254 research outputs found
Spatiotemporal evolution of early innate immune responses triggered by neural stem cell grafting
No full textINTRODUCTION: Transplantation of neural stem cells (NSCs) is increasingly suggested to become part of future therapeutic approaches to improve functional outcome of various central nervous system disorders. However, recently it has become clear that only a small fraction of grafted NSCs display long-term survival in the (injured) adult mouse brain. Given the clinical invasiveness of NSC grafting into brain tissue, profound characterisation and understanding of early post-transplantation events is imperative to claim safety and efficacy of cell-based interventions.
METHODS: Here, we applied in vivo bioluminescence imaging (BLI) and post-mortem quantitative histological analysis to determine the localisation and survival of grafted NSCs at early time points post-transplantation.
RESULTS: An initial dramatic cell loss (up to 80% of grafted cells) due to apoptosis could be observed within the first 24 hours post-implantation, coinciding with a highly hypoxic NSC graft environment. Subsequently, strong spatiotemporal microglial and astroglial cell responses were initiated, which stabilised by day 5 post-implantation and remained present during the whole observation period. Moreover, the increase in astrocyte density was associated with a high degree of astroglial scarring within and surrounding the graft site. During the two-week follow up in this study, the NSC graft site underwent extensive remodelling with NSC graft survival further declining to around 1% of the initial number of grafted cells.
CONCLUSIONS: The present study quantitatively describes the early post-transplantation events following NSC grafting in the adult mouse brain and warrants that such intervention is directly associated with a high degree of cell loss, subsequently followed by strong glial cell responses
JCB909055 Supplemental Material - Supplemental material for CCR2 deficiency in monocytes impairs angiogenesis and functional recovery after ischemic stroke in mice
No full textSupplemental material, JCB909055 Supplemental Material for CCR2 deficiency in monocytes impairs angiogenesis and functional recovery after ischemic stroke in mice by Jordi Pedragosa, Francesc Miró-Mur, Amaia Otxoa-de-Amezaga, Carles Justicia, Francisca Ruíz-Jaén, Peter Ponsaerts, Manolis Pasparakis and Anna M Planas in Journal of Cerebral Blood Flow & Metabolism</p
Modelling neuroinflammation in a dish using murine iPSC-derived microglia and macrophages
No full textAbstract: Neuroinflammation is defined as the development of an inflammatory response in the central nervous system (CNS) upon trauma or disease. The post-insult neuroinflammatory environment is generally colonized by two myeloid cell populations: endogenous parenchymal microglia and blood-derived monocytes that infiltrate the CNS in response to a compromised blood-brain-barrier. Both these cell populations are characterized by multiple functional states and can either display highly pro-inflammatory properties or promote the resolution of inflammation and provide support for tissue regeneration. Steering the activation of microglia and CNS infiltrating monocytes in favour of the latter, hold great promises as a future treatment option for CNS pathologies. Our research group has previously demonstrated that local administration of the immune-modulating cytokine interleukin 13 (IL13) drives microglia and infiltrating monocytes toward the anti-inflammatory phenotype and improves disease outcome in several mouse models of CNS disease. Unfortunately, as the existing cell culture systems to evaluate microglia/monocyte immune properties often overlook the important contribution of CNS environmental signalling to neuroinflammatory processes, a comprehensive in vitro analysis of microglia and monocyte/macrophage behaviour was not possible. The main aim of this PhD research was to establish and validate a novel in vitro platform to investigate similarities and differences in immune reactivity for both microglia and monocytes under pro-inflammatory stimulation, as well as following immunomodulatory treatment with IL13. In the first part of this doctoral thesis, cellular models of murine induced pluripotent stem cell (iPSC)-derived microglia and iPSC-derived macrophages were generated and characterized. Analysis of their transcriptome profile and evaluation of their phenotypical and functional properties following classical and alternative immune stimulation revealed strong similarities with endogenous microglia and brain-infiltrating monocytes/macrophages, respectively, thus confirming the cellular identity of in vitro differentiated iPSC-microglia and iPSC-macrophages. Furthermore, we provide evidence that a brain-like culture milieu strongly modulates the phenotypic and inflammatory properties of iPSC-microglia and iPSC-macrophages, suggesting that CNS-specific environmental cues play an important role in the restrained immune activation potential of murine microglia as compared to murine monocytes. These findings imply that the development of novel immunomodulating therapeutic approaches will need to (re)consider both cell types independently and in combination within a neural environment. In the second part of this doctoral thesis, we aimed at unravelling the immune mechanism(s) responsible for the observed in vivo clinical and/or histopathological benefits following IL13-mediated therapeutic intervention. To this end, we explored how pro-inflammatory activation of iPSC-microglia and iPSC-macrophage can be modulated by IL13. Interestingly, our results indicate that IL13 has divergent signalling outcome in microglia as compared to macrophages. While iPSC-macrophages profoundly inhibited the release of pro-inflammatory mediators upon IL13 administration, iPSC-microglia cultures exacerbated inflammation-induced oxidative stress in the presence of IL13. This striking observation was additionally confirmed in vivo following intracerebral delivery of IL13, thus emphasizing that IL13 might operate through multiple and even opposite mechanisms within the inflamed CNS and not simply via a binary activation as previously postulated. In conclusion, the technological and scientific findings reported in this doctoral thesis reveal the value of iPSC-derived neuro-immune cell culture models as an in vitro tool to unravel the different contribution of microglia and monocytes to the development and resolution of neuroinflammatory responses, as well as to investigate novel therapeutic interventions for inflammation-associated CNS injury or disease
Development, characterization and application of luminescent human iPSC-derived neurospheroids in ischemic stroke research
No full textAbstract: Although stroke is one of the world\u2019s leading causes of death and disability, and more than a thousand candidate neuroprotective drugs have been proposed based on extensive in vitro and animal-based research, an effective neuroprotective/restorative therapy for ischemic stroke patients is still missing. Especially the high attrition rate of neuroprotective compounds in clinical studies should make us question the ability of in vitro stroke models currently used for ischemic stroke research to recapitulate human ischemic responses with sufficient fidelity. The ischemic stroke field would greatly benefit from the implementation a physiologically relevant human in vitro stroke model. The development and application of human iPSC-derived 3D neurospheroid models represent an appropriate approach to fulfill this need. In a first part, a luminescent human iPSC-derived neurospheroid model enabling the real-time read-out of neural viability after ischemia-like conditions was developed and characterized. By depriving neurospheroids from oxygen and glucose, the ability of the applied bioluminescent system to detect neurotoxicity was demonstrated. Moreover, differences in behaviour after oxygen-glucose deprivation between different ages of neurospheroids were observed, whereby 1-week-old but not 4-week-old neurospheroids displayed spontaneous recovery. This underscores the need for more mature neurospheroids in in vitro stroke research that more faithfully recapitulate the in vivo adult situation. Furthermore, evaluation of the pan-caspase inhibitor Z-VAD-FMK in the established model demonstrated its inability to increase overall neural survival in neurospheroids in contrast to a 2D culture of the same hiPSC-derived neural stem cells, where neuroprotection was observed. This exemplifies how the increased complexity of spheroid models can result in a different outcome when testing neuroprotective compounds. In a second part, the foundation was laid to further increase the complexity and predictivity of the developed human neurospheroid model by generating more mature, multicellular neurospheroids. In a first set of explorative experiments, culture conditions were optimized in order to obtain neurospheroids with increased maturity and the presence of astrocytes. Hereby, increasing the culture time of the neurospheroids markedly increased neuronal maturity as well as the spontaneous development of astrocytes. It was also noted that culture of neurospheroids in selected differentiation media did not give rise to astrocytes for the evaluated time points despite their ability to display faster differentiation and maturation. In a second set of explorative experiments, the integration of hematopoietic progenitors cells into neurospheroids was explored for the future creation of an immune-competent, microglia-enriched neurospheroid model for ischemic stroke. Comparison of different time points for the addition of hematopoietic progenitor cells to neurospheroids, showed higher integration efficiency when added to pre-established neurospheroids
Immunomodulatory Therapy for Spinal Cord Injury
No full textSCI is a devastating pathology which has a significant impact on life expectancy and quality, and also bears considerable economic burden. Despite considerable progress in palliative care, there is currently no therapeutic intervention available which leads to functional recovery. Therefore, there is an urgent need to develop new strategies and therapies. Inflammatory responses are a major component of the secondary injury phase and play a key role in regulating the pathogenesis of acute and chronic SCI. However, the continual and dual role of the neuroinflammatory response leaves it difficult to decide upon a single modulatory strategy. Additionally, the role of certain immunomodulatory cytokines which are up- or down-regulated in response to SCI still remains unclear. In this thesis, we focus on the type-2 cytokines: IL-25 and IL-13 and investigate their therapeutic potential for treatment of SCI. Understanding the interaction of these factors with the surrounding microenvironment and exploiting their modulatory effects on cells such as microglia and macrophages, may provide a vital therapeutic tool in developing strategies for treating CNS trauma
Towards improved understanding of neuroglobins significance in cytoprotection and neurodevelopment using overexpression and knockout models
No full textImmunomodulatory Therapy for Spinal Cord Injury
No full textSCI is a devastating pathology which has a significant impact on life expectancy and quality, and also bears considerable economic burden. Despite considerable progress in palliative care, there is currently no therapeutic intervention available which leads to functional recovery. Therefore, there is an urgent need to develop new strategies and therapies. Inflammatory responses are a major component of the secondary injury phase and play a key role in regulating the pathogenesis of acute and chronic SCI. However, the continual and dual role of the neuroinflammatory response leaves it difficult to decide upon a single modulatory strategy. Additionally, the role of certain immunomodulatory cytokines which are up- or down-regulated in response to SCI still remains unclear. In this thesis, we focus on the type-2 cytokines: IL-25 and IL-13 and investigate their therapeutic potential for treatment of SCI. Understanding the interaction of these factors with the surrounding microenvironment and exploiting their modulatory effects on cells such as microglia and macrophages, may provide a vital therapeutic tool in developing strategies for treating CNS trauma
Interleukin-13 as a master regulator of alternative microglia/macrophage activation : modulation of allograft- and pathology-associated immune responses
No full textAntiviral immune response dynamics during varicella-zoster virus infection in a human iPSC-derived neuronal model and in herpes zoster patients
No full textAbstract: Varicella-zoster virus (VZV) naturally infects more than 95% of the population which results in varicella (i.e., chickenpox). Upon infection, VZV particles may access sensory nerve endings in the skin and can be transported to neural ganglia, via retrograde axonal transport, where VZV latency is established. In more than one out of four people, VZV will eventually reactivate from this latent state causing herpes zoster (HZ, i.e. shingles). In addition, a substantial part of HZ patients suffers from long-lasting pain after the rash has disappeared, adding to the burden of disease. Although effective vaccines against HZ are now available, vaccination coverage worldwide is relatively low. Hence, VZV and HZ remain a major burden for the foreseeable future. The humanotropic and neurotropic nature of VZV is a major hurdle in the field which hampers the advancement of our understanding of VZV infection dynamics and pathogenesis. Due to VZV\u2019s strictly humanotropic character, no small animal model can fully recapitulate VZV disease. To overcome this, we aimed to develop a human iPSC-derived neuronal model in a chambered system that separates cell bodies and axon termini, which allows mimicking the natural route of VZV infection via axon termini. We showed that, following VZV infection, activation of interferon-stimulated genes (ISGs) depends on exogenous interferon-\u3b1 (IFN\u3b1). Indeed, VZV infection of hiPSC-neurons via their axon termini, resulted in VZV spread throughout the neuronal cultures, without the production of IFN\u3b1 or ISGs which have direct antiviral effector functions. In contrast, exogenous treatment of hiPSC-neurons with IFN\u3b1 resulted in a reduction of VZV spread and in the upregulation of ISGs. Taken together, we suggest that whilst hiPSC-neurons are good IFN\u3b1-responders, they seem poor IFN\u3b1-producers thereby being unable to limit VZV spread in hiPSC-neuronal cultures. Importantly, this may imply that other cell types within the nervous system are essential as IFN\u3b1-producers. Hence, we believe that future studies investigating innate immune responses to VZV infection of neuronal cells should be carried out by including additional immune cell types in the cultures. In the second part of this thesis, we investigated the adaptive immune response dynamics during VZV reactivation in HZ patients. We found that VZV-specific antibody titers were still significantly higher one year after the HZ episode as compared to controls, raising the possibility that continuous subclinical VZV reactivation may contribute to high VZV IgG titers in HZ patients. In the last part of this thesis, transcriptomic analyses on whole blood from HZ patients and controls were carried out. Our data showed activation of several host immune pathways during VZV reactivation, especially related to the type I IFN response, but also related to adaptive immune responses. In addition, we found that upregulation of complement component 4 binding protein alpha (C4BPA), a major inhibitor of the complement system, may be a potential risk factor for the development of HZ but this needs further investigation. Indeed, a genome-wide association study could reveal if genetic variants are implicated in HZ disease
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