RESUMO
BACKGROUND: Although effective in reducing relapse rate and delaying progression, current therapies for multiple sclerosis (MS) do not completely halt disease progression. T cell autoimmunity to myelin antigens is considered one of the main mechanisms driving MS. It is characterized by autoreactivity to disease-initiating myelin antigen epitope(s), followed by a cascade of epitope spreading, which are both strongly patient-dependent. Targeting a variety of MS-associated antigens by myelin antigen-presenting tolerogenic dendritic cells (tolDC) is a promising treatment strategy to re-establish tolerance in MS. Electroporation with mRNA encoding myelin proteins is an innovative technique to load tolDC with the full spectrum of naturally processed myelin-derived epitopes. METHODS: In this study, we generated murine tolDC presenting myelin oligodendrocyte glycoprotein (MOG) using mRNA electroporation and we assessed the efficacy of MOG mRNA-electroporated tolDC to dampen pathogenic T cell responses in experimental autoimmune encephalomyelitis (EAE). For this, MOG35-55-immunized C57BL/6 mice were injected intravenously at days 13, 17, and 21 post-disease induction with 1α,25-dihydroxyvitamin D3-treated tolDC electroporated with MOG-encoding mRNA. Mice were scored daily for signs of paralysis. At day 25, myelin reactivity was evaluated following restimulation of splenocytes with myelin-derived epitopes. Ex vivo magnetic resonance imaging (MRI) was performed to assess spinal cord inflammatory lesion load. RESULTS: Treatment of MOG35-55-immunized C57BL/6 mice with MOG mRNA-electroporated or MOG35-55-pulsed tolDC led to a stabilization of the EAE clinical score from the first administration onwards, whereas it worsened in mice treated with non-antigen-loaded tolDC or with vehicle only. In addition, MOG35-55-specific pro-inflammatory pathogenic T cell responses and myelin antigen epitope spreading were inhibited in the peripheral immune system of tolDC-treated mice. Finally, magnetic resonance imaging analysis of hyperintense spots along the spinal cord was in line with the clinical score. CONCLUSIONS: Electroporation with mRNA is an efficient and versatile tool to generate myelin-presenting tolDC that are capable to stabilize the clinical score in EAE. These results pave the way for further research into mRNA-electroporated tolDC treatment as a patient-tailored therapy for MS.
Assuntos
Células Dendríticas/metabolismo , Eletroporação/métodos , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/terapia , Glicoproteína Mielina-Oligodendrócito/metabolismo , RNA Mensageiro/metabolismo , Animais , Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/imunologia , Feminino , Humanos , Tolerância Imunológica/fisiologia , Células K562 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/administração & dosagem , Glicoproteína Mielina-Oligodendrócito/imunologia , RNA Mensageiro/administração & dosagem , RNA Mensageiro/imunologiaRESUMO
The establishment and validation of reliable induced pluripotent stem cell (iPSC)-derived in vitro models to study microglia and monocyte/macrophage immune function holds great potential for fundamental and translational neuro-immunology research. In this study, we first demonstrate that ramified CX3CR1+ iPSC-microglia (cultured within a neural environment) and round-shaped CX3CR1- iPSC-macrophages can easily be differentiated from newly established murine CX3CR1eGFP/+CCR2RFP/+ iPSC lines. Furthermore, we show that obtained murine iPSC-microglia and iPSC-macrophages are distinct cell populations, even though iPSC-macrophages may upregulate CX3CR1 expression when cultured within a neural environment. Next, we characterized the phenotypical and functional properties of murine iPSC-microglia and iPSC-macrophages following classical and alternative immune polarisation. While iPSC-macrophages could easily be triggered to adopt a classically-activated or alternatively-activated phenotype following, respectively, lipopolysaccharideâ¯+â¯interferon γ or interleukin 13 (IL13) stimulation, iPSC-microglia and iPSC-macrophages cultured within a neural environment displayed a more moderate activation profile as characterised by the absence of MHCII expression upon classical immune polarisation and the absence of Ym1 expression upon alternative immune polarisation. Finally, extending our preceding in vivo studies, this striking phenotypical divergence was also observed for resident microglia and infiltrating monocytes within highly inflammatory cortical lesions in CX3CR1eGFP/+CCR2RFP/+ mice subjected to middle cerebral arterial occlusion (MCAO) stroke and following IL13-mediated therapeutic intervention thereon. In conclusion, our study demonstrates that the applied murine iPSC-microglia and iPSC-macrophage culture models are able to recapitulate in vivo microglia and monocyte/macrophage ontogeny and corresponding phenotypical/functional properties upon classical and alternative immune polarisation, and therefore represent a valuable in vitro platform to further study and modulate microglia and (infiltrating) monocyte immune responses under neuro-inflammatory conditions within a neural environment.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Neuroimunomodulação/fisiologia , Animais , Receptor 1 de Quimiocina CX3C/metabolismo , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Feminino , Células-Tronco Pluripotentes Induzidas/fisiologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Monócitos/metabolismo , Neuroimunomodulação/imunologia , Fenótipo , Receptores CCR2/metabolismoRESUMO
BACKGROUND: Subtle adjustment of the activation status of CNS resident microglia and peripheral macrophages, to promote their neuroprotective and neuroregenerative functions, may facilitate research towards curing neurodegenerative disorders. In the present study, we investigated whether targeted intracerebral delivery of the anti-inflammatory cytokine interleukin (IL)13, by means of transplanting IL13-expressing mesenchymal stem cells (IL13-MSCs), can promote a phenotypic switch in both microglia and macrophages during the pro-inflammatory phase in a mouse model of ischemic stroke. METHODS: We used the CX3CR1eGFP/+ CCR2RFP/+ transgenic mouse model to separately recognize brain-resident microglia from infiltrated macrophages. Quantitative immunohistochemical analyses were applied to characterize polarization phenotypes of both cell types. RESULTS: Distinct behaviors of both cell populations were noted dependent on the anatomical site of the lesion. Immunohistochemistry revealed that mice grafted with IL13-MSCs, in contrast to non-grafted and MSC-grafted control mice, were able to drive recruited microglia and macrophages into an alternative activation state, as visualized by a significant increase of Arg-1 and a noticeable decrease of MHC-II expression at day 14 after ischemic stroke. Interestingly, both Arg-1 and MHC-II were expressed more abundantly in macrophages than in microglia, further confirming the distinct behavior of both cell populations. CONCLUSIONS: The current data highlight the importance of controlled and localized delivery of the anti-inflammatory cytokine IL13 for modulation of both microglia and macrophage responses after ischemic stroke, thereby providing pre-clinical rationale for the application of L13-MSCs in future investigations of neurodegenerative disorders.
Assuntos
Anti-Inflamatórios/uso terapêutico , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/terapia , Interleucina-13/uso terapêutico , Macrófagos/efeitos dos fármacos , Microglia/efeitos dos fármacos , Animais , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/fisiologia , Infarto da Artéria Cerebral Média/diagnóstico por imagem , Infarto da Artéria Cerebral Média/fisiopatologia , Interleucina-13/genética , Interleucina-13/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Macrófagos/metabolismo , Transplante de Células-Tronco Mesenquimais/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Movimento/fisiologia , Força Muscular , Propriocepção , RNA Mensageiro/metabolismo , Receptores CCR2/genética , Receptores CCR2/metabolismo , Tato/fisiologia , Transdução GenéticaRESUMO
Transplantation of mesenchymal stem cells (MSCs) into injured or diseased tissue-for the in situ delivery of a wide variety of MSC-secreted therapeutic proteins-is an emerging approach for the modulation of the clinical course of several diseases and traumata. From an emergency point-of-view, allogeneic MSCs have numerous advantages over patient-specific autologous MSCs since "off-the-shelf" cell preparations could be readily available for instant therapeutic intervention following acute injury. Although we confirmed the in vitro immunomodulatory capacity of allogeneic MSCs on antigen-presenting cells with standard coculture experiments, allogeneic MSC grafts were irrevocably rejected by the host's immune system upon either intramuscular or intracerebral transplantation. In an attempt to modulate MSC allograft rejection in vivo, we transduced MSCs with an interleukin-13 (IL13)-expressing lentiviral vector. Our data clearly indicate that prolonged survival of IL13-expressing allogeneic MSC grafts in muscle tissue coincided with the induction of an alternatively activated macrophage phenotype in vivo and a reduced number of alloantigen-reactive IFNγ- and/or IL2-producing CD8(+) T cells compared to nonmodified allografts. Similarly, intracerebral IL13-expressing MSC allografts also exhibited prolonged survival and induction of an alternatively activated macrophage phenotype, although a peripheral T cell component was absent. In summary, this study demonstrates that both innate and adaptive immune responses are effectively modulated in vivo by locally secreted IL13, ultimately resulting in prolonged MSC allograft survival in both muscle and brain tissue. Stem Cells 2016;34:1971-1984.
Assuntos
Sobrevivência de Enxerto/imunologia , Interleucina-13/farmacologia , Isoantígenos/imunologia , Ativação Linfocitária/efeitos dos fármacos , Macrófagos/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Linfócitos T/imunologia , Aloenxertos/efeitos dos fármacos , Aloenxertos/imunologia , Animais , Formação de Anticorpos/efeitos dos fármacos , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Engenharia Genética , Imunomodulação/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Microglia/efeitos dos fármacos , Microglia/patologia , Linfócitos T/efeitos dos fármacosRESUMO
Detrimental inflammatory responses in the central nervous system are a hallmark of various brain injuries and diseases. With this study we provide evidence that lentiviral vector-mediated expression of the immune-modulating cytokine interleukin 13 (IL-13) induces an alternative activation program in both microglia and macrophages conferring protection against severe oligodendrocyte loss and demyelination in the cuprizone mouse model for multiple sclerosis (MS). First, IL-13 mediated modulation of cuprizone induced lesions was monitored using T2 -weighted magnetic resonance imaging and magnetization transfer imaging, and further correlated with quantitative histological analyses for inflammatory cell influx, oligodendrocyte death, and demyelination. Second, following IL-13 immune gene therapy in cuprizone-treated eGFP+ bone marrow chimeric mice, we provide evidence that IL-13 directs the polarization of both brain-resident microglia and infiltrating macrophages towards an alternatively activated phenotype, thereby promoting the conversion of a pro-inflammatory environment toward an anti-inflammatory environment, as further evidenced by gene expression analyses. Finally, we show that IL-13 immune gene therapy is also able to limit lesion severity in a pre-existing inflammatory environment. In conclusion, these results highlight the potential of IL-13 to modulate microglia/macrophage responses and to improve disease outcome in a mouse model for MS. GLIA 2016;64:2181-2200.
Assuntos
Doenças Desmielinizantes/terapia , Encefalite/terapia , Terapia Genética/métodos , Interleucina-13 , Macrófagos/efeitos dos fármacos , Microglia/efeitos dos fármacos , Animais , Antígenos de Diferenciação/metabolismo , Transplante de Medula Óssea , Cuprizona/toxicidade , Citocinas/genética , Citocinas/metabolismo , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/diagnóstico por imagem , Modelos Animais de Doenças , Encefalite/induzido quimicamente , Encefalite/diagnóstico por imagem , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interleucina-13/genética , Interleucina-13/metabolismo , Interleucina-13/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibidores da Monoaminoxidase/toxicidade , Proteínas da Mielina/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transdução GenéticaRESUMO
BACKGROUND: Promoting the neuroprotective and repair-inducing effector functions of microglia and macrophages, by means of M2 polarisation or alternative activation, is expected to become a new therapeutic approach for central nervous system (CNS) disorders in which detrimental pro-inflammatory microglia and/or macrophages display a major contribution to the neuropathology. In this study, we present a novel in vivo approach using intracerebral grafting of mesenchymal stem cells (MSC) genetically engineered to secrete interleukin 13 (IL13-MSC). METHODS: In the first experimental setup, control MSC and IL13-MSC were grafted in the CNS of eGFP+ bone marrow chimaeric C57BL/6 mice to histologically evaluate IL13-mediated expression of several markers associated with alternative activation, including arginase1 and Ym1, on MSC graft-recognising microglia and MSC graft-infiltrating macrophages. In the second experimental setup, IL13-MSC were grafted on the right side (or on both the right and left sides) of the splenium of the corpus callosum in wild-type C57BL/6 mice and in C57BL/6 CX3CR1eGFP/+CCR2RFP/+ transgenic mice. Next, CNS inflammation and demyelination was induced by means of a cuprizone-supplemented diet. The influence of IL13-MSC grafting on neuropathological alterations was monitored by non-invasive T 2-weighted magnetic resonance imaging (MRI) and quantitative histological analyses, as compared to cuprizone-treated mice with control MSC grafts and/or cuprizone-treated mice without MSC injection. RESULTS: In the first part of this study, we demonstrate that MSC graft-associated microglia and MSC graft-infiltrating macrophages are forced into alternative activation upon grafting of IL13-MSC, but not upon grafting of control MSC. In the second part of this study, we demonstrate that grafting of IL13-MSC, in addition to the recruitment of M2 polarised macrophages, limits cuprizone-induced microgliosis, oligodendrocyte death and demyelination. Furthermore, we here demonstrate that injection of IL13-MSC at both sides of the splenium leads to a superior protective effect as compared to a single injection at one side of the splenium. CONCLUSIONS: Controlled and localised production of IL13 by means of intracerebral MSC grafting has the potential to modulate cell graft- and pathology-associated microglial/macrophage responses, and to interfere with oligodendrocyte death and demyelinating events in the cuprizone mouse model.
Assuntos
Cuprizona/toxicidade , Doenças Desmielinizantes , Gliose/etiologia , Interleucina-13/metabolismo , Transplante de Células-Tronco Mesenquimais , Inibidores da Monoaminoxidase/toxicidade , Oligodendroglia/patologia , Animais , Linhagem Celular Transformada , Citocinas/genética , Citocinas/metabolismo , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/diagnóstico por imagem , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/cirurgia , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imageamento por Ressonância Magnética , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Básica da Mielina/metabolismo , Oligodendroglia/efeitos dos fármacosRESUMO
Conventional MRI is frequently used during the diagnosis of multiple sclerosis but provides only little additional pathological information. Proton MRS ((1) H-MRS), however, provides biochemical information on the lesion pathology by visualization of a spectrum of metabolites. In this study we aimed to better understand the changes in metabolite concentrations following demyelination of the white matter. Therefore, we used the cuprizone model, a well-established mouse model to mimic type III human multiple sclerosis demyelinating lesions. First, we identified CX3 CL1/CX3 CR1 signaling as a major regulator of microglial activity in the cuprizone mouse model. Compared with control groups (heterozygous CX3 CR1(+/-) C57BL/6 mice and wild type CX3 CR1(+/+) C57BL/6 mice), microgliosis, astrogliosis, oligodendrocyte cell death and demyelination were shown to be highly reduced or absent in CX3 CR1(-/-) C57BL/6 mice. Second, we show that (1) H-MRS metabolite spectra are different when comparing cuprizone-treated CX3 CR1(-/-) mice showing mild demyelination with cuprizone-treated CX3 CR1(+/+) mice showing severe demyelination and demyelination-associated inflammation. Following cuprizone treatment, CX3 CR1(+/+) mice show a decrease in the Glu, tCho and tNAA concentrations as well as an increased Tau concentration. In contrast, following cuprizone treatment CX3 CR1(-/-) mice only showed a decrease in tCho and tNAA concentrations. Therefore, (1) H-MRS might possibly allow us to discriminate demyelination from demyelination-associated inflammation via changes in Tau and Glu concentration. In addition, the observed decrease in tCho concentration in cuprizone-induced demyelinating lesions should be further explored as a possible diagnostic tool for the early identification of human MS type III lesions.
Assuntos
Doenças Desmielinizantes/patologia , Gliose/patologia , Imageamento por Ressonância Magnética , Neuroimagem/métodos , Espectroscopia de Prótons por Ressonância Magnética , Animais , Ácido Aspártico/análogos & derivados , Ácido Aspártico/análise , Química Encefálica , Colina/análise , Creatina/análise , Cuprizona/toxicidade , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/diagnóstico , Dipeptídeos/análise , Modelos Animais de Doenças , Feminino , Gliose/induzido quimicamente , Gliose/diagnóstico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oligodendroglia/patologia , Fosfocreatina/análiseRESUMO
Although implantation of cellular material in the central nervous system (CNS) is a key direction in CNS regenerative medicine, this approach is currently limited by the occurrence of strong endogenous immune cell responses. In a model of mesenchymal stem cell (MSC) grafting in the CNS of immune-competent mice, we previously described that MSC grafts become highly surrounded and invaded by Iba1(+) myeloid cells (microglia and/or macrophages). Here, following grafting of blue fluorescent protein (BFP)-expressing MSC in the CNS of CX3CR1(+/-) and CX3CR1(-/-) mice, our results indicate: (1) that the observed inflammatory response is independent of the fractalkine signalling axis, and (2) that a significant spatial distribution of Iba1(+) inflammatory cells occurs, in which Iba1(+) CX3CR1(+) myeloid cells mainly surround the MSC graft and Iba1(+) CX3CR1(-) myeloid cells mainly invade the graft at 10 days post transplantation. Although Iba1(+) CX3CR1(+) myeloid cells are considered to be of resident microglial origin, Iba1(+) CX3CR1(-) myeloid cells are most likely of peripheral monocyte/macrophage origin. In order to confirm the latter, we performed MSC-BFP grafting experiments in the CNS of eGFP(+) bone marrow chimeric C57BL/6 mice. Analysis of MSC-BFP grafts in the CNS of these mice confirmed our observation that peripheral monocytes/macrophages invade the MSC graft and that resident microglia surround the MSC graft site. Furthermore, analysis of major histocompatibility complex class II (MHCII) expression revealed that mainly macrophages, but not microglia, express this M1 pro-inflammatory marker in the context of MSC grafting in the CNS. These results again highlight the complexity of cell implantation immunology in the CNS.
Assuntos
Encéfalo/metabolismo , Macrófagos/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Microglia/metabolismo , Animais , Encéfalo/imunologia , Encéfalo/patologia , Receptor 1 de Quimiocina CX3C , Proteínas de Ligação ao Cálcio/metabolismo , Expressão Gênica , Genes Reporter , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Imunofenotipagem , Macrófagos/imunologia , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Microglia/imunologia , Células Mieloides/metabolismo , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Receptores de HIV/genética , Receptores de HIV/metabolismo , Transdução de Sinais , Transdução Genética , Quimeras de TransplanteRESUMO
SRPK1, the prototype of the serine/arginine family of kinases, has been implicated in the regulation of multiple cellular processes such as pre-mRNA splicing, chromatin structure, nuclear import and germ cell development. SRPK1a is a much less studied isoform of SRPK1 that contains an extended N-terminal domain and so far has only been detected in human testis. In the present study we show that SRPK1 is the predominant isoform in K562 cells, with the ratio of the two isoforms being critical in determining cell fate. Stable overexpression of SRPK1a induces erythroid differentiation of K562 cells. The induction of globin synthesis was accompanied by a marked decrease in proliferation and a significantly reduced clonogenic potential. Small interfering RNA-mediated down-regulation of SRPK1 in K562 cells results similarly in a decrease in proliferative capacity and induction of globin synthesis. A decreased SRPK1/SRPK1a ratio is also observed upon hemin/DMSO-induced differentiation of K562 cells as well as in normal human erythroid progenitor cells. Mass spectrometric analysis of SRPK1a-associated proteins identified multiple classes of RNA-binding proteins including RNA helicases, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, and mRNA-associated proteins. Several of the SRPK1a-copurifying proteins have been previously identified in ribosomal and pre-ribosomal complexes, thereby suggesting that SRPK1a may play an important role in linking ribosomal assembly and/or function to erythroid differentiation in human leukaemic cells.
Assuntos
Diferenciação Celular , Eritrócitos/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Bases , Primers do DNA , Humanos , Células K562 , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/fisiologiaRESUMO
Currently, much attention is given to the development of cellular therapies for treatment of central nervous system (CNS) injuries. Diverse cell implantation strategies, either to directly replace damaged neural tissue or to create a neuroregenerative environment, are proposed to restore impaired brain function. However, because of the complexity of the CNS, it is now becoming clear that the contribution of cell implantation into the brain will mainly act in a supportive manner. In addition, given the time dependence of neural development during embryonic and post-natal life, cellular implants, either self or non-self, will most likely have to interact for a sustained period of time with both healthy and injured neural tissue. The latter also implies potential recognition of cellular implants by the innate immune system of the brain. In this review, we will emphasize on preclinical observations in rodents, regarding the recognition and immunogenicity of autologous, allogeneic and xenogeneic cellular implants in the CNS of immune-competent hosts. Taken together, we here suggest that a profound study of the interaction between cellular grafts and the brain's innate immune system will be inevitable before clinical cell transplantation in the CNS can be performed successfully.
Assuntos
Encéfalo/imunologia , Transplante de Células , Imunidade Inata/imunologia , Imunologia de Transplantes/imunologia , Animais , Terapia Baseada em Transplante de Células e Tecidos , Doenças do Sistema Nervoso Central/terapia , HumanosRESUMO
Premenopausal bilateral ovariectomy is considered to be one of the risk factors of Alzheimer's disease (AD). However, the underlying mechanisms remain unclear. Here, we aimed to investigate long-term neurological consequences of ovariectomy in a rodent AD model, TG2576 (TG), and wild-type mice (WT) that underwent an ovariectomy or sham-operation, using in vivo MRI biomarkers. An increase in osmoregulation and energy metabolism biomarkers in the hypothalamus, a decrease in white matter integrity, and a decrease in the resting-state functional connectivity was observed in ovariectomized TG mice compared to sham-operated TG mice. In addition, we observed an increase in functional connectivity in ovariectomized WT mice compared to sham-operated WT mice. Furthermore, genotype (TG vs. WT) effects on imaging markers and GFAP immunoreactivity levels were observed, but there was no effect of interaction (Genotype × Surgery) on amyloid-beta-and GFAP immunoreactivity levels. Taken together, our results indicated that both genotype and ovariectomy alters imaging biomarkers associated with AD.
Assuntos
Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/psicologia , Química Encefálica , Função Executiva , Ovariectomia/efeitos adversos , Substância Branca/metabolismo , Substância Branca/fisiopatologia , Doença de Alzheimer/etiologia , Doença de Alzheimer/genética , Animais , Biomarcadores , Modelos Animais de Doenças , Genótipo , Imageamento por Ressonância Magnética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Placebos , Fatores de Risco , Fatores de Tempo , Substância Branca/diagnóstico por imagemRESUMO
BACKGROUND: Cell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system (CNS). However, in many pre-clinical cell therapy studies, reporter gene-assisted imaging of cellular implants in the CNS and potential reporter gene and/or cell-based immunogenicity, still remain challenging research topics. RESULTS: In this study, we performed cell implantation experiments in the CNS of immunocompetent mice using autologous (syngeneic) luciferase-expressing bone marrow-derived stromal cells (BMSC-Luc) cultured from ROSA26-L-S-L-Luciferase transgenic mice, and BMSC-Luc genetically modified using a lentivirus encoding the enhanced green fluorescence protein (eGFP) and the puromycin resistance gene (Pac) (BMSC-Luc/eGFP/Pac). Both reporter gene-modified BMSC populations displayed high engraftment capacity in the CNS of immunocompetent mice, despite potential immunogenicity of introduced reporter proteins, as demonstrated by real-time bioluminescence imaging (BLI) and histological analysis at different time-points post-implantation. In contrast, both BMSC-Luc and BMSC-Luc/eGFP/Pac did not survive upon intramuscular cell implantation, as demonstrated by real-time BLI at different time-points post-implantation. In addition, ELISPOT analysis demonstrated the induction of IFN-gamma-producing CD8+ T-cells upon intramuscular cell implantation, but not upon intracerebral cell implantation, indicating that BMSC-Luc and BMSC-Luc/eGFP/Pac are immune-tolerated in the CNS. However, in our experimental transplantation model, results also indicated that reporter gene-specific immune-reactive T-cell responses were not the main contributors to the immunological rejection of BMSC-Luc or BMSC-Luc/eGFP/Pac upon intramuscular cell implantation. CONCLUSION: We here demonstrate that reporter gene-modified BMSC derived from ROSA26-L-S-L-Luciferase transgenic mice are immune-tolerated upon implantation in the CNS of syngeneic immunocompetent mice, providing a research model for studying survival and localisation of autologous BMSC implants in the CNS by real-time BLI and/or histological analysis in the absence of immunosuppressive therapy.
Assuntos
Células da Medula Óssea/citologia , Encéfalo , Luciferases/genética , Células Estromais/transplante , Tolerância ao Transplante , Animais , Encéfalo/metabolismo , Células Cultivadas , Diagnóstico por Imagem , Genes Reporter , Luciferases/metabolismo , Substâncias Luminescentes/metabolismo , Medições Luminescentes , Masculino , Camundongos , Camundongos Transgênicos , Modelos AnimaisRESUMO
Alzheimer's disease (AD) is the most common form of dementia in the elderly. According to the amyloid hypothesis, the accumulation and deposition of amyloid-beta (Aß) peptides play a key role in AD. Soluble Aß (sAß) oligomers were shown to be involved in pathological hypersynchronisation of brain resting-state networks in different transgenic developmental-onset mouse models of amyloidosis. However, the impact of protein overexpression during brain postnatal development may cause additional phenotypes unrelated to AD. To address this concern, we investigated sAß effects on functional resting-state networks in transgenic mature-onset amyloidosis Tet-Off APP (TG) mice. TG mice and control littermates were raised on doxycycline (DOX) diet from 3d up to 3 m of age to suppress transgenic Aß production. Thereafter, longitudinal resting-state functional MRI was performed on a 9.4 T MR-system starting from week 0 (3 m old mice) up to 28w post DOX treatment. Ex-vivo immunohistochemistry and ELISA analysis was performed to assess the development of amyloid pathology. Functional Connectivity (FC) analysis demonstrated early abnormal hypersynchronisation in the TG mice compared to the controls at 8w post DOX treatment, particularly across regions of the default mode-like network, known to be affected in AD. Ex-vivo analyses performed at this time point confirmed a 20-fold increase in total sAß levels preceding the apparition of Aß plaques and inflammatory responses in the TG mice compared to the controls. On the contrary at week 28, TG mice showed an overall hypoconnectivity, coinciding with a widespread deposition of Aß plaques in the brain. By preventing developmental influence of APP and/or sAß during brain postnatal development, we demonstrated FC abnormalities potentially driven by sAß neurotoxicity on resting-state neuronal networks in mature-induced TG mice. Thus, the Tet-Off APP mouse model could be a powerful tool while used as a mature-onset model to shed light into amyloidosis mechanisms in AD.
Assuntos
Peptídeos beta-Amiloides , Amiloidose/diagnóstico por imagem , Encéfalo/diagnóstico por imagem , Modelos Animais de Doenças , Rede Nervosa/diagnóstico por imagem , Fatores Etários , Peptídeos beta-Amiloides/metabolismo , Amiloidose/metabolismo , Animais , Encéfalo/metabolismo , Feminino , Imageamento por Ressonância Magnética/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Rede Nervosa/metabolismo , SolubilidadeRESUMO
BACKGROUND: Bone marrow-derived stromal cells (MSC) are attractive targets for ex vivo cell and gene therapy. In this context, we investigated the feasibility of a plasmid-based strategy for genetic modification of human (h)MSC with enhanced green fluorescent protein (EGFP) and neurotrophin (NT)3. Three genetically modified hMSC lines (EGFP, NT3, NT3-EGFP) were established and used to study cell survival and transgene expression following transplantation in rat spinal cord. RESULTS: First, we demonstrate long-term survival of transplanted hMSC-EGFP cells in rat spinal cord under, but not without, appropriate immune suppression. Next, we examined the stability of EGFP or NT3 transgene expression following transplantation of hMSC-EGFP, hMSC-NT3 and hMSC-NT3-EGFP in rat spinal cord. While in vivo EGFP mRNA and protein expression by transplanted hMSC-EGFP cells was readily detectable at different time points post-transplantation, in vivo NT3 mRNA expression by hMSC-NT3 cells and in vivo EGFP protein expression by hMSC-NT3-EGFP cells was, respectively, undetectable or declined rapidly between day 1 and 7 post-transplantation. Further investigation revealed that the observed in vivo decline of EGFP protein expression by hMSC-NT3-EGFP cells: (i) was associated with a decrease in transgenic NT3-EGFP mRNA expression as suggested following laser capture micro-dissection analysis of hMSC-NT3-EGFP cell transplants at day 1 and day 7 post-transplantation, (ii) did not occur when hMSC-NT3-EGFP cells were transplanted subcutaneously, and (iii) was reversed upon re-establishment of hMSC-NT3-EGFP cell cultures at 2 weeks post-transplantation. Finally, because we observed a slowly progressing tumour growth following transplantation of all our hMSC cell transplants, we here demonstrate that omitting immune suppressive therapy is sufficient to prevent further tumour growth and to eradicate malignant xenogeneic cell transplants. CONCLUSION: In this study, we demonstrate that genetically modified hMSC lines can survive in healthy rat spinal cord over at least 3 weeks by using adequate immune suppression and can serve as vehicles for transgene expression. However, before genetically modified hMSC can potentially be used in a clinical setting to treat spinal cord injuries, more research on standardisation of hMSC culture and genetic modification needs to be done in order to prevent tumour formation and transgene silencing in vivo.
Assuntos
Transplante de Medula Óssea/métodos , Terapia Genética/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Neurotrofina 3/genética , Neurotrofina 3/metabolismo , Plasmídeos/genética , Medula Espinal/metabolismo , Medula Espinal/cirurgia , Animais , Sobrevivência Celular , Células Cultivadas , Terapia Combinada , Plasmídeos/administração & dosagem , Ratos , Transfecção/métodos , Transgenes/genéticaRESUMO
Over the last 30 years, numerous allogeneic and xenogeneic cell grafts have been transplanted into the central nervous system (CNS) of mice and men in an attempt to cure neurological diseases. In the early studies, human or porcine embryonic neural cells were grafted in the striatum of animals or patients in an attempt to replace lost neurons. Although the immune-privileged status of the brain as a recipient organ was widely accepted, it rapidly became evident that CNS-grafted allogeneic and xenogeneic cells could be recognized and rejected by the immune system, resulting in poor neural graft survival and limited functional recovery. Since then, the CNS transplantation field has witnessed a sharp rise in the number of studies in which allogeneic and xenogeneic neural or mesenchymal stem cells (NSCs or MSCs, respectively) are transplanted, predominantly aiming at providing trophic stimulation and promoting endogenous repair of the brain. Interestingly, in many recent NSC and MSC-based publications functional improvement was used as the principal measure to evaluate the success of cell transplantation, while the fate of transplanted cells remained largely unreported. In this review, we first attempt to understand why primary neural cell isolates were largely substituted for NSCs and MSCs in cell grafting studies. Next, we review the current knowledge on the immune mechanisms involved in the recognition and rejection of allogeneic and xenogeneic cellular grafts in the CNS. Finally, we propose strategies to reduce graft immunogenicity and to improve graft survival in order to design improved cell-based CNS therapies. Stem Cells Translational Medicine 2017;6:1434-1441.
Assuntos
Imunidade Adaptativa/fisiologia , Sistema Nervoso Central/citologia , Sistema Nervoso Central/imunologia , Imunidade Inata/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Neurais/citologia , Imunidade Adaptativa/imunologia , Animais , Humanos , Imunidade Inata/imunologia , Células-Tronco Mesenquimais/fisiologia , Células-Tronco Neurais/fisiologiaRESUMO
Over the past two decades, several cell types with fibroblast-like morphology, including mesenchymal stem/stromal cells, but also other adult, embryonic and extra-embryonic fibroblast-like cells, have been brought forward in the search for cellular therapies to treat severe brain injuries and/or diseases. Although current views in regenerative medicine are highly focused on the immune modulating and regenerative properties of stromal cell transplantation in vivo, many open questions remain regarding their true mode of action. In this perspective, this study integrates insights gathered over the past 10 years to formulate a unifying model of the cellular events that accompany fibroblast-like cell grafting in the rodent brain. Cellular interactions are discussed step-by-step, starting from the day of implantation up to 10 days after transplantation. During the short period that precedes stable settlement of autologous/syngeneic stromal cell grafts, there is a complex interplay between hypoxia-mediated cell death of grafted cells, neutrophil invasion, microglia and macrophage recruitment, astrocyte activation and neo-angiogenesis within the stromal cell graft site. Consequently, it is speculated that regenerative processes following cell therapeutic intervention in the CNS are not only modulated by soluble factors secreted by grafted stromal cells (bystander hypothesis), but also by in vivo inflammatory processes following stromal cell grafting. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Sistema Nervoso Central/imunologia , Sistema Nervoso Central/patologia , Inflamação/patologia , Animais , Humanos , Modelos Biológicos , Células Estromais/citologia , Células Estromais/imunologia , Células Estromais/transplanteRESUMO
Transplantation of neural stem cells (NSC) in diseased or injured brain tissue is widely studied as a potential treatment for various neurological pathologies. However, effective cell replacement therapy relies on the intrinsic capacity of cellular grafts to overcome hypoxic and/or immunological barriers after transplantation. In this context, it is hypothesized that structural support for grafted NSC will be of utmost importance. With this study, we present a novel decellularization protocol for 1.5 mm thick mouse brain sections, resulting in the generation of acellular three-dimensional (3D) brain sections. Next, the obtained 3D brain sections were seeded with murine NSC expressing both the eGFP and luciferase reporter proteins (NSC-eGFP/Luc). Using real-time bioluminescence imaging, the survival and growth of seeded NSC-eGFP/Luc cells was longitudinally monitored for 1-7 weeks in culture, indicating the ability of the acellular brain sections to support sustained ex vivo growth of NSC. Next, the organization of a 3D maze-like cellular structure was examined using confocal microscopy. Moreover, under mitogenic stimuli (EGF and hFGF-2), most cells in this 3D culture retained their NSC phenotype. Concluding, we here present a novel protocol for decellularization of mouse brain sections, which subsequently support long-term 3D culture of undifferentiated NSC.
Assuntos
Encéfalo/citologia , Técnicas de Cultura de Células/métodos , Células-Tronco Neurais/citologia , Animais , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Luciferases/metabolismo , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/metabolismo , FenótipoRESUMO
While multiple rodent preclinical studies, and to a lesser extent human clinical trials, claim the feasibility, safety, and potential clinical benefit of cell grafting in the central nervous system (CNS), currently only little convincing knowledge exists regarding the actual fate of the grafted cells and their effect on the surrounding environment (or vice versa). Our preceding studies already indicated that only a minor fraction of the initially grafted cell population survives the grafting process, while the surviving cell population becomes invaded by highly activated microglia/macrophages and surrounded by reactive astrogliosis. In the current study, we further elaborate on early cellular and inflammatory events following syngeneic grafting of eGFP(+) mouse embryonic fibroblasts (mEFs) in the CNS of immunocompetent mice. Based on obtained quantitative histological data, we here propose a detailed mathematically derived working model that sequentially comprises hypoxia-induced apoptosis of grafted mEFs, neutrophil invasion, neoangiogenesis, microglia/macrophage recruitment, astrogliosis, and eventually survival of a limited number of grafted mEFs. Simultaneously, we observed that the cellular events following mEF grafting activates the subventricular zone neural stem and progenitor cell compartment. This proposed model therefore further contributes to our understanding of cell graft-induced cellular responses and will eventually allow for successful manipulation of this intervention.
Assuntos
Sistema Nervoso Central/metabolismo , Inflamação , Ventrículos Laterais/citologia , Neutrófilos/imunologia , Animais , Apoptose , Proliferação de Células , Células Cultivadas , Sistema Nervoso Central/imunologia , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/transplante , Genes Reporter , Sobrevivência de Enxerto , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipóxia , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Modelos Biológicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neutrófilos/citologiaRESUMO
Although intracerebral transplantation of various fibroblast(-like) cell populations has been shown feasible, little is known about the actual in vivo remodeling of these cellular grafts and their environment. In this study, we aimed to compare the in vitro and in vivo behavior of two phenotypically similar-but developmentally distinct-fibroblast-like cell populations, namely, mouse embryonic fibroblasts (mEFs) and mouse fetal membrane-derived stromal cells (mFMSCs). While both mEFs and mFMSCs are readily able to reduce TNF-α secretion by LPS/IFN-γ-activated BV-2 microglia, mFMSCs and mEFs display strikingly opposite behavior with regard to VEGF production under normal and inflammatory conditions. Whereas mFMSCs downregulate VEGF production upon coculture with LPS/IFN-γ-activated BV-2 microglia, mEFs upregulate VEGF production in the presence of LPS/IFN-γ-activated BV-2 microglia. Subsequently, in vivo grafting of mFMSCs and mEFs revealed no difference in microglial and astroglial responses toward the cellular grafts. However, mFMSC grafts displayed a lower degree of neoangiogenesis compared to mEF grafts, thereby potentially explaining the lower cell number able to survive in mFMSC grafts. In summary, our results suggest that physiological differences between fibroblast-like cell populations might lie at the basis of variations in histopathological and/or clinical outcome following cell grafting in mouse brain.
Assuntos
Encéfalo/patologia , Embrião de Mamíferos/citologia , Membranas Extraembrionárias/citologia , Fibroblastos/transplante , Células Estromais/transplante , Animais , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Imunofenotipagem , Interferon gama/farmacologia , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Células Estromais/citologia , Células Estromais/metabolismo , Transplante Homólogo , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Preclinical animal studies involving intracerebral (stem) cell grafting are gaining popularity in many laboratories due to the reported beneficial effects of cell grafting on various diseases or traumata of the central nervous system (CNS). In this chapter, we describe a histological workflow to characterize and quantify cellular events following neural and fibroblast(-like) stem cell grafting in healthy and demyelinated CNS tissue. First, we provide standardized protocols to isolate and culture eGFP(+) neural and fibroblast(-like) stem cells from embryonic mouse tissue. Second, we describe flow cytometric procedures to determine cell viability, eGFP transgene expression, and the expression of different stem cell lineage markers. Third, we explain how to induce reproducible demyelination in the CNS of mice by means of cuprizone administration, a validated mouse model for human multiple sclerosis. Fourth, the technical procedures for cell grafting in the CNS are explained in detail. Finally, an optimized and validated workflow for the quantitative histological analysis of cell graft survival and endogenous astroglial and microglial responses is provided.