Abstract

Multiple sclerosis (MS) is characterized by macrophage accumulation and inflammatory infiltrates into the CNS contributing to demyelination. Because purinergic P2X7 receptor (P2X7R) is known to be abundantly expressed on cells of the hematopoietic lineage and of the nervous system, we further investigated its phenotypic expression in MS and experimental autoimmune encephalomyelitis conditions. By quantitative reverse transcription polymerase chain reaction and flow cytometry, we analyzed the P2X7R expression in human mononuclear cells of peripheral blood from stable and acute relapsing-remitting MS phases. Human monocytes were also challenged in vitro with pro-inflammatory stimuli such as the lipopolysaccharide, or the P2X7R preferential agonist 2′(3′)-O-(4 Benzoylbenzoyl)adenosine 5′-triphosphate, before evaluating P2X7R protein expression. Finally, by immunohistochemistry and immunofluorescence confocal analysis, we investigated the P2X7R expression in frontal cortex from secondary progressive MS cases. We demonstrated that P2X7R is present and inhibited on peripheral monocytes isolated from MS donors during the acute phase of the disease, moreover it is down-regulated in human monocytes after pro-inflammatory stimulation in vitro. P2X7R is instead up-regulated on astrocytes in the parenchyma of frontal cortex from secondary progressive MS patients, concomitantly with monocyte chemoattractant protein-1 chemokine, while totally absent from microglia/macrophages or oligodendrocytes, despite the occurrence of inflammatory conditions. Our results suggest that inhibition of P2X7R on monocytes and up-regulation in astrocytes might contribute to sustain inflammatory mechanisms in MS. By acquiring further knowledge about P2X7R dynamics and identifying P2X7R as a potential marker for the disease, we expect to gain insights into the molecular pathways of MS.

Highlights

  • Peripheral and central mechanisms provide insights into the cellular basis of neuroinflammation that leads to severe demyelination and neurodegeneration in multiple sclerosis (MS)

  • Given the abundant expression of P2X7 receptor (P2X7R) on monocytes [38,39,40], in this work we firstly confirmed its presence in circulating monocytes from healthy donors (HD) and demonstrated its down-regulation in stable and acute MS patients compared with HD, by quantitative reverse transcription polymerase chain reaction (RT-qPCR) (Figure 1A)

  • FACS analysis confirmed a significant decrease of cluster of differentiation 14 (CD14)/P2X7R-positive monocytes only in MS acute patients compared with stable MS and HD conditions (Figure 1B)

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Summary

Introduction

Peripheral and central mechanisms provide insights into the cellular basis of neuroinflammation that leads to severe demyelination and neurodegeneration in multiple sclerosis (MS). Astrocytes appear to have a dual role in MS, depending on the disease status and lesion topography, and contributing in both pathogenic alterations and beneficial repair [2,3,4,5,6] In examining those mechanisms that converge in causing inflammatory demyelination, the analysis of frontal cortex constitutes a convenient experimental platform, because profuse lesions in cerebral cortex constitute a significant proportion of MS pathology, and characterize the evolution from a relapsing/ remitting early phase into a secondary progressive MS (SPMS) [7,8,9]. It was reported that P2X7R knockdown causes a more severe EAE and that lymphocyte from P2X7R−/− mice proliferate more vigorously in vitro, producing reduced levels of IFN-γ and NO, suggesting an important role for this receptor in MS lymphocyte homeostasis [33]

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