Publicação
Balance between pro-inflammatory/anti-inflammatory indicators of SOD1G93A microglia in steady conditions and modification by immunomodulation
| Resumo: | Amyotrophic Lateral Sclerosis (ALS) is the third most common neurodegenerative disease, mostly sporadic, with limited identified targets, biomarkers and therapeutic options. The most widely used animal model and experimental cellular models to study ALS pathological mechanisms are based on mutations in the anti-oxidant protein SOD1, particularly that of G93A. ALS affects mainly motor neurons, but it is widely accepted that immune unbalance plays a crucial role in the ALS disease, and microglial dysfunction is described to be associated with neuronal injury influencing disease onset and progression. As the immune cells of the central nervous system, microglia produce inflammatory responses towards an insult by secreting pro-inflammatory mediators to the extracellular milieu in the form of soluble factors, or in membrane-bound vesicles called exosomes, an important component in intercellular communication and in disease dissemination. In this thesis we aimed to better understand the role of microglia in ALS disease using the mutant SOD1G93A microglia, and assessing their reactivity upon the immunostimulation by lipopolysaccharide (LPS), and immunomodulation by glycoursodeoxycholic acid (GUDCA) and vinyl sulfone (VS), having in mind the goal of fighting ALS neurodegeneration. For that, we assessed microglia function/dysfunction and reactivity after human SOD1 overexpression in the N9 cell line, either wild type (hSOD1WT) or mutated in G93A (hSOD1G93A), alone or treated with LPS, and when exposed to GUDCA and VS, known for their potential anti-inflammatory effects. Data showed that overexpression of hSOD1WT in N9 cells leads to a decrease in all analyzed pro- and anti-inflammatory markers, whereas hSOD1G93A increases both pro-inflammatory TNF-α, IL-1β, MHCII and HMGB1 gene expression levels, together with anti-inflammatory Arg1 and SOCS1 indicators, and reduces iNOS, Fizz1, IL-10, TLR4, miR-125b and miR-21. Interestingly we found an elevated cargo of miR-155 and miR-146a in hSOD1G93A microglia-derived exosomes. Upon LPS exposure, all cells switched from ramified into amoeboid morphology. LPS-treated transgenic microglia showed equivalent pro-inflammatory markers, when compared to LPS-treated naïve cells. However, they revealed decreased levels of the anti-inflammatory Arg1, Fizz1 and IL-10, thus reducing the ability to later balance the microglia reactivity to the insult. Surprisingly, cells also evidenced reduced miR-155 expression, what may even compromise an adequate pro-inflammatory response. In contrast with hSOD1WT cells, SOD1G93A microglia displayed decreased gene expression of S100B and equal of TNF-α mRNA, when compared to naïve cells. Additionally, the ability of ingesting a high number of beads (≥ 11) was found diminished. Treatment with GUDCA or VS decreased the cell body area of reactive microglia, and SOCS1 and Arg1 mRNA expression. Nevertheless, both immunomodulators increased TLR4, as well as reduced IL-1β and S100B gene expression, which may represent benefits for response to selected insults, while protecting from destructive secondary damage, respectively. In addition, though it decreased cellular MFG-E8 and enhanced miR-125b in exosomes, GUDCA markedly increased the cellular gene expression of the anti-inflammatory IL-10. On the other hand, VS was the only one able to reduce the pro-inflammatory MMP-9 activity and to elevate the exosomal cargo in the anti-inflammatory miR-21. In conclusion, this work demonstrates the advantageous hSOD1WT overexpression in balancing pro- and anti-inflammatory mediators in microglial cells, but overall that upregulation of hSOD1G93A increases their reactivity and may have a detrimental role in reducing their wound repair ability after insult, thus causing homeostatic imbalance between anti-inflammatory and pro-inflammatory gene expression mediators. In addition, the study also highlights that, although with different potential roles, both VS and GUDCA may have benefits over specific hSOD1G93A polarized microglia subtypes. |
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| Autores principais: | Ezequiel, Catarina Alexandra Barbosa |
| Assunto: | hSOD1G93A microglia activation glycoursodeoxycholic acid vinyl sulfone mIRNAs lipopolysaccharide Teses de mestrado - 2017 |
| Ano: | 2017 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Amyotrophic Lateral Sclerosis (ALS) is the third most common neurodegenerative disease, mostly sporadic, with limited identified targets, biomarkers and therapeutic options. The most widely used animal model and experimental cellular models to study ALS pathological mechanisms are based on mutations in the anti-oxidant protein SOD1, particularly that of G93A. ALS affects mainly motor neurons, but it is widely accepted that immune unbalance plays a crucial role in the ALS disease, and microglial dysfunction is described to be associated with neuronal injury influencing disease onset and progression. As the immune cells of the central nervous system, microglia produce inflammatory responses towards an insult by secreting pro-inflammatory mediators to the extracellular milieu in the form of soluble factors, or in membrane-bound vesicles called exosomes, an important component in intercellular communication and in disease dissemination. In this thesis we aimed to better understand the role of microglia in ALS disease using the mutant SOD1G93A microglia, and assessing their reactivity upon the immunostimulation by lipopolysaccharide (LPS), and immunomodulation by glycoursodeoxycholic acid (GUDCA) and vinyl sulfone (VS), having in mind the goal of fighting ALS neurodegeneration. For that, we assessed microglia function/dysfunction and reactivity after human SOD1 overexpression in the N9 cell line, either wild type (hSOD1WT) or mutated in G93A (hSOD1G93A), alone or treated with LPS, and when exposed to GUDCA and VS, known for their potential anti-inflammatory effects. Data showed that overexpression of hSOD1WT in N9 cells leads to a decrease in all analyzed pro- and anti-inflammatory markers, whereas hSOD1G93A increases both pro-inflammatory TNF-α, IL-1β, MHCII and HMGB1 gene expression levels, together with anti-inflammatory Arg1 and SOCS1 indicators, and reduces iNOS, Fizz1, IL-10, TLR4, miR-125b and miR-21. Interestingly we found an elevated cargo of miR-155 and miR-146a in hSOD1G93A microglia-derived exosomes. Upon LPS exposure, all cells switched from ramified into amoeboid morphology. LPS-treated transgenic microglia showed equivalent pro-inflammatory markers, when compared to LPS-treated naïve cells. However, they revealed decreased levels of the anti-inflammatory Arg1, Fizz1 and IL-10, thus reducing the ability to later balance the microglia reactivity to the insult. Surprisingly, cells also evidenced reduced miR-155 expression, what may even compromise an adequate pro-inflammatory response. In contrast with hSOD1WT cells, SOD1G93A microglia displayed decreased gene expression of S100B and equal of TNF-α mRNA, when compared to naïve cells. Additionally, the ability of ingesting a high number of beads (≥ 11) was found diminished. Treatment with GUDCA or VS decreased the cell body area of reactive microglia, and SOCS1 and Arg1 mRNA expression. Nevertheless, both immunomodulators increased TLR4, as well as reduced IL-1β and S100B gene expression, which may represent benefits for response to selected insults, while protecting from destructive secondary damage, respectively. In addition, though it decreased cellular MFG-E8 and enhanced miR-125b in exosomes, GUDCA markedly increased the cellular gene expression of the anti-inflammatory IL-10. On the other hand, VS was the only one able to reduce the pro-inflammatory MMP-9 activity and to elevate the exosomal cargo in the anti-inflammatory miR-21. In conclusion, this work demonstrates the advantageous hSOD1WT overexpression in balancing pro- and anti-inflammatory mediators in microglial cells, but overall that upregulation of hSOD1G93A increases their reactivity and may have a detrimental role in reducing their wound repair ability after insult, thus causing homeostatic imbalance between anti-inflammatory and pro-inflammatory gene expression mediators. In addition, the study also highlights that, although with different potential roles, both VS and GUDCA may have benefits over specific hSOD1G93A polarized microglia subtypes. |
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