RESUMO
Dendritic cells (DCs) vaccine is a potential tool for oncoimmunotherapy. However, it is known that this therapeutic strategy has failed in solid tumors, making the development of immunoadjuvants highly relevant. Recently, we demonstrated that Phoneutria nigriventer spider venom (PnV) components are cytotoxic to glioblastoma (GB) and activate macrophages for an antitumor profile. However, the effects of these molecules on the adaptive immune response have not yet been evaluated. This work aimed to test PnV and its purified fractions in DCs in vitro. For this purpose, bone marrow precursors were collected from male C57BL6 mice, differentiated into DCs and treated with venom or PnV-isolated fractions (F1-molecules < 3 kDa, F2-3 to 10 kDa and F3->10 kDa), with or without costimulation with human GB lysate. The results showed that mainly F1 was able to activate DCs, increasing the activation-dependent surface marker (CD86) and cytokine release (IL-1ß, TNF-α), in addition to inducing a typical morphology of mature DCs. From the F1 purification, a molecule named LW9 was the most effective, and mass spectrometry showed it to be a peptide. The present findings suggest that this molecule could be an immunoadjuvant with possible application in DC vaccines for the treatment of GB.
Assuntos
Glioblastoma , Venenos de Aranha , Camundongos , Masculino , Humanos , Animais , Glioblastoma/terapia , Venenos de Aranha/farmacologia , Camundongos Endogâmicos C57BL , Diferenciação Celular , Células DendríticasRESUMO
The pleiotropic role of the major histocompatibility complex class I (MHC-I) reflects the close association between the nervous and immune systems. In turn, MHC-I upregulation postinjury is associated with a better regenerative outcome in isogenic mice following peripheral nerve damage. In the present work, we compared the time course of neuronal, glial, and sensorimotor recovery (1, 3, 5, 7, and 28 days after lesiondal) following unilateral sciatic nerve crush in A/J and C57BL/6J mice. The A/J strain showed higher expression of MHC-I (7 dal, ** p < 0.01), Iba-1 (microglial reaction, 7 dal, *** p < 0.001), and GFAP (astrogliosis, 5 dal, * p < 0.05) than the C57BL/6J counterpart. Synaptic coverage (synaptophysin) was equivalent in both strains over time. In addition, mRNA expression of microdissected spinal motoneurons revealed an increase in cytoskeleton-associated molecules (cofilin, shp2, and crmp2, * p < 0.05), but not trkB, in C57BL/6J mice. Gait recovery, studied by the sciatic functional index, was faster in the A/J strain, despite the equivalent results of C57BL/6J at 28 days after injury. A similar recovery was also seen for the nociceptive threshold (von Frey test). Interestingly, when evaluating proprioceptive recovery, C57BL/6J animals showed an enlarged base of support, indicating abnormal ambulation postinjury. Overall, the present results reinforce the role of MHC-I expression in the plasticity of the nervous system following axotomy, which in turn correlates with the variable recovery capacity among strains of mice.
Assuntos
Nervo Isquiático , Medula Espinal , Camundongos , Animais , Camundongos Endogâmicos C57BL , Medula Espinal/metabolismo , Axotomia/métodos , Compressão Nervosa , Gliose/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Camundongos EndogâmicosRESUMO
Major histocompatibility complex class I (MHC-I) has been implicated in several types of neuroplasticity phenomena. Interferon beta-1b (IFN-ß) increases MHC-I expression by motoneurons after sciatic nerve crush in mice, improving axonal growth and functional recovery. Additionally, IFN-ß induces glial hypertrophy associated with upregulation of glial fibrillary acidic protein (GFAP) and MHC-I in murine astrocytes in vitro. As knowledge about MHC-I and its role in synaptic plasticity in human astrocytes (HAs) is scarce, we investigated these aspects in mature HAs obtained from the neocortex of patients undergoing surgery due to hippocampal sclerosis. Cells were exposed to media in the absence (0 IU/ml) or presence of IFN-ß for 5 days (500 IU/ml). Beta-2 microglobulin (ß2m), a component of the MHC-I, GFAP and vimentin proteins, was quantified by flow cytometry (FC) and increased by 100%, 60% and 46%, respectively, after IFN-ß exposure. We also performed qRT-PCR gene expression analyses for ß2m, GFAP, vimentin, and pro- and anti-inflammatory cytokines. Our data showed that IFN-ß-treated astrocytes displayed ß2m and GFAP gene upregulation. Additionally, they presented a proinflammatory profile with increase in the IL-6 and IL-1ß genes and a tendency to upregulate TNF-α. Moreover, we evaluated the effect of HAs conditioned medium (CM) on the formation/maintenance of neurites/synapses by the PC12 lineage. Synaptophysin protein expression was quantified by FC. The CM of IFN-ß-activated astrocytes was not harmful to PC12 neurites, and there was no change in synaptophysin protein expression. Therefore, IFN-ß activated HAs by increasing GFAP, vimentin and MHC-I protein expression. Like MHC-I modulation and astrocyte activation may be protective after peripheral nerve damage and in some neurodegenerative conditions, this study opens perspectives on the pathophysiological roles of astroglial MHC-I in the human CNS.
Assuntos
Astrócitos , Interferon beta , Humanos , Animais , Camundongos , Astrócitos/metabolismo , Sinaptofisina/genética , Sinaptofisina/metabolismo , Sinaptofisina/farmacologia , Vimentina/genética , Vimentina/metabolismo , Vimentina/farmacologia , Interferon beta/genética , Interferon beta/metabolismo , Interferon beta/farmacologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Complexo Principal de Histocompatibilidade , FenótipoRESUMO
Immunomodulation has been considered an important approach in the treatment of malignant tumours. However, the modulation of innate immune cells remains an underexplored tool. Studies from our group demonstrated that the Phoneutria nigriventer spider venom (PnV) administration increased the infiltration of macrophage in glioblastoma, in addition to decreasing the tumour size in a preclinical model. The hypothesis that PnV would be modulating the innate immune system led us to the main objective of the present study: to elucidate the effects of PnV and its purified fractions on cultured macrophages. Results showed that PnV and the three fractions activated macrophages differentiated from bone marrow precursors. Further purification generated 23 subfractions named low weight (LW-1 to LW-12) and high weight (HW-1 to HW-11). LW-9 presented the best immunomodulatory effect. Treated cells were more phagocytic, migrated more, showed an activated morphological profile and induced an increased cytotoxic effect of macrophages on tumour cells. However, while M1-controls (LPS) increased IL-10, TNF-alpha and IL-6 release, PnV, fractions and subfractions did not alter any cytokine, with the exception of LW-9 that stimulated IL-10 production. These findings suggest that molecules present in LW-9 have the potential to be used as immunoadjuvants in the treatment of cancer.
Assuntos
Adjuvantes Imunológicos/farmacologia , Glioblastoma/terapia , Imunoterapia , Macrófagos/efeitos dos fármacos , Venenos de Aranha/farmacologia , Animais , Células Cultivadas , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , CamundongosRESUMO
The objective of this study was to evaluate the effects of red Light Emiting Diode (red LED) irradiation on fibroblasts in adipose-derived mesenchymal stem cells (ASC) co-culture on the scratch assay. We hypothesized that red LED irradiation could stimulate paracrine secretion of ASC, contributing to the activation of genes and molecules involved in cell migration and tissue repair. ASC were co-cultured with NIH/3T3 fibroblasts through direct contact and subjected to red LED irradiation (1.45 J/cm2/5min6s) after the scratch assay, during 4 days. Four groups were established: fibroblasts (F), fibroblasts + LED (FL), fibroblasts + ASC (FC) and fibroblasts + LED + ASC (FLC). The analyzes were based on Ctgf and Reck expression, quantification of collagen types I and III, tenomodulin, VEGF, TGF-ß1, MMP-2 and MMP-9, as well as viability analysis and cell migration. Higher Ctgf expression was observed in FC compared to F. Group FC presented higher amount of tenomodulin and VEGF in relation to the other groups. In the cell migration analysis, a higher number of cells was observed in the scratched area of the FC group on the 4th day. There were no differences between groups considering cell viability, Reck expression, amount of collagen types I and III, MMP-2 and TGF-ß1, whereas TGF-ß1 was not detected in the FC group and the MMP-9 in none of the groups. Our hypothesis was not supported by the results because the red LED irradiation decreased the healing response of ASC. An inhibitory effect of the LED irradiation associated with ASC co-culture was observed with reduction of the amount of TGF-ß1, VEGF and tenomodulin, possibly involved in the reduced cell migration. In turn, the ASC alone seem to have modulated fibroblast behavior by increasing Ctgf, VEGF and tenomodulin, leading to greater cell migration. In conclusion, red LED and ASC therapy can have independent effects on fibroblast wound healing, but the combination of both does not have a synergistic effect. Therefore, future studies with other parameters of red LED associated with ASC should be tested aiming clinical application for tissue repair.
RESUMO
Peripheral nerve injuries severely impair patients' quality of life as full recovery is seldom achieved. Upon axonal disruption, the distal nerve stump undergoes fragmentation, and myelin breaks down; the subsequent regeneration progression is dependent on cell debris removal. In addition to tissue clearance, macrophages release angiogenic and neurotrophic factors that contribute to axon growth. Based on the importance of macrophages for nerve regeneration, especially during the initial response to injury, we treated mice with granulocyte-macrophage colony-stimulating factor (GM-CSF) at various intervals after sciatic nerve crushing. Sciatic nerves were histologically analyzed at different time intervals after injury for the presence of macrophages and indicators of regeneration. Functional recovery was followed by an automated walking track test. We found that GM-CSF potentiated early axon growth, as indicated by the enhanced expression of growth-associated protein at 7 days postinjury. Inducible nitric oxide synthase expression increased at the beginning and at the end of the regenerative process, suggesting that nitric oxide is involved in axon growth and pruning. As expected, GM-CSF treatment stimulated macrophage infiltration, which increased at 7 and 14 days; however, it did not improve myelin clearance. Instead, GM-CSF stimulated early brain-derived neurotrophic factor (BDNF) production, which peaked at 7 days. Locomotor recovery pattern was not improved by GM-CSF treatment. The present results suggest that GM-CSF may have beneficial effects on early axonal regeneration.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Locomoção/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Bainha de Mielina/metabolismo , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Traumatismos dos Nervos Periféricos/metabolismo , Recuperação de Função Fisiológica/efeitos dos fármacos , Nervo Isquiático/lesões , Degeneração Walleriana/tratamento farmacológico , Degeneração Walleriana/metabolismoRESUMO
MHC-I molecules are involved in the antigenic presentation of cytosol-derived peptides to CD8T lymphocytes. In the nervous system, MHC-I expression is low to absent, occurring only during certain phases of development and aging or after injuries. The involvement of MHC-I in synaptic plasticity has been reported and, following lesion, astrocytes become reactive, limiting tissue damage. Such cells also attempt to restore homeostasis by secreting cytokines and neurotrophic factors. Moreover, astrocytes modulate synapse function, by taking up and releasing neurotransmitters and by limiting the synaptic cleft. Thus, the aim of the present study was to evaluate if astrocyte activation and reactivity are related to MHC I expression and if astrogliosis can be downregulated by silencing MHC-I mRNA synthesis. Given that, we evaluated astrocyte reactivity and synaptogenesis in co-cultures of astrocytes and spinal neurons under MHC-I RNA interference. For that, the MHC-I ß2-microglobulin subunit (ß2m) was knocked-down by siRNA in co-cultures (ß2m expression <60%, p<0.001). As measured by qRT-PCR, silencing of ß2m decreased expression of the astrocytic marker GFAP (<60%, p<0.001), as well as neurotrophic factors (BDNF and GDNF) and pro-inflammatory cytokines (TNF-α, IL-1, IL-6, IL-12 and IL-17). No significant changes in synaptic stability indicate that neuron-neuron interaction was preserved after ß2m silencing. Overall, the present data reinforce the importance of MHC-I expression for generation of astrogliosis, what may, in turn, become a target for future CNS/PNS therapies following injury.
Assuntos
Astrócitos/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Técnicas de Cocultura , Citocinas/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Gliose , Antígenos de Histocompatibilidade Classe I/genética , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Interferência de RNA , RNA Mensageiro/genética , Medula Espinal/citologia , Medula Espinal/metabolismo , Sinapses/fisiologia , Microglobulina beta-2/genéticaRESUMO
Major histocompatibility complex class one (MHC-I) antigen-presenting molecules participate in central nervous system (CNS) synaptic plasticity, as does the paired immunoglobulin-like receptor B (PirB), an MHC-I ligand that can inhibit immune-cells and bind to myelin axon growth inhibitors. Based on the dual roles of both molecules in the immune and nervous systems, we evaluated their expression in the central and peripheral nervous system (PNS) following sciatic nerve injury in mice. Increased PirB and MHC-I protein and gene expression is present in the spinal cord one week after nerve transection, PirB being mostly expressed in the neuropile region. In the crushed nerve, MHC-I protein levels increased 2 weeks after lesion (wal) and progressively decreased over the next eight weeks. The same kinetics were observed for infiltrating cytotoxic T lymphocytes (CTLs) but not for PirB expression, which continuously increased. Both MHC-I and PirB were found in macrophages and Schwann cells but rarely in axons. Interestingly, at 8 wal, PirB was mainly restricted to the myelin sheath. Our findings reinforce the participation of MHC-I and PirB in CNS plasticity events. In contrast, opposing expression levels of these molecules were found in the PNS, so that MHC-I and PirB seem to be mostly implicated in antigen presentation to CTLs and axon myelination, respectively.
Assuntos
Anti-Inflamatórios/uso terapêutico , Encefalomielite Autoimune Experimental/tratamento farmacológico , Primaquina/uso terapêutico , Análise de Variância , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Fatores de Transcrição Forkhead/metabolismo , Adjuvante de Freund/toxicidade , Proteína Glial Fibrilar Ácida/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Glicoproteína Mielina-Oligodendrócito/toxicidade , Proteínas do Tecido Nervoso/metabolismo , Fragmentos de Peptídeos/toxicidade , Índice de Gravidade de Doença , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismoRESUMO
The thymus plays an important role shaping the T cell repertoire in the periphery, partly, through the elimination of inflammatory auto-reactive cells. It has been shown that, during Plasmodium berghei infection, the thymus is rendered atrophic by the premature egress of CD4+CD8+ double-positive (DP) T cells to the periphery. To investigate whether autoimmune diseases are affected after Plasmodium berghei NK65 infection, we immunized C57BL/6 mice, which was previously infected with P. berghei NK65 and treated with chloroquine (CQ), with MOG35-55 peptide and the clinical course of Experimental Autoimmune Encephalomyelitis (EAE) was evaluated. Our results showed that NK65+CQ+EAE mice developed a more severe disease than control EAE mice. The same pattern of disease severity was observed in MOG35-55-immunized mice after adoptive transfer of P. berghei-elicited splenic DP-T cells. The higher frequency of IL-17+- and IFN-γ+-producing DP lymphocytes in the Central Nervous System of these mice suggests that immature lymphocytes contribute to disease worsening. To our knowledge, this is the first study to integrate the possible relationship between malaria and multiple sclerosis through the contribution of the thymus. Notwithstanding, further studies must be conducted to assert the relevance of malaria-induced thymic atrophy in the susceptibility and clinical course of other inflammatory autoimmune diseases.