Unravelling the immune signature of herpes zoster: Insights into pathophysiology and the HLA risk profile.

The varicella-zoster virus (VZV) infects over 95% of the population. VZV reactivation causes herpes zoster (HZ), known as shingles, primarily affecting the elderly and immunocompromised individuals. However, HZ can also occur in otherwise healthy individuals. We analyzed the immune signature and risk profile in HZ patients using a genome-wide association study across different UK Biobank HZ cohorts. Additionally, we conducted one of the largest HZ HLA association studies to date, coupled with transcriptomic analysis of pathways underlying HZ susceptibility. Our findings highlight the significance of the MHC locus for HZ development, identifying five protective and four risk HLA alleles. This demonstrates that HZ susceptibility is largely governed by variations in the MHC. Furthermore, functional analyses revealed the upregulation of type I interferon and adaptive immune responses. These findings provide fresh molecular insights into the pathophysiology and the activation of innate and adaptive immune responses triggered by symptomatic VZV reactivation.

Transcriptomic and proteomic profiling of bi-partite and tri-partite murine iPSC-derived neurospheroids under steady-state and inflammatory condition.

induced-pluripotent stem cell (iPSC)-derived neurospheroid (NSPH) models are an emerging in vitro toolkit to study the influence of inflammatory triggers on neurodegeneration and repair in a 3D neural environment. In contrast to their human counterpart, the absence of murine iPSC-derived NSPHs for profound characterisation and validation studies is a major experimental research gap, even though they offer the only possibility to truly compare or validate in vitro NSPH responses with in vivo brain responses. To contribute to these developments, we here describe the generation and characterisation of 5-week-old CX3CR1eGFP+/- CCR2RFP+/- murine (m)iPSC-derived bi-partite (neurons + astrocytes) and tri-partite (neurons + astrocytes + microglia) NSPH models that can be subjected to cellular activation following pro-inflammatory stimulation. First, cytokine analysis demonstrates that both bi-partite and tri-partite NSPHs can be triggered to release IL6 and CXCL10 following three days of stimulation with, respectively, TNFα + IL1ß + IFNγ and LPS + IFNγ. Additionally, immunocytochemical analysis for G3BP1 and PABPC1 revealed the development of stress granules in both bi-partite and tri-partite NSPHs after 3 days of stimulation. To further investigate the observed signs of inflammatory response and cellular stress, we performed an untargeted transcriptomic and proteomic analysis of bi- and tri-partite NSPHs under steady-state and inflammatory conditions. Here, using the combined differential gene and protein expression profiles between unstimulated and stimulated NSPHs, Ingenuity Pathway Analysis (IPA) confirms the activation of canonical pathways associated with inflammation and cellular stress in both bi-partite and tri-partite NSPHs. Moreover, our multi-omics analysis suggests a higher level of downstream inflammatory responses, impairment of homeostatic and developmental processes, as well as activation of cell death processes in stimulated tri-partite NSPHs compared to bi-partite NSPHs. Concluding, these results emphasise the advantages of including microglia in NSPH research to study inflammation-induced neurodegeneration in a 3D neural environment.

Transmembrane protein 119 is neither a specific nor a reliable marker for microglia.

Microglia are the resident innate immune cells of the central nervous system (CNS) parenchyma. To determine the impact of microglia on disease development and progression in neurodegenerative and neuroinflammatory diseases, it is essential to distinguish microglia from peripheral macrophages/monocytes, which are eventually equally recruited. It has been suggested that transmembrane protein 119 (TMEM119) serves as a reliable microglia marker that discriminates resident microglia from blood-derived macrophages in the human and murine brain. Here, we investigated the validity of TMEM119 as a microglia marker in four in vivo models (cuprizone intoxication, experimental autoimmune encephalomyelitis (EAE), permanent filament middle cerebral artery occlusion (fMCAo), and intracerebral 6-hydroxydopamine (6-OHDA) injections) as well as post mortem multiple sclerosis (MS) brain tissues. In all applied animal models and post mortem MS tissues, we found increased densities of ionized calcium-binding adapter molecule 1+ (IBA1+ ) cells, paralleled by a significant decrease in TMEM119 expression. In addition, other cell types in peripheral tissues (i.e., follicular dendritic cells and brown adipose tissue) were also found to express TMEM119. In summary, this study demonstrates that TMEM119 is not exclusively expressed by microglia nor does it label all microglia, especially under cellular stress conditions. Since novel transgenic lines have been developed to label microglia using the TMEM119 promotor, downregulation of TMEM119 expression might interfere with the results and should, thus, be considered when working with these transgenic mouse models.

Macrophage-based delivery of interleukin-13 improves functional and histopathological outcomes following spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) elicits a robust neuroinflammatory reaction which, in turn, exacerbates the initial mechanical damage. Pivotal players orchestrating this response are macrophages (Mφs) and microglia. After SCI, the inflammatory environment is dominated by pro-inflammatory Mφs/microglia, which contribute to secondary cell death and prevent regeneration. Therefore, reprogramming Mφ/microglia towards a more anti-inflammatory and potentially neuroprotective phenotype has gained substantial therapeutic interest in recent years. Interleukin-13 (IL-13) is a potent inducer of such an anti-inflammatory phenotype. In this study, we used genetically modified Mφs as carriers to continuously secrete IL-13 (IL-13 Mφs) at the lesion site. METHODS: Mφs were genetically modified to secrete IL-13 (IL-13 Mφs) and were phenotypically characterized using qPCR, western blot, and ELISA. To analyze the therapeutic potential, the IL-13 Mφs were intraspinally injected at the perilesional area after hemisection SCI in female mice. Functional recovery and histopathological improvements were evaluated using the Basso Mouse Scale score and immunohistochemistry. Neuroprotective effects of IL-13 were investigated using different cell viability assays in murine and human neuroblastoma cell lines, human neurospheroids, as well as murine organotypic brain slice cultures. RESULTS: In contrast to Mφs prestimulated with recombinant IL-13, perilesional transplantation of IL-13 Mφs promoted functional recovery following SCI in mice. This improvement was accompanied by reduced lesion size and demyelinated area. The local anti-inflammatory shift induced by IL-13 Mφs resulted in reduced neuronal death and fewer contacts between dystrophic axons and Mφs/microglia, suggesting suppression of axonal dieback. Using IL-4Rα-deficient mice, we show that IL-13 signaling is required for these beneficial effects. Whereas direct neuroprotective effects of IL-13 on murine and human neuroblastoma cell lines or human neurospheroid cultures were absent, IL-13 rescued murine organotypic brain slices from cell death, probably by indirectly modulating the Mφ/microglia responses. CONCLUSIONS: Collectively, our data suggest that the IL-13-induced anti-inflammatory Mφ/microglia phenotype can preserve neuronal tissue and ameliorate axonal dieback, thereby promoting recovery after SCI.

Promising Strategies for the Development of Advanced In Vitro Models with High Predictive Power in Ischaemic Stroke Research.

Although stroke is one of the world's 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 ischaemic stroke patients is still missing. In particular, the high attrition rate of neuroprotective compounds in clinical studies should make us question the ability of in vitro models currently used for ischaemic stroke research to recapitulate human ischaemic responses with sufficient fidelity. The ischaemic stroke field would greatly benefit from the implementation of more complex in vitro models with improved physiological relevance, next to traditional in vitro and in vivo models in preclinical studies, to more accurately predict clinical outcomes. In this review, we discuss current in vitro models used in ischaemic stroke research and describe the main factors determining the predictive value of in vitro models for modelling human ischaemic stroke. In light of this, human-based 3D models consisting of multiple cell types, either with or without the use of microfluidics technology, may better recapitulate human ischaemic responses and possess the potential to bridge the translational gap between animal-based in vitro and in vivo models, and human patients in clinical trials.

Application of Perinatal Derivatives on Oncological Preclinical Models: A Review of Animal Studies.

The increasing cancer incidence has certified oncological management as one of the most critical challenges for the coming decades. New anticancer strategies are still needed, despite the significant advances brought to the forefront in the last decades. The most recent, promising therapeutic approaches have benefitted from the application of human perinatal derivatives (PnD), biological mediators with proven benefits in several fields beyond oncology. To elucidate preclinical results and clinic outcomes achieved in the oncological field, we present a narrative review of the studies resorting to animal models to assess specific outcomes of PnD products. Recent preclinical evidence points to promising anticancer effects offered by PnD mediators isolated from the placenta, amniotic membrane, amniotic fluid, and umbilical cord. Described effects include tumorigenesis prevention, uncontrolled growth or regrowth inhibition, tumor homing ability, and adequate cell-based delivery capacity. Furthermore, PnD treatments have been described as supportive of chemotherapy and radiological therapies, particularly when resistance has been reported. However, opposite effects of PnD products have also been observed, offering support and trophic effect to malignant cells. Such paradoxical and dichotomous roles need to be intensively investigated. Current hypotheses identify as explanatory some critical factors, such as the type of the PnD biological products used or the manufacturing procedure to prepare the tissue/cellular treatment, the experimental design (including human-relevant animal models), and intrinsic pathophysiological characteristics. The effective and safe translation of PnD treatments to clinical practice relies on the collaborative efforts of all researchers working with human-relevant oncological preclinical models. However, it requires proper guidelines and consensus compiled by experts and health workers who accurately describe the methodology of tissue collection, PnD isolation, manufacturing, preservation, and delivery to the final user.

Murine induced pluripotent stem cell-derived neuroimmune cell culture models emphasize opposite immune-effector functions of interleukin 13-primed microglia and macrophages in terms of neuroimmune toxicity.

Cellular models of induced pluripotent stem cell (iPSC)-derived microglia and macrophages are an emerging toolbox to investigate neuroinflammation in vitro. We previously demonstrated that murine iPSC-microglia and iPSC-macrophages display phenotypical activation properties highly comparable to microglia and macrophages in vivo. Here we extended the characterization of iPSC-microglia and iPSC-macrophages with the analysis of their transcriptome profile. Next, these cellular models were employed to evaluate neuroimmune toxicity in vitro and to investigate the immune-modulatory properties of interleukin 13 (IL13), a cytokine known for its ability to protect against neuroinflammation-induced pathology by modulating microglia and macrophage activation. iPSC-microglia and iPSC-macrophages, in co-culture with astrocyte-committed neural stem cells (NSC), were (pre)treated with IL13 and stimulated with lipopolysaccharide (LPS) and interferon γ (IFNγ), to assess how IL13 modulates their inflammatory response. Additionally, the use of luciferase-expressing NSC (Luc-NSC) allowed real-time monitoring of immune-mediated neurotoxicity. Despite the known anti-inflammatory properties of IL13, iPSC-microglia primed with IL13 before LPS + IFNγ stimulation significantly increased NO secretion. This was associated with a marked reduction of the luminescence signal produced by Luc-NSC. Interestingly, we observed that IL13 signaling has a divergent functional outcome in microglia as compared to macrophages, as for the latter no major alterations in NO release and Luc-NSC viability were observed upon IL13 (pre)treatment. Finally, the striking IL13-induced upregulation of NO secretion by microglia under pro-inflammatory conditions was confirmed in vivo, where intracerebral delivery of IL13 increased inducible nitric oxide synthase mRNA expression. Concluding, we applied iPSC-derived neuroimmune cell culture models to identify distinct neuroimmune (toxicity) responses of microglia and macrophages to IL13-based immune modulation.

Focal white matter lesions induce long-lasting axonal degeneration, neuroinflammation and behavioral deficits.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) with episodes of inflammatory demyelination and remyelination. While remyelination has been linked with functional recovery in MS patients, there is evidence of ongoing tissue damage despite complete myelin repair. In this study, we investigated the long-term consequences of an acute demyelinating white matter CNS lesion. For this purpose, acute demyelination was induced by 5-week-cuprizone intoxication in male C57BL/6 J mice, and the tissues were examined after a 7-month recovery period. While myelination and oligodendrocyte densities appeared normal, ongoing axonal degeneration and glia cell activation were found in the remyelinated corpus callosum. Neuropathologies were paralleled by subtle gait abnormalities evaluated using DigiGait™ high speed ventral plane videography. Gene array analyses revealed increased expression levels of various inflammation related genes, among protein kinase c delta (PRKCD). Immunofluorescence stains revealed predominant microglia/macrophages PRKCD expression in both, cuprizone tissues and post-mortem MS lesions. These results support the hypothesis that chronic microglia/macrophages driven tissue injury represents a key aspect of progressive neurodegeneration and functional decline in MS.

Cochlin Deficiency Protects Aged Mice from Noise-Induced Hearing Loss.

Several studies have shown that type IV fibrocytes, located in the spiral ligament, degenerate first after noise exposure. Interestingly, this is the region where Coch expression is most abundant. As it is suggested that cochlin plays a role in our innate immune system, our goal is to investigate hearing thresholds and inner ear inflammation after noise exposure in Coch knockout (Coch-/-) mice compared to Coch wildtype (Coch+/+) mice. Animals were randomly allocated to a noise exposure group and a control group. Vestibular and auditory testing was performed at 48 h and one week after noise exposure. Whole mount staining and cryosectioning of the cochlea was performed in order to investigate hair cells, spiral ganglion neurons, inner ear inflammation, Coch expression and fibrocyte degeneration. Hearing assessment revealed that Coch+/+ mice had significantly larger threshold shifts than Coch-/- mice after noise exposure. We were unable to identify any differences in hair cells, neurons, fibrocytes and influx of macrophages in the inner ear between both groups. Interestingly, Coch expression was significantly lower in the group exposed to noise. Our results indicate that the absence of Coch has a protective influence on hearing thresholds after noise exposure, but this is not related to reduced inner ear inflammation in the knockout.

Neuroprotective modulation of microglia effector functions following priming with interleukin 4 and 13: current limitations in understanding their mode-of-action.

In recent years the long-standing theory of microglia's properties for dual polarization towards a pro- or anti-inflammatory phenotype has been deeply challenged. Furthermore, the elucidation of microglia ontogenesis exposed intrinsic differences between microglia and peripheral myeloid cells, thereby further underscoring the need to re-evaluate microglia-specific activation behavior, especially within an inflamed central nervous system (CNS) environment. This review critically summarizes recent literature on the in vitro and in vivo response of murine microglia to the immune-modulatory cytokines interleukin 4 (IL4) and interleukin 13 (IL13), i.e. those driving the so-called anti-inflammatory phenotype. Here we highlight several pivotal factors that may influence experimental outcome and/or interpretation of in vitro and in vivo studies evaluating microglia's phenotypical and functional properties upon IL4/IL13 treatment. Finally, the current therapeutic relevance of IL4/IL13-induced microglia activation in both acute and chronic CNS disorders is discussed.

Clinical and immunological control of experimental autoimmune encephalomyelitis by tolerogenic dendritic cells loaded with MOG-encoding mRNA.

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.

Murine iPSC-derived microglia and macrophage cell culture models recapitulate distinct phenotypical and functional properties of classical and alternative neuro-immune polarisation.

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, lipopolysaccharide + 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.

Targeted intracerebral delivery of the anti-inflammatory cytokine IL13 promotes alternative activation of both microglia and macrophages after stroke.

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.

Combination of cuprizone and experimental autoimmune encephalomyelitis to study inflammatory brain lesion formation and progression.

Brain-intrinsic degenerative cascades are a proposed factor driving inflammatory lesion formation in multiple sclerosis (MS) patients. We recently described a model combining noninflammatory cytodegeneration (via cuprizone) with the classic active experimental autoimmune encephalomyelitis (Cup/EAE model), which exhibits inflammatory forebrain lesions. Here, we describe the histopathological characteristics and progression of these Cup/EAE lesions. We show that inflammatory lesions develop at various topographical sites in the forebrain, including white matter tracts and cortical and subcortical grey matter areas. The lesions are characterized by focal demyelination, discontinuation of the perivascular glia limitans, focal axonal damage, and neutrophil granulocyte extravasation. Transgenic mice with enhanced green fluorescent protein-expressing microglia and red fluorescent protein-expressing monocytes reveal that both myeloid cell populations contribute to forebrain inflammatory infiltrates. EAE-triggered inflammatory cerebellar lesions were augmented in mice pre-intoxicated with cuprizone. Gene expression studies suggest roles of the chemokines Cxcl10, Ccl2, and Ccl3 in inflammatory lesion formation. Finally, follow-up experiments in Cup/EAE mice with chronic disease revealed that forebrain, but not spinal cord, lesions undergo spontaneous reorganization and repair. This study underpins the significance of brain-intrinsic degenerative cascades for immune cell recruitment and, in consequence, MS lesion formation.

In Vivo Interleukin-13-Primed Macrophages Contribute to Reduced Alloantigen-Specific T Cell Activation and Prolong Immunological Survival of Allogeneic Mesenchymal Stem Cell Implants.

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.

Multipotent adult progenitor cells improve the hematopoietic function in myelodysplasia.

BACKGROUND AIMS: Myelodysplastic syndromes (MDS) are a group of clonal stem cell disorders affecting the normal hematopoietic differentiation process and leading to abnormal maturation and differentiation of all blood cell lineages. Treatment options are limited, and there is an unmet medical need for effective therapies for patients with severe cytopenias. METHODS: We demonstrate that multipotent adult progenitor cells (MAPC) improve the function of hematopoietic progenitors derived from human MDS bone marrow (BM) by significantly increasing the frequency of primitive progenitors as well as the number of myeloid colonies. RESULTS: This effect was more pronounced in a non-contact culture, indicating the importance of soluble factors produced by the MAPC cells. Moreover, the cells did not stimulate the growth of the abnormal MDS clone, as shown by fluorescent in situ hybridization analysis on BM cells from patients with a known genetic abnormality. We also demonstrate that MAPC cells can provide stromal support for patient-derived hematopoietic cells. When MAPC cells were intravenously injected into a mouse model of MDS, they migrated to the site of injury and increased the hematopoietic function in diseased mice. DISCUSSION: The preclinical studies undertaken here indicate an initial proof of concept for the use of MAPC cell therapy in patients with MDS-related severe and symptomatic cytopenias and should pave the way for further investigation in clinical trials.

Intracerebral delivery of the M2 polarizing cytokine interleukin 13 using mesenchymal stem cell implants in a model of temporal lobe epilepsy in mice.

OBJECTIVES: Neuroinflammation plays a critical role in the pathophysiology of mesial temporal lobe epilepsy. We aimed to evaluate whether intracerebral transplantation of interleukin 13-producing mesenchymal stem cells (IL-13 MSCs) induces an M2 microglia/macrophage activation phenotype in the hippocampus with an epileptogenic insult, thereby providing a neuroprotective environment with reduced epileptogenesis. METHODS: Genetically engineered syngeneic IL-13 MSCs or vehicle was injected within the hippocampus 1 week before the intrahippocampal kainic acid-induced status epilepticus (SE) in C57BL/6J mice. Neuroinflammation was evaluated at disease onset as well as during the chronic epilepsy period (9 weeks). In addition, continuous video-electroencephalography (EEG) (vEEG) monitoring was obtained during the chronic epilepsy period (between 6 and 9 weeks after SE). RESULTS: Evaluation of vEEG recordings suggested that IL-13 MSC grafts did not affect the severity and duration of SE or the seizure burden during the chronic epilepsy period, when compared to the vehicle treated SE mice. An M2-activation phenotype was induced in microglia/macrophages that infiltrated the -13 MSC graft site, as evidenced by the arginase1 expression at the graft site at both the 2-week and 9-week time-points. However, M2-activated immune cells were rarely observed outside the graft site and, accordingly, the neuroinflammatory response or cell loss related to SE induction was not altered by IL-13 MSC grafting. Moreover, an increase in the proportion of F4/80+ cells was observed in the IL-13 MSC group compared to the controls. SIGNIFICANCE: Our data suggest that MSC-based IL-13 delivery to induce M2 glial activation does not provide any neuroprotective or disease-modifying effects in a mouse model of epilepsy. Moreover, use of cell grafting to deliver bioactive compounds for modulating neuroinflammation may have confounding effects in disease pathology of epilepsy due to the additional immune response generated by the grafted cells.

Interleukin-13 immune gene therapy prevents CNS inflammation and demyelination via alternative activation of microglia and macrophages.

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.

Interleukin-25 is detrimental for recovery after spinal cord injury in mice.

BACKGROUND: The cytokine, interleukin (IL)-25, is thought to be critically involved in inducing a type 2 immune response which may contribute to regeneration after central nervous system (CNS) trauma. We investigated whether applying recombinant IL-25, locally or systemically, in a mouse model of spinal cord injury (SCI) improves functional and histological recovery. FINDINGS: Repeated systemic administration of IL-25 did not influence functional recovery following SCI. In contrast, a single local administration of IL-25 significantly worsened locomotor outcome, which was evident from a decreased Basso mouse scale (BMS) score compared with phosphate-buffered saline (PBS)-treated controls. This was accompanied by a significant increase in lesion size, demyelination, and T helper cell infiltration. CONCLUSIONS: These data show for the first time that IL-25 is either ineffective when applied systemically or detrimental to spinal cord recovery when applied locally. Our findings question the potential neuroprotective role of IL-25 following CNS trauma.

Intracerebral transplantation of interleukin 13-producing mesenchymal stem cells limits microgliosis, oligodendrocyte loss and demyelination in the cuprizone mouse model.

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.