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Protocol to assess the tolerogenic properties of adoptively transferred dendritic cells during murine experimental autoimmune encephalomyelitis

ABSTRACT: Summary By their capacity to induce peripheral T cell tolerance, dendritic cells (DCs) present a promising target cell and therapeutic strategy for treatment of several autoimmune diseases including multiple sclerosis (MS). This protocol describes how to determine the tolerogenic capacities of DCs in the context of the murine MS model, experimental autoimmune encephalomyelitis (EAE). We provide a step-by-step instruction for EAE induction, antigen-loaded bone-marrow-derived-DC (BM-DC) generation, adoptive cell transfer, and analysis of DC-mediated changes in regulatory T cell populations. For complete details on the use and execution of this protocol, please refer to Vogel et al. (2022). Graphical abstract Highlights • Protocol to induce EAE in mice and score for disease phenotypes• Generation and adoptive transfer of MOG35-55-loaded BM-DCs• Protocol to assess the tolerogenic properties of transferred DCs• Flow cytometric analysis of Tregs in secondary lymphoid organs Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. By their capacity to induce peripheral T cell tolerance, dendritic cells (DCs) present a promising target cell and therapeutic strategy for treatment of several autoimmune diseases including multiple sclerosis (MS). This protocol describes how to determine the tolerogenic capacities of DCs in the context of the murine MS model, experimental autoimmune encephalomyelitis (EAE). We provide a step-by-step instruction for EAE induction, antigen-loaded bone-marrow-derived-DC (BM-DC) generation, adoptive cell transfer, and analysis of DC-mediated changes in regulatory T cell populations.

SUBMITTER: Vogel A

PROVIDER: S-EPMC9440594 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Project description:BackgroundAlthough 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.MethodsIn 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.ResultsTreatment 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.ConclusionsElectroporation 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.

Project description:AimsDaphnetin, a coumarin derivative extracted from Daphne odora var. marginata, has been reported to have antiinflammatory and immunosuppressive properties. Our previous study indicated that it was able to remarkably suppress the neuroinflammation and suggested its potential application in treating neuroinflammatory diseases. Multiple sclerosis (MS), a Th cell-mediated autoimmune disease, is the most common inflammatory demyelinating disease of the central nervous system (CNS). We examined whether daphnetin treatment can protect mice against experimental autoimmune encephalomyelitis (EAE), an animal model for MS.MethodsTo assess the effect of daphnetin in neuroinflammatory diseases, the EAE mice were established and treated with daphnetin at 8 mg/kg for 28 days. The severity of neuroinflammation and demyelination in the spinal cords was examined histopathologically. Infiltration of CD4(+) T cells into the CNS was assessed by immunohistochemistry, and the cytokine production was determined by ELISA. Meanwhile, the effect of daphnetin on the activity of dendritic cells (DCs) was evaluated, as assessed by DCs' capability to express surface markers, secrete cytokines, and activate naïve CD4(+) T cells. Furthermore, we explored the molecular mechanisms whereby DAPH regulated DCs' activity and thereby CD4(+) T cell responses.ResultsThe administration of daphnetin markedly alleviated the clinical symptoms of EAE and reduced the CNS inflammation and demyelination in experimental mice. Th1 and Th17 cell responses were profoundly repressed in daphnetin-treated EAE mice. Mechanistically, daphnetin treatment significantly repressed the activation, maturation, and antigen-presenting capability of DCs. NF-κB signaling was significantly reduced in daphnetin-treated DCs, along with a concomitant induction of heme oxygenase-1, a negative regulator of inflammatory signaling.ConclusionsOur findings for the first time demonstrate the property of daphnetin in regulating DCs' function and subsequently Th development. Given the low or absent toxicity associated with daphnetin, our data may suggest a novel safe and effective approach to control autoimmune neuroinflammation.

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Protocol to assess the tolerogenic properties of adoptively transferred dendritic cells during murine experimental autoimmune encephalomyelitis

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