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Plovamer acetate, but not glatiramer acetate, induces an anti-inflammatory phenotype in human monocytes
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Abstracts
469 Plovamer acetate, but not glatiramer acetate, induces an anti-inflammatory phenotype in human monocytes Chui-se Tham, Mike Webb, Kenneth Crook, Ursula Boschert Neurology eTIP, EMD Serono Research & Development Institute, Billerica, United States Background: Plovamer acetate (PA) is a copolymer mixture of 4 amino acids of defined ratio rationally designed to have improved efficacy over glatiramer acetate (GA). Aside from binding to major histocompatibility complex (MHC) II molecules, both copolymers (PA and GA) have been proposed to act directly on monocytes through an MHC II-independent mechanism. Objective: To compare the effect of PA versus GA in the in vitro polarization of human monocytes to M1 and M2 macrophage subsets. Methods: Monocytes from human blood were polarized for 6 days towards M1 and M2 phenotypes in a medium containing granulocyte macrophage colony-stimulating factor and macrophage colonystimulating factor, respectively. M1 cultures were activated using interferon gamma plus lipopolysaccharide, and M2a or M2c using interleukin (IL)-4 or IL-10, respectively. RNA was isolated and multiple transcripts were analyzed to demonstrate M1/M2 polarization. The effect of exposure to PA or GA during the period of polarization was then assessed. Results: Human monocytes were successfully polarized to M1/M2 subsets as indicated by the pattern of transcripts induced after activation. CD206 and CD163 expression was detected by fluorescence-activated cell sorting on M2 cells, reliably discriminating them from M1 cells. Released cytokines tumor necrosis factor alpha (TNFalpha), IL12p70, and IL-1beta served as markers of activated M1 cells, and these, together with COX2 and CD80, were also detected by qPCR in M1 cells. PA inhibited the expression of TNFalpha, COX2, and IL12p40 by M1-activated cells, while GA either increased the expression or had no effect. Conversely, PA significantly potentiated the expression of CD163 and CD209 by M2activated cells, while GA had no effect. Conclusion: M1/M2 polarizing cultures were successfully established with the use of a variety of markers to distinguish the different lineages. When PA or GA was included during the polarization period, PA had a negative effect on the expression of M1-associated markers and a positive effect on the expression of M2-associated markers. GA, in contrast, enhanced M1 markers and had no effect on M2 markers. Together, these findings suggest that unlike GA, PA suppresses the establishment of a proinflammatory M1 phenotype, while enhancing an anti-inflammatory M2 phenotype, supporting the development of PA as a disease-modifying drug.
In recent years, improving data have shown that under different pathological conditions, microglia can be either supportive for neurogenesis and homeostasis or even detrimental for neural repair. Even if the factors leading microglia to assume its different phenotypes are still unknown, it is tempting to speculate that its heterogeneity may reflect the variety of roles that microglia plays physiologically. According with very recent data, microglia develops from a specific precursor within the yolk sac and once reached the brain give rise to a self-renewal population independent from the monocyte-macrophage lineage. Microglia enters the developing brain from E8.5 to E9.5, just before the neurogenesis will start, suggesting a supportive role on developing neurons thus placing the embryonic environment as the best setting to investigate its protective or supportive phenotype. The first aim of our project was to identify a unique microglia gene expression profile during development. We compared brain's microglia gene expression at the middle of embryonic life, at E14, and then soon after birth at P1 with their myeloid counterparts taken from the liver at the very same stages. We performed a whole genome microarray on ex vivo sorted CD45 low/CD11b+ cells extracted from the brain and liver at E14 and at P1. We found only 385 genes differentially expressed between embryonic microglia compared to postnatal one but up to 2824 genes differentially expressed between the brain and liver (p b 0.01), among which 1022 were exclusively expressed on brain E14.5 and P1 microglia but absent in the liver counterpart. We then performed a whole genome microarray on adult brain microglia that we compared to the ones obtained from the developmental stages. Interesting, we found that microglia gene expression differs greatly between developmental and adult phase. We also identify 98 genes that are consistently expressed by microglia throughout every stages and absent from the myeloid counterpart and could represent a "core" of microglia specific genes. Our next step will be to evaluate microglia gene expression profile at different phases of experimental autoimmune encephalomyelitis (EAE) to see whether at some point its profile might resemble the one observed during development.
doi:10.1016/j.jneuroim.2014.08.224
611 Inflammatory dysregulation of blood monocytes in Parkinson's disease patients Veselin Grozdanova, Corinna Blierderhaeusera, Valerie Rotha, Kathrin Fundel-Clemensb, Lisa Zondlera, Wolfgang P. Ruf a, Bastian Hengererb, Jan Kassubeka, Albert C. Ludolpha, Jochen H. Weishaupta, Karin M. Danzera Neurology, Ulm University, Ulm, Germany; bDiv. Research Germany, Boehringer Ingelheim Pharma GmbH, Biberach/Riss, Germany
a
doi:10.1016/j.jneuroim.2014.08.223
552 Microglia's gene expression at different stages of evolution and under pathological conditions Francesca Grassivaro, Ramesh Menon, Cinthia Farina, Luca Muzio, Gianvito Martino Institute of Experimental Neurology (INSPE), University Vita Salute-San Raffaele, Milan, Italy
Despite extensive effort on studying inflammatory processes in the CNS of Parkinson's disease (PD) patients, implications of the peripheral immune system are still poorly understood. In particular, the role of peripheral blood monocytes remains elusive. Here, we set out to obtain a comprehensive picture of circulating myeloid cells in PD patients. We applied a human primary monocyte culture system and flow cytometry based techniques to functionally characterize monocytes from PD patients during disease. We found an enrichment of classical pro-inflammatory monocytes in the blood of PD patients along with an increase in the monocyte-recruiting chemoattractant protein CCL2. Monocytes from PD patients display a
Abstracts
469 Plovamer acetate, but not glatiramer acetate, induces an anti-inflammatory phenotype in human monocytes Chui-se Tham, Mike Webb, Kenneth Crook, Ursula Boschert Neurology eTIP, EMD Serono Research & Development Institute, Billerica, United States Background: Plovamer acetate (PA) is a copolymer mixture of 4 amino acids of defined ratio rationally designed to have improved efficacy over glatiramer acetate (GA). Aside from binding to major histocompatibility complex (MHC) II molecules, both copolymers (PA and GA) have been proposed to act directly on monocytes through an MHC II-independent mechanism. Objective: To compare the effect of PA versus GA in the in vitro polarization of human monocytes to M1 and M2 macrophage subsets. Methods: Monocytes from human blood were polarized for 6 days towards M1 and M2 phenotypes in a medium containing granulocyte macrophage colony-stimulating factor and macrophage colonystimulating factor, respectively. M1 cultures were activated using interferon gamma plus lipopolysaccharide, and M2a or M2c using interleukin (IL)-4 or IL-10, respectively. RNA was isolated and multiple transcripts were analyzed to demonstrate M1/M2 polarization. The effect of exposure to PA or GA during the period of polarization was then assessed. Results: Human monocytes were successfully polarized to M1/M2 subsets as indicated by the pattern of transcripts induced after activation. CD206 and CD163 expression was detected by fluorescence-activated cell sorting on M2 cells, reliably discriminating them from M1 cells. Released cytokines tumor necrosis factor alpha (TNFalpha), IL12p70, and IL-1beta served as markers of activated M1 cells, and these, together with COX2 and CD80, were also detected by qPCR in M1 cells. PA inhibited the expression of TNFalpha, COX2, and IL12p40 by M1-activated cells, while GA either increased the expression or had no effect. Conversely, PA significantly potentiated the expression of CD163 and CD209 by M2activated cells, while GA had no effect. Conclusion: M1/M2 polarizing cultures were successfully established with the use of a variety of markers to distinguish the different lineages. When PA or GA was included during the polarization period, PA had a negative effect on the expression of M1-associated markers and a positive effect on the expression of M2-associated markers. GA, in contrast, enhanced M1 markers and had no effect on M2 markers. Together, these findings suggest that unlike GA, PA suppresses the establishment of a proinflammatory M1 phenotype, while enhancing an anti-inflammatory M2 phenotype, supporting the development of PA as a disease-modifying drug.
In recent years, improving data have shown that under different pathological conditions, microglia can be either supportive for neurogenesis and homeostasis or even detrimental for neural repair. Even if the factors leading microglia to assume its different phenotypes are still unknown, it is tempting to speculate that its heterogeneity may reflect the variety of roles that microglia plays physiologically. According with very recent data, microglia develops from a specific precursor within the yolk sac and once reached the brain give rise to a self-renewal population independent from the monocyte-macrophage lineage. Microglia enters the developing brain from E8.5 to E9.5, just before the neurogenesis will start, suggesting a supportive role on developing neurons thus placing the embryonic environment as the best setting to investigate its protective or supportive phenotype. The first aim of our project was to identify a unique microglia gene expression profile during development. We compared brain's microglia gene expression at the middle of embryonic life, at E14, and then soon after birth at P1 with their myeloid counterparts taken from the liver at the very same stages. We performed a whole genome microarray on ex vivo sorted CD45 low/CD11b+ cells extracted from the brain and liver at E14 and at P1. We found only 385 genes differentially expressed between embryonic microglia compared to postnatal one but up to 2824 genes differentially expressed between the brain and liver (p b 0.01), among which 1022 were exclusively expressed on brain E14.5 and P1 microglia but absent in the liver counterpart. We then performed a whole genome microarray on adult brain microglia that we compared to the ones obtained from the developmental stages. Interesting, we found that microglia gene expression differs greatly between developmental and adult phase. We also identify 98 genes that are consistently expressed by microglia throughout every stages and absent from the myeloid counterpart and could represent a "core" of microglia specific genes. Our next step will be to evaluate microglia gene expression profile at different phases of experimental autoimmune encephalomyelitis (EAE) to see whether at some point its profile might resemble the one observed during development.
doi:10.1016/j.jneuroim.2014.08.224
611 Inflammatory dysregulation of blood monocytes in Parkinson's disease patients Veselin Grozdanova, Corinna Blierderhaeusera, Valerie Rotha, Kathrin Fundel-Clemensb, Lisa Zondlera, Wolfgang P. Ruf a, Bastian Hengererb, Jan Kassubeka, Albert C. Ludolpha, Jochen H. Weishaupta, Karin M. Danzera Neurology, Ulm University, Ulm, Germany; bDiv. Research Germany, Boehringer Ingelheim Pharma GmbH, Biberach/Riss, Germany
a
doi:10.1016/j.jneuroim.2014.08.223
552 Microglia's gene expression at different stages of evolution and under pathological conditions Francesca Grassivaro, Ramesh Menon, Cinthia Farina, Luca Muzio, Gianvito Martino Institute of Experimental Neurology (INSPE), University Vita Salute-San Raffaele, Milan, Italy
Despite extensive effort on studying inflammatory processes in the CNS of Parkinson's disease (PD) patients, implications of the peripheral immune system are still poorly understood. In particular, the role of peripheral blood monocytes remains elusive. Here, we set out to obtain a comprehensive picture of circulating myeloid cells in PD patients. We applied a human primary monocyte culture system and flow cytometry based techniques to functionally characterize monocytes from PD patients during disease. We found an enrichment of classical pro-inflammatory monocytes in the blood of PD patients along with an increase in the monocyte-recruiting chemoattractant protein CCL2. Monocytes from PD patients display a