Abstract
BackgroundMicroglia are major players in the pathogenesis of multiple sclerosis (MS) and may play a dual role in disease progression. The activation status of microglia in vivo is highly dynamic and occurs as a continuum, with the pro-inflammatory and anti-inflammatory phenotypes on either end of this spectrum. Little is known about in vivo dynamics of microglia phenotypes in MS due to the lack of diagnostic tools. Positron emission tomography (PET) imaging is a powerful non-invasive technique that allows real-time imaging of microglia activation phenotypes in the central nervous system, depending on the availability of selective PET tracers. Our objective is to investigate and characterize the expression of the purinergic receptors P2Y12R and P2X7R as potential targets for PET tracer development and subsequent PET imaging in order to evaluate the dynamics of microglia status in vivo.MethodsWe used immunohistochemical analysis to explore the expression of P2Y12R and P2X7R in experimental autoimmune encephalomyelitis (EAE) post-mortem tissues and different stages of well-characterized MS lesions. We evaluated by quantitative real-time polymerase chain reaction the expression of P2Y12R and P2X7R in human polarized microglia, and we performed autoradiography binding assay with radiolabeled P2Y12R and P2X7R antagonists using MS and rat EAE tissues.ResultsHere, we demonstrate that P2X7R is associated with a pro-inflammatory phenotype of human microglia in vitro, and is highly expressed in microglia in MS lesions as well as during the peak of EAE. In contrast, P2Y12R was associated with an anti-inflammatory phenotype in human microglia in vitro and was expressed at lower levels in active inflammatory MS lesions compared to normal-appearing white matter (NAWM) and similarly in EAE, while its expression increased in the remission phase of EAE. Binding of radiolabeled tracers specific for P2Y12R and P2X7R on ex vivo tissues validated the value of these receptors as PET imaging targets for microglia phenotypes in vivo.ConclusionOur results suggest that P2Y12R and P2X7R are excellent targets for PET imaging to discriminate distinct microglia phenotypes in MS. Ultimately, this may provide insight into the role of microglia in disease progression and monitor novel treatment strategies to alter microglia phenotype.
Highlights
Microglia are major players in the pathogenesis of multiple sclerosis (MS) and may play a dual role in disease progression
Dynamic of expression of P2Y12R and P2X7R in human polarized microglia To investigate the regulation of P2Y12R and P2X7R expression, human microglia were freshly isolated from 10 different adult post-mortem brains, cultured for 7 days and polarized into an inflammatory phenotype with IFN-γ/LPS or anti-inflammatory phenotype with IL-4 as described previously [3] (Additional file 1: Figure S1)
The increased expression of P2X7R in pro-inflammatory microglia and P2Y12R in anti-inflammatory microglia was observed at the protein level as shown by western blot from the same donors (Fig. 1d, e)
Summary
Microglia are major players in the pathogenesis of multiple sclerosis (MS) and may play a dual role in disease progression. The activation status of microglia in vivo is highly dynamic and occurs as a continuum, with the pro-inflammatory and anti-inflammatory phenotypes on either end of this spectrum. Anti-inflammatory microglia and macrophages are predominant in the recovery phase where they contribute to phagocytosis of myelin debris and associated axonal regeneration [11, 12]. This protective role was corroborated by several studies showing that injection of macrophages polarized towards a more anti-inflammatory phenotype inhibited or ameliorated EAE associated clinical signs in mice [13, 14]
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