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Cell-Based Delivery of Interleukin-13 Directs Alternative Activation of Macrophages Resulting in Improved Functional Outcome after Spinal Cord Injury.

ABSTRACT: The therapeutic effects of mesenchymal stem cell (MSC) transplantation following spinal cord injury (SCI) to date have been limited. Therefore, we aimed to enhance the immunomodulatory properties of MSCs via continuous secretion of the anti-inflammatory cytokine interleukin-13 (IL-13). By using MSCs as carriers of IL-13 (MSC/IL-13), we investigated their therapeutic potential, compared with non-engineered MSCs, in a mouse model of SCI. We show that transplanted MSC/IL-13 significantly improve functional recovery following SCI, and also decrease lesion size and demyelinated area by more than 40%. Further histological analyses in CX₃CR1^EGFP/+ CCR2^RFP/+ transgenic mice indicated that MSC/IL-13 significantly decrease the number of resident microglia and increase the number of alternatively activated macrophages. In addition, the number of macrophage-axon contacts in MSC/IL-13-treated mice was decreased by 50%, suggesting a reduction in axonal dieback. Our data provide evidence that transplantation of MSC/IL-13 leads to improved functional and histopathological recovery in a mouse model of SCI.

SUBMITTER: Dooley D

PROVIDER: S-EPMC5161742 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Cell-Based Delivery of Interleukin-13 Directs Alternative Activation of Macrophages Resulting in Improved Functional Outcome after Spinal Cord Injury.

Dooley Dearbhaile D Lemmens Evi E Vangansewinkel Tim T Le Blon Debbie D Hoornaert Chloé C Ponsaerts Peter P Hendrix Sven S

Stem cell reports 20161201 6

The therapeutic effects of mesenchymal stem cell (MSC) transplantation following spinal cord injury (SCI) to date have been limited. Therefore, we aimed to enhance the immunomodulatory properties of MSCs via continuous secretion of the anti-inflammatory cytokine interleukin-13 (IL-13). By using MSCs as carriers of IL-13 (MSC/IL-13), we investigated their therapeutic potential, compared with non-engineered MSCs, in a mouse model of SCI. We show that transplanted MSC/IL-13 significantly improve fu ...[more]

PMID: 27974221

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Project description:BackgroundMicroglia and macrophages (MG/M?) have a diverse range of functions depending on unique cytokine stimuli, and contribute to neural cell death, repair, and remodeling during central nervous system diseases. While IL-1 has been shown to exacerbate inflammation, it has also been recognized to enhance neuroregeneration. We determined the activating phenotype of MG/M? and the impact of IL-1 in an in vivo spinal cord injury (SCI) model of IL-1 knock-out (KO) mice. Moreover, we demonstrated the contribution of IL-1 to both the classical and alternative activation of MG in vitro using an adult MG primary culture.MethodsSCI was induced by transection of the spinal cord between the T9 and T10 vertebra in wild-type and IL-1 KO mice. Locomotor activity was monitored and lesion size was determined for 14 days. TNF? and Ym1 levels were monitored to determine the MG/M? activating phenotype. Primary cultures of MG were produced from adult mice, and were exposed to IFN? or IL-4 with and without IL-1?. Moreover, cultures were exposed to IL-4 and/or IL-13 in the presence and absence of IL-1?.ResultsThe locomotor activity and lesion area of IL-1 KO mice improved significantly after SCI compared with wild-type mice. TNF? production was significantly suppressed in IL-1 KO mice. Also, Ym1, an alternative activating MG/M? marker, did not increase in IL-1 KO mice, suggesting that IL-1 contributes to both the classical and alternative activation of MG/M?. We treated primary MG cultures with IFN? or IL-4 in the presence and absence of IL-1?. Increased nitric oxide and TNF? was present in the culture media and increased inducible NO synthase was detected in cell suspensions following co-treatment with IFN? and IL-1?. Expression of the alternative activation markers Ym1 and arginase-1 was increased after exposure to IL-4 and further increased after co-treatment with IL-4 and IL-1?. The phenotype was not observed after exposure of cells to IL-13.ConclusionsWe demonstrate here in in vivo experiments that IL-1 suppressed SCI in a process mediated by the reduction of inflammatory responses. Moreover, we suggest that IL-1 participates in both the classical and alternative activation of MG in in vivo and in vitro systems.

Project description:Background: Microglia and macrophages adopt a pro-inflammatory phenotype after spinal cord injury (SCI), what is thought to contribute to secondary tissue degeneration. We previously reported that this is due, in part, to the low levels of anti-inflammatory cytokines, such as IL-4. Since IL-13 and IL-4 share receptors and both cytokines drive microglia and macrophages towards an anti-inflammatory phenotype in vitro, here we studied whether administration of IL-13 and IL-4 after SCI leads to beneficial effects. Methods: We injected mice with recombinant IL-13 or IL-4 at 48 h after SCI and assessed their effects on microglia and macrophage phenotype and functional outcomes. We also performed RNA sequencing analysis of macrophages and microglia sorted from the injured spinal cords of mice treated with IL-13 or IL-4 and evaluated the metabolic state of these cells by using Seahorse technology. Results: We observed that IL-13 induced the expression of anti-inflammatory markers in microglia and macrophages after SCI but, in contrast to IL-4, it failed to mediate functional recovery. We found that these two cytokines induced different gene signatures in microglia and macrophages after SCI and that IL-4, in contrast to IL-13, shifted microglia and macrophage metabolism from glycolytic to oxidative phosphorylation. These findings were further confirmed by measuring the metabolic profile of these cells. Importantly, we also revealed that macrophages stimulated with IL-4 or IL-13 are not deleterious to neurons, but they become cytotoxic when oxidative metabolism is blocked. This suggests that the metabolic shift, from glycolysis to oxidative phosphorylation, is required to minimize the cytotoxic responses of microglia and macrophages. Conclusions: These results reveal that the metabolic fitness of microglia and macrophages after SCI contributes to secondary damage and that strategies aimed at boosting oxidative phosphorylation might be a novel approach to minimize the deleterious actions of microglia and macrophages in neurotrauma.

Project description:BackgroundSpinal 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.MethodsMφs 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.ResultsIn 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.ConclusionsCollectively, 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.

Project description:BackgroundAfter spinal cord injury (SCI), dysregulated or nonresolving inflammatory processes can severely disturb neuronal homeostasis and drive neurodegeneration. Although mesenchymal stromal cell (MSC)-based therapies have showed certain therapeutic efficacy, no MSC therapy has reached its full clinical goal. In this study, we examine interleukin-10 (IL10) genetically modified clinical-grade MSCs (IL10-MSCs) and evaluate their clinical safety, effectiveness, and therapeutic mechanism in a completely transected SCI mouse model.MethodsWe established stable IL10-overexpressing human umbilical-cord-derived MSCs through electric transduction and screened out clinical-grade IL10-MSCs according to the criteria of cell-based therapeutic products, which were applied to mice with completely transected SCI by repeated tail intravenous injections. Then we comprehensively investigated the motor function, histological structure, and nerve regeneration in SCI mice, and further explored the potential therapeutic mechanism after IL10-MSC treatment.ResultsIL10-MSC treatment markedly reinforced locomotor improvement, accompanied with decreased lesion volume, regeneration of axons, and preservation of neurons, compared with naïve unmodified MSCs. Further, IL10-MSC transplantation increased the ratio of microglia to infiltrated alternatively activated macrophages (M2), and reduced the ratio of classically activated macrophages (M1) at the injured spinal cord, meanwhile increasing the percentage of Treg and Th2 cells, and reducing the percentage of Th1 cells in the peripheral circulatory system. In addition, IL10-MSC administration could prevent apoptosis and promote neuron differentiation of neural stem cells (NSCs) under inflammatory conditions in vitro.ConclusionsIL10-MSCs exhibited a reliable safety profile and demonstrated promising therapeutic efficacy in SCI compared with naïve MSCs, providing solid support for future clinical application of genetically engineered MSCs.

Project description:Background: The recruitment of immune system cells into the central nervous system (CNS) has a profound effect on the outcomes of injury and disease. Glia-derived chemoattractants, including chemokines, play a pivotal role in this process. In addition, cytokines and chemokines influence the phenotype of infiltrating immune cells. Depending on the stimuli present in the local milieu, infiltrating macrophages acquire the classically activated M1 or alternatively activated M2 phenotypes. The polarization of macrophages into detrimental M1 versus beneficial M2 phenotypes significantly influences CNS pathophysiology. Earlier studies indicated that a toll-like receptor 9 (TLR9) antagonist modulates astrocyte-derived cytokine and chemokine release. However, it is not known whether these molecular changes affect astrocyte-induced chemotaxis and polarization of macrophages. The present studies were undertaken to address these issues.Methods: The chemotaxis and polarization of mouse peritoneal macrophages by spinal cord astrocytes were evaluated in a Transwell co-culture system. Arrays and ELISA were utilized to quantify chemokines in the conditioned medium (CM) of pure astrocyte cultures. Immunostaining for M1- and M2-specific markers characterized the macrophage phenotype. The percentage of M2 macrophages at the glial scar was determined by stereological approaches in mice sustaining a mid-thoracic spinal cord contusion injury (SCI) and intrathecally treated with oligodeoxynucleotide 2088 (ODN 2088), the TLR9 antagonist. Statistical analyses used two-tailed independent-sample t-test and one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. A p value <?0.05 was considered to be statistically significant.Results: ODN 2088-treated astrocytes significantly increased the chemotaxis of peritoneal macrophages via release of chemokine (C-C motif) ligand 1 (CCL1). Vehicle-treated astrocytes polarized macrophages into the M2 phenotype and ODN 2088-treated astrocytes promoted further M2 polarization. Reduced CCL2 and CCL9 release by astrocytes in response to ODN 2088 facilitated the acquisition of the M2 phenotype, suggesting that CCL2 and CCL9 are negative regulators of M2 polarization. The percentage of M2 macrophages at the glial scar was higher in mice sustaining a SCI and receiving ODN 2088 treatment as compared to vehicle-treated injured controls.Conclusions: TLR9 antagonism could create a favorable environment during SCI by supporting M2 macrophage polarization and chemotaxis via modulation of astrocyte-to-macrophage signals.

Dataset Information

Cell-Based Delivery of Interleukin-13 Directs Alternative Activation of Macrophages Resulting in Improved Functional Outcome after Spinal Cord Injury.

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Cell-Based Delivery of Interleukin-13 Directs Alternative Activation of Macrophages Resulting in Improved Functional Outcome after Spinal Cord Injury.

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