Contributions
Abstract: P402
Type: Poster
Abstract Category: Pathology and pathogenesis of MS - Experimental models
Multiple sclerosis (MS) is a chronic demyelinating disorder of the central nervous system (CNS) leading to progressive neurological disability. In rodent models of de/remyelination the removal of myelin debris via phagocytosis facilitates axonal regeneration and re-modelling and creates an environment that is permissive for remyelination and repair. It is generally believed that the prime suspects for carrying out this phagocytosis are microglia and infiltrating peripheral macrophages. Therefore modulating functional polarity of these cells to enhance phagocytic efficacy may present a promising strategy in the treatment of MS. Utilising pHrodo® technology we set up a high throughput in vitro assay to quantitatively measure and compare myelin phagocytosis in unstimulated “M0”, pro-inflammatory “M1” (48h interferon-gamma (IFN-γ) and lipopolysaccharide (LPS) stimulation) and anti-inflammatory “M2” (48h interleukin (IL)-4 and IL-13 stimulation) polarised mouse microglia. In addition we used flow cytometry to study the impact of myelin on cell polarity based on the expression of cell surface markers: cluster of differentiation (CD) -86, major histocompatibility complex class II (MHCII) and CD206. We examined a range of myelin doses (1, 5, 10 and 15µg/well) and found that myelin exposure induced a dose-dependent cell death, which was potentiated in M1 polarised cells. Based on these data we screened the effects of myelin on microglial phenotype using the lowest dose of myelin (5µg/well) that showed reduced impact on cell survival. We report that myelin induced changes in the expression of cell surface markers of M0 microglia and up-regulated CD206, a marker associated with an alternative M2 phenotype. The expression profile in M2 polarised cells was similar to that of M0 cells, whereas M1 cells sustained their pro-inflammatory phenotype and showed greater expression of MHCII compared to M0 and M2 cells. We also demonstrated that M2 polarised microglia exhibited a 3-4 fold greater capacity to phagocytose myelin debris at 24-30 hours than compared to M1 polarised cells. In conclusion, we have established a plate based assay enabling the quantification of myelin phagocytosis in primary murine microglia and have shown that an alternatively activated phenotype may be necessary for the effective clearance of myelin debris. Phenotypic profiling indicates that myelin exposure also induces an M2 switch, which may be inhibited in a pro-inflammatory environment.
Disclosure:
Louise Nicol: Nothing to disclose
Guglielmo Rosignoli: Nothing to disclose
Peter Thornton: Nothing to disclose
John Stockley: Nothing to disclose
Beatriz Gomez Perez-Nievas: Nothing to disclose
Josquin Nys: Nothing to disclose
Matthew Sleeman: Nothing to disclose
Clare Jones: Nothing to disclose
Abstract: P402
Type: Poster
Abstract Category: Pathology and pathogenesis of MS - Experimental models
Multiple sclerosis (MS) is a chronic demyelinating disorder of the central nervous system (CNS) leading to progressive neurological disability. In rodent models of de/remyelination the removal of myelin debris via phagocytosis facilitates axonal regeneration and re-modelling and creates an environment that is permissive for remyelination and repair. It is generally believed that the prime suspects for carrying out this phagocytosis are microglia and infiltrating peripheral macrophages. Therefore modulating functional polarity of these cells to enhance phagocytic efficacy may present a promising strategy in the treatment of MS. Utilising pHrodo® technology we set up a high throughput in vitro assay to quantitatively measure and compare myelin phagocytosis in unstimulated “M0”, pro-inflammatory “M1” (48h interferon-gamma (IFN-γ) and lipopolysaccharide (LPS) stimulation) and anti-inflammatory “M2” (48h interleukin (IL)-4 and IL-13 stimulation) polarised mouse microglia. In addition we used flow cytometry to study the impact of myelin on cell polarity based on the expression of cell surface markers: cluster of differentiation (CD) -86, major histocompatibility complex class II (MHCII) and CD206. We examined a range of myelin doses (1, 5, 10 and 15µg/well) and found that myelin exposure induced a dose-dependent cell death, which was potentiated in M1 polarised cells. Based on these data we screened the effects of myelin on microglial phenotype using the lowest dose of myelin (5µg/well) that showed reduced impact on cell survival. We report that myelin induced changes in the expression of cell surface markers of M0 microglia and up-regulated CD206, a marker associated with an alternative M2 phenotype. The expression profile in M2 polarised cells was similar to that of M0 cells, whereas M1 cells sustained their pro-inflammatory phenotype and showed greater expression of MHCII compared to M0 and M2 cells. We also demonstrated that M2 polarised microglia exhibited a 3-4 fold greater capacity to phagocytose myelin debris at 24-30 hours than compared to M1 polarised cells. In conclusion, we have established a plate based assay enabling the quantification of myelin phagocytosis in primary murine microglia and have shown that an alternatively activated phenotype may be necessary for the effective clearance of myelin debris. Phenotypic profiling indicates that myelin exposure also induces an M2 switch, which may be inhibited in a pro-inflammatory environment.
Disclosure:
Louise Nicol: Nothing to disclose
Guglielmo Rosignoli: Nothing to disclose
Peter Thornton: Nothing to disclose
John Stockley: Nothing to disclose
Beatriz Gomez Perez-Nievas: Nothing to disclose
Josquin Nys: Nothing to disclose
Matthew Sleeman: Nothing to disclose
Clare Jones: Nothing to disclose