Induction of NTPDase1/CD39 by Reactive Microglia and Macrophages Is Associated With the Functional State During EAE.

Marija Jakovljevic,Iva Bozic,Irena Lavrnja,Jean Sévigny,Ana Milosevic,Sanja Pekovic,Nadezda Nedeljkovic,Danijela Laketa,Danijela Savic,Ivana Bjelobaba

doi:10.3389/fnins.2019.00410

Abstract

Purinergic signaling is critically involved in neuroinflammation associated with multiple sclerosis (MS) and its major inflammatory animal model, experimental autoimmune encephalomyelitis (EAE). Herein, we explored the expression of ectonucleoside triphosphate diphosphohydrolase1 (NTPDase1/CD39) in the spinal cord, at the onset (Eo), peak (Ep), and end (Ee) of EAE. Several-fold increase in mRNA and in NTPDase1 protein levels were observed at Eo and Ep. In situ hybridization combined with fluorescent immunohistochemistry showed that reactive microglia and infiltrated mononuclear cells mostly accounted for the observed increase. Colocalization analysis revealed that up to 80% of Iba1 immunoreactivity and ∼50% of CD68 immunoreactivity was colocalized with NTPDase1, while flow cytometric analysis revealed that ∼70% of mononuclear infiltrates were NTPDase1+ at Ep. Given the main role of NTPDase1 to degrade proinflammatory ATP, we hypothesized that the observed up-regulation of NTPDase1 may be associated with the transition between proinflammatory M1-like to neuroprotective M2-like phenotype of microglia/macrophages during EAE. Functional phenotype of reactive microglia/macrophages that overexpress NTPDase1 was assessed by multi-image colocalization analysis using iNOS and Arg1 as selective markers for M1 and M2 reactive states, respectively. At the peak of EAE NTPDase1 immunoreactivity showed much higher co-occurrence with Arg1 immunoreactivity in microglia and macrophages, compared to iNOS, implying its stronger association with M2-like reactive phenotype. Additionally, in ∼80% of CD68 positive cells NTPDase1 was coexpressed with Arg1 compared to negligible fraction coexpresing iNOS and ∼15% coexpresing both markers, additionally indicating prevalent association of NTPDase1 with M2-like microglial/macrophages phenotype at Ep. Together, our data suggest an association between NTPDase1 up-regulation by reactive microglia and infiltrated macrophages and their transition toward antiinflammatory phenotype in EAE.

Highlights

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS), characterized by focal neurodegenerative and demyelinating lesions (Lassmann, 2018)
The aim of the present study was to identify cell types that are responsible for massive up-regulation of NTPDase1 in the spinal cord of animals affected by EAE, and the potential pathophysiological significance of such changes
The expression of expression profile of NTPDase1 at mRNA (Entpd1) mRNA and NTPDase1 protein increased with the onset of symptoms, and reached a maximum at the peak of the disease, being about 3- and 2-fold higher than in control, respectively

Summary

Introduction

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS), characterized by focal neurodegenerative and demyelinating lesions (Lassmann, 2018). The disease-associated tissue injury is the consequence of neuroinflammation, conducted by CNS resident reactive microglia and astrocytes, and infiltrated cells, mainly Th1, Th17 and monocytes/macrophages (Lassmann, 2014). The accumulated data obtained in EAE model, point to microglia/macrophages and astrocytes as critical players in neuroinflammation associated with MS/EAE (Mensah-Brown et al, 2011; Bjelobaba et al, 2018). In MS, the subsequent cross-talk between reactive microglia/macrophages and activated astrocytes usually leads to self-sustained chronic neuroinflammation and progressive/irreversible neurodegeneration (Brosnan and Raine, 2013; Lassmann, 2018), whereas in the acute EAE models, the full-blown neuroinflammation turns into resolution, with ensuing myelin repair and tissue recovery. The accumulated data show that the course of the pathogenic event, chronic vs. acute, depends above all, on the functional state of activated microglia and astrocytes (Brosnan and Raine, 2013; Cherry et al, 2014)

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