebrate regeneration, since contrary to adult mammals, it is able to faithfully regenerate cardiac tissue. Interestingly, the role that autophagy may play in zebrafish heart regeneration has not been studied yet. In the present work, we hypothesize that, in the context of a well-established injury model of ventricular apex resection, autophagy plays a critical role during cardiac regeneration and its regulation can directly affect the zebrafish regenerative potential. We studied the autophagy events occurring upon injury using electron microscopy, in vivo tracking of autophagy markers, and protein analysis. Additionally, using pharmacological tools, we investigated how rapamycin, an inducer of autophagy, affects regeneration relevant processes. Our results show that a tightly regulated autophagic response is triggered upon injury and during the early stages of the regeneration process. Furthermore, treatment with rapamycin caused an impairment in the cardiac regeneration outcome. These findings are reminiscent of the pathophysiological description of an injured human heart and hence put forward the zebrafish as a model to study the poorly understood double-sword effect that autophagy has in cardiac homeostasis.
Assuntos
Autofagia/fisiologia , Coração/fisiologia , Regeneração/fisiologia , Animais , Proliferação de Células/fisiologia , Ventrículos do Coração/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
The study of spinal cord regeneration using diverse animal models, which range from null to robust regenerative capabilities, is imperative for understanding how regeneration evolved and, eventually, to treat spinal cord injury and paralysis in humans. In this study, we used electroablation to fully transect the spinal cord of zebrafish larvae (3 days postfertilization) and examined regeneration of the tissue over time. We used transgenic lines to follow immune cells, oligodendrocytes, and neurons in vivo during the entire regenerative process. We observed that immune cells are recruited to the injury site, oligodendrocytes progenitor cells (olig2-expressing cells) invade, and axons cross the gap generated upon damage from anterior to reinnervate caudal structures. Together with the recovery of cell types and structures, a complete reversal of paralysis was observed in the lesioned larvae indicating functional regeneration. Finally, using transplantation to obtain mosaic larvae with single-labeled neurons, we show that severed spinal axons exhibited varying regenerative capabilities and plasticity depending on their original dorsoventral position in the spinal cord.
Assuntos
Neurogênese/fisiologia , Regeneração da Medula Espinal/fisiologia , Animais , Larva , Peixe-ZebraRESUMO
The role of macrophages during regeneration in zebrafish has been well-documented. Nevertheless, new evidence indicates that zebrafish macrophages are a heterogeneous population of cells, and that they can play different roles during immune responses and in tissue restoration after damage and infection. In this work, we first aimed to classify zebrafish macrophages according to their distribution in the larva during homeostasis and after tissue damage, distinguishing peripheral, and hematopoietic tissue resident macrophages. We discovered differences between the migratory behavior of these two macrophage populations both before and after tissue damage, triggered by the amputation of the tail fin. Further, we found a specific role for peripheral tissue-resident macrophages, and we propose that these cells contribute to tail fin regeneration by down-regulating inflammatory mediators such as interleukin-1b (il1b) and by diminishing reactive oxygen species (ROS) in the damage site. Our work suggests that specific macrophage populations recruited after tissue damage in zebrafish larvae can display different functions during both inflammation and tissue regeneration.
Assuntos
Inflamação/imunologia , Macrófagos/fisiologia , Regeneração/fisiologia , Animais , Homeostase , Interleucina-1beta/fisiologia , Proteínas Tirosina Quinases/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Receptores Proteína Tirosina Quinases , Peixe-Zebra , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Neutrophils are a major component of the innate immune response and the most abundant circulating cell type in humans and zebrafish. The CXCL12/CXCR4 ligand receptor pair plays a key role in neutrophil homeostasis, controlling definitive hematopoiesis and neutrophil release into circulation. Neutrophils overexpressing CXCR4 respond by migrating towards sources of CXCL12, which is abundant in hematopoietic tissues. However, the physiological role of CXCL12/CXCR4 signaling during inflammatory responses remains unknown. Here, we show that zebrafish mutants lacking functional CXCL12a or CXCR4b show disrupted granulopoiesis in the kidney and increased number of circulating neutrophils. Additionally, CXCL12a and CXCR4b mutants display exacerbated recruitment of neutrophils to wounds and not to infections, and migrating neutrophils to wounds show increased directionality. Our results show that CXCL12a/CXCR4b signaling antagonizes wound-induced inflammatory signals by retaining neutrophils in hematopoietic tissues as a part of a balance between both inflammatory and anti-inflammatory cues, whose dynamic levels control neutrophils complex migratory behavior.
Assuntos
Quimiocina CXCL12/imunologia , Hematopoese/imunologia , Neutrófilos/imunologia , Receptores CXCR4/imunologia , Proteínas de Peixe-Zebra/imunologia , Peixe-Zebra/imunologia , Animais , Animais Geneticamente Modificados , Células Cultivadas , Embrião não Mamífero/citologia , Embrião não Mamífero/imunologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Inflamação , Larva/imunologia , Larva/metabolismo , Neutrófilos/metabolismo , Neutrófilos/patologia , Peixe-Zebra/metabolismoRESUMO
Tolerogenic dendritic cells (TolDCs) are promising tools for therapy of autoimmune diseases, such as rheumatoid arthritis (RA). Here, we characterize monocyte-derived TolDCs from RA patients modulated with dexamethasone and activated with monophosphoryl lipid A (MPLA), referred to as MPLA-tDCs, in terms of gene expression, phenotype, cytokine profile, migratory properties, and T cell-stimulatory capacity in order to explore their suitability for cellular therapy. MPLA-tDCs derived from RA patients displayed an anti-inflammatory profile with reduced expression of co-stimulatory molecules and high IL-10/IL-12 ratio, but were capable of migrating toward the lymphoid chemokines CXCL12 and CCL19. These MPLA-tDCs induced hyporesponsiveness of autologous CD4+ T cells specific for synovial antigens in vitro. Global transcriptome analysis confirmed a unique transcriptional profile of MPLA-tDCs and revealed that RA-associated genes, which were upregulated in untreated DCs from RA patients, returned to expression levels of healthy donor-derived DCs after treatment with dexamethasone and MPLA. Thus, monocyte-derived DCs from RA patients have the capacity to develop tolerogenic features at transcriptional as well as at translational level, when modulated with dexamethasone and MPLA, overcoming disease-related effects. Furthermore, the ability of MPLA-tDCs to impair T cell responses to synovial antigens validates their potential as cellular treatment for RA.
RESUMO
In vertebrates, damage to mechanosensory hair cells elicits an inflammatory response, including rapid recruitment of macrophages and neutrophils. While hair cells in amniotes usually become permanently lost, they readily regenerate in lower vertebrates such as fish. Damage to hair cells of the fish lateral line is followed by inflammation and rapid regeneration; however the role of immune cells in this process remains unknown. Here, we show that recruited macrophages are required for normal regeneration of lateral line hair cells after copper damage. We found that genetic ablation or local ablation using clodronate liposomes of macrophages recruited to the site of injury, significantly delays hair cell regeneration. Neutrophils, on the other hand, are not needed for this process. We anticipate our results to be a starting point for a more detailed description of extrinsic signals important for regeneration of mechanosensory cells in vertebrates. J. Cell. Biochem. 117: 1880-1889, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Estruturas Animais/fisiologia , Cobre/toxicidade , Macrófagos/imunologia , Mecanotransdução Celular/imunologia , Neurônios Aferentes/imunologia , Regeneração/imunologia , Peixe-Zebra/imunologia , Animais , Neutrófilos/imunologiaRESUMO
Neuronal death in Parkinson's disease (PD) is often preceded by axodendritic tree retraction and loss of neuronal functionality. The presence of non-functional but live neurons opens therapeutic possibilities to recover functionality before clinical symptoms develop. Considering that iron accumulation and oxidative damage are conditions commonly found in PD, we tested the possible neuritogenic effects of iron chelators and antioxidant agents. We used three commercial chelators: DFO, deferiprone and 2.2'-dypyridyl, and three 8-hydroxyquinoline-based iron chelators: M30, 7MH and 7DH, and we evaluated their effects in vitro using a mesencephalic cell culture treated with the Parkinsonian toxin MPP+ and in vivo using the MPTP mouse model. All chelators tested promoted the emergence of new tyrosine hydroxylase (TH)-positive processes, increased axodendritic tree length and protected cells against lipoperoxidation. Chelator treatment resulted in the generation of processes containing the presynaptic marker synaptophysin. The antioxidants N-acetylcysteine and dymetylthiourea also enhanced axodendritic tree recovery in vitro, an indication that reducing oxidative tone fosters neuritogenesis in MPP+-damaged neurons. Oral administration to mice of the M30 chelator for 14 days after MPTP treatment resulted in increased TH- and GIRK2-positive nigra cells and nigrostriatal fibers. Our results support a role for oral iron chelators as good candidates for the early treatment of PD, at stages of the disease where there is axodendritic tree retraction without neuronal death.
Assuntos
Antioxidantes/farmacologia , Quelantes de Ferro/farmacologia , Intoxicação por MPTP/tratamento farmacológico , Fibras Nervosas/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/antagonistas & inibidores , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , 2,2'-Dipiridil/farmacologia , Animais , Deferiprona , Desferroxamina/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/agonistas , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/biossíntese , Hidroxiquinolinas/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Intoxicação por MPTP/metabolismo , Intoxicação por MPTP/patologia , Masculino , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/metabolismo , Mesencéfalo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fibras Nervosas/metabolismo , Fibras Nervosas/patologia , Neuritos/metabolismo , Neuritos/patologia , Cultura Primária de Células , Piridonas/farmacologia , Ratos , Ratos Sprague-Dawley , Sinaptofisina/agonistas , Sinaptofisina/biossíntese , Tirosina 3-Mono-Oxigenase/biossínteseRESUMO
IL-33 is a known member of the IL-1 cytokine superfamily classically named "atypical" due to its diverse functions. The receptor for this cytokine is the ST2 chain (or IL-1RL1), part of the IL-1R family, and the accessory chain IL-1R. ST2 can be found as both soluble and membrane-bound forms, property that explains, at least in part, its wide range of functions. IL-33 has increasingly gained our attention as a potential target to modulate immune responses. At the beginning, it was known as one of the participants during the development of allergic states and other Th2-mediated responses and it is now accepted that IL-33 contributes to Th1-driven pathologies as demonstrated in animal models of experimental autoimmune encephalomyelitis (EAE), collagen-induced arthritis, and trinitrobenzene sulfonic acid-induced experimental colitis, among others. Interestingly, current data are placing IL-33 as a novel regulator of immune tolerance by affecting regulatory T cells (Tregs); although the mechanism is not fully understood, it seems that dendritic cells and myeloid suppressor-derived cells may be cooperating in the generation and/or establishment of IL-33-mediated tolerance. Here, we review the most updated literature on IL-33, its role on T cell biology, and its impact in immune tolerance.
RESUMO
Interleukin-33 (IL-33) has been a focus of study because of its variety of functions shaping CD4(+) T-cell biology. In the present work, we evaluated the modulatory effect of IL-33 on suppressor cells in an in vivo transplantation model. C57BL/6 wild-type mice were grafted with syngeneic or allogeneic skin transplants and treated with exogenous IL-33 daily. After 10 days of treatment, we analysed draining lymph node cellularity and found in allogeneic animals an increment in myeloid-derived suppressor cells, which co-express MHC-II, and become enriched upon IL-33 treatment. In line with this observation, inducible nitric oxide synthase and arginase 1 expression were also increased in allogeneic animals upon IL-33 administration. In addition, IL-33 treatment up-regulated the number of Foxp3(+) regulatory T (Treg) cells in the allogeneic group, complementing the healthier integrity of the allografts and the increased allograft survival. Moreover, we demonstrate that IL-33 promotes CD4(+) T-cell expansion and conversion of CD4(+) Foxp3(-) T cells into CD4(+) Foxp3(+) Treg cells in the periphery. Lastly, the cytokine pattern of ex vivo-stimulated draining lymph nodes indicates that IL-33 dampens interferon-γ and IL-17 production, stimulating IL-10 secretion. Altogether, our work complements previous studies on the immune-modulatory activity of IL-33, showing that this cytokine affects myeloid-derived suppressor cells at the cell number and gene expression levels. More importantly, our research demonstrates for the first time that IL-33 allows for in vivo Foxp3(+) Treg cell conversion and favours an anti-inflammatory or tolerogenic state by skewing cytokine production. Therefore, our data suggest a potential use of IL-33 to prevent allograft rejection, bringing new therapeutics to the transplantation field.
Assuntos
Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Interleucinas/farmacologia , Transplante de Pele , Linfócitos T Reguladores/imunologia , Animais , Arginase/biossíntese , Diferenciação Celular/imunologia , Proliferação de Células , Fatores de Transcrição Forkhead/imunologia , Antígenos de Histocompatibilidade Classe II/biossíntese , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Interferon gama/biossíntese , Interleucina-10/biossíntese , Interleucina-10/metabolismo , Interleucina-17/biossíntese , Interleucina-33 , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/imunologia , Óxido Nítrico Sintase Tipo II/biossíntese , Pele/imunologia , Linfócitos T Reguladores/citologia , Células Th1/citologia , Células Th1/imunologia , Células Th17/citologia , Células Th17/imunologia , Transplante IsogênicoRESUMO
Retinoic acid (RA), a vitamin A metabolite, has been attributed to relevant functions in adaptive immunity. On T cells, the disruption on RA signaling alters both CD4+ and CD8+ T cells effector function. In this study, we evaluated the contribution of RA synthesis during the immune response using an in vivo skin transplantation model. Our data indicates that the frequency and number of cells containing an active retinaldehyde dehydrogenase (RALDH), a key enzyme for RA synthesis, is increased during skin transplant rejection. In addition, we found that the expression of the mRNA coding for the isoform RALDH2 is up-regulated on graft rejecting draining lymph nodes (dLNs) cells. Lastly, we observed that IFN-γ and IL-17 production by ex vivo re-stimulated dLNs cells is greatly increased during rejection, which it turns depends on RA synthesis, as shown in experiments using a specific RALDH inhibitor. Altogether, our data demonstrate that RA synthesis is incremented during the immune response against an allograft, and also indicates that the synthesis of RA is required for cytokine production by dLNs resident T cells.
Assuntos
Aloenxertos/imunologia , Citocinas/biossíntese , Rejeição de Enxerto/imunologia , Retinal Desidrogenase/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Aloenxertos/metabolismo , Animais , Ativação Enzimática , Expressão Gênica , Rejeição de Enxerto/genética , Camundongos , Modelos Animais , Retinal Desidrogenase/genética , Transplante de Pele , Transplante Homólogo , Tretinoína/metabolismoRESUMO
During allograft rejection, several immune cell types, including dendritic cells, CD4(+) and CD8(+) T cells among others, recirculate between the graft and the nearest draining lymph node, resulting in immunity against the 'foreign' tissue. Regulatory CD4(+) T cells are critical for controlling the magnitude of the immune response and may act to promote or maintain tolerance. They are characterized by the expression of CD25 and Foxp3, and more recently, Neuropilin-1 (Nrp1). The role of these suppressor cells during allograft rejection is not well understood. Our work shows that during graft rejection, there is an increase in the frequency of total CD4(+) T cells expressing Nrp1, but the expression of this molecule is downregulated in the regulatory CD4(+) T-cell compartment. Interestingly, the expression of the transcription factor Eos, which renders cell function stability, is also reduced. In adoptive transfer experiments, we observed that during allograft rejection: (i) natural regulatory CD4(+) T cells maintain high levels of Nrp1 expression, (ii) effector CD4(+) T cells (Nrp1(-)) become Nrp1(+)Eos(+) and (iii) the transfer of regulatory CD4(+) T cells (Nrp1(+)) can promote allograft survival, and also enhance the gain of Nrp1 and Eos on T-effector cells. Together, these data suggest that rejection occurs, at least in part, through the loss of Nrp1 expression on regulatory CD4(+) T cells, their stability or both. Additionally, the transfer of regulatory CD4(+) T cells (based on Nrp1 expression) permits the acceptance of the allograft, placing Nrp1 as a new target for immune therapy.
Assuntos
Aloenxertos/imunologia , Sobrevivência de Enxerto/imunologia , Neuropilina-1/metabolismo , Transplante de Pele , Linfócitos T Reguladores/imunologia , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA , Regulação para Baixo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Linfócitos T Reguladores/metabolismoRESUMO
In the immune system, Neuropilin-1 (Nrp1) is a molecule that plays an important role in establishing the immunological synapse between dendritic cells (DCs) and T cells. Recently, Nrp1 has been identified as a marker that seems to distinguish natural T regulatory (nTreg) cells, generated in the thymus, from inducible T regulatory (iTreg) cells raised in the periphery. Given the crucial role of both nTreg and iTreg cells in the generation and maintenance of immune tolerance, the ability to phenotypically identify each of these cell populations in vivo is needed to elucidate their biological properties. In turn, these properties have the potential to be developed for therapeutic use to promote immune tolerance. Here we describe the nature and functions of Nrp1, including its potential use as a therapeutic target in transplantation tolerance.
RESUMO
Centipedes are venomous arthropods responsible for a significant number of non-lethal human envenomations. Despite this, information about the composition and function of their venom contents is scarce. In this study, we have used a 'structure to function' proteomic approach combining two-dimensional chromatography (2D-LC), electrospray ionization quadrupole/time-of-flight mass spectrometry (ESI-Q-TOF/MS), N-terminal sequencing and similarity searching to better understand the complexities of the venoms from two Brazilian centipede species: Scolopendra viridicornis nigra and Scolopendra angulata. Comparisons between the LC profiles and the mass compositions of the venoms of the two species are provided. The observed molecular masses ranged from 3019.62 to 20996.94Da in S. viridicornis nigra (total: 62 molecular masses) and from 1304.73 to 22639.15Da in S. angulata (total: 65 molecular masses). Also, the N-termini of representatives of 10 protein/peptide families were successfully sequenced where nine of them showed no significant similarity to other protein sequences deposited in the Swiss-Prot database. A screening for insecto-toxic activities in fractions from S. viridicornis venom has also been performed. Six out of the 12 tested fractions were responsible for clear toxic effects in house flies. This work demonstrates that centipede venoms might be a neglected but important source of new bioactive compounds.
Assuntos
Venenos de Artrópodes/química , Artrópodes , Sequência de Aminoácidos , Animais , Venenos de Artrópodes/toxicidade , Cromatografia Líquida , Dípteros/efeitos dos fármacos , Feminino , Masculino , Dados de Sequência Molecular , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade , Testes de ToxicidadeRESUMO
Tetrodotoxin (TTX) is one of the most potent toxin already isolated, which occurs in a wide range of marine as well as terrestrial animals such as in newts and anurans. In this work, the occurrence of TTX and analogues was examined in three brachycephalid species: Brachycephalus ephippium, B. nodoterga and B. pernix using LC-FLD and LC-MS/MS. In toxicity assay (intra-peritonial injection in mice) B. nodoterga extracts were non-toxic, while B. pernix extract exhibit the highest toxicity among the studied species. Skin showed the highest toxic, followed by the liver. Retention time data in the LC-FLD system indicated the presence of TTX, 4-epiTTX, 4,9-anhydroTTX and TDA, SIM data confirmed the presence of these compounds and revealed other analogs such as 11-norTTX-6(S)-ol, 5-deoxyTTX, 11-deoxyTTX, 11-oxoTTX, 6-epiTTX. Two new components were also identified by mass spectrometry (348 and 330Da). These unknown compounds have daughter ions similar to TTX, suggesting new putative TTX analogues.
Assuntos
Venenos de Anfíbios/química , Anuros/fisiologia , Tetrodotoxina/análogos & derivados , Tetrodotoxina/isolamento & purificação , Venenos de Anfíbios/toxicidade , Animais , Relação Dose-Resposta a Droga , Camundongos , Estrutura Molecular , Tetrodotoxina/toxicidadeRESUMO
11-oxoTTX is an analogue 4-5 times more toxic than TTX itself, been rare even in marine animals. Two ions at m/z 320 and 336 corresponding to TTX and 11-oxoTTX (M+H(+)), respectively, were detected in the Brachycephalidae frog Brachycephalus ephippium extracts. The fragment ion pattern of 11-oxoTTX is similar to that TTX, although its possible to verify some specific fragments.
Assuntos
Venenos de Anfíbios/química , Anuros/metabolismo , Tetrodotoxina/análogos & derivados , Tetrodotoxina/análise , Extratos de Tecidos/química , Animais , Cromatografia Líquida de Alta Pressão , Estrutura Molecular , Padrões de Referência , Pele/química , Tetrodotoxina/química , Tetrodotoxina/toxicidadeRESUMO
Brachycephalus ephippium is a diurnal frog, that shows aposematic colouration and inhabits Atlantic forest leaf litter in south-eastern Brazil. The presence of tetrodotoxin (TTX) in the skin, liver and ovaries of B. ephippium was demonstrated. The skin (260 M.U./g) exhibited the highest toxicity followed by liver (177 M.U./g). TTX and its analogues, tetrodonic acid, 4-epitetrodotoxin and 4,9 anhydrotetrodotoxin were isolated and identified by HPLC followed by fluorimetric analysis. TTX and 11-nortetrodotoxin-6(S)-ol had their presence confirmed by mass spectrometry (MALDI-TOF). The results confirm Brachycephalidae as a fourth family of anurans containing TTX.