Down-regulation of NTPDase2 and ADP-sensitive P2 Purinoceptors Correlate with Severity of Symptoms during Experimental Autoimmune Encephalomyelitis

Abstract

The present study explores tissue and cellular distribution of ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and the gene and protein expression in rat spinal cord during the course of experimental autoimmune encephalomyelitis (EAE). Given that NTPDase2 hydrolyzes ATP with a transient accumulation of ADP, the expression of ADP-sensitive P2 purinoceptors was analyzed as well. The autoimmune disease was actively induced in Dark Agouti female rats and the changes were analyzed 10, 15 and 29 days after the induction. These selected time points correspond to the onset (Eo), peak (Ep) and recovery (Er) from EAE. In control animals, NTPDase2 was confined in the white matter, in most of the glial fibrillary acidic protein (GFAP)-immunoreactive (ir) astrocytes and in a considerable number of nestin-ir cells, while the other cell types were immunonegative. Immunoreactivity corresponding to NTPDase2 decreased significantly at Eo and Ep and then returned to the baseline levels at Er. The preservation of the proportion of GFAP single-labeled and GFAP/NTPDase2 double-labeled elements along the course of EAE indicated that changes in NTPDase2-ir occurred at fibrous astrocytes that typically express NTPDase2 in normal conditions. Significant downregulation of P2Y1 and P2Y12 receptor proteins at Eo and several-fold induction of P2Y12 and P2Y13 receptor proteins at Ep and/or Er were observed implying that the pathophysiological process in EAE may be linked to ADP signaling. Cell-surface expression of NTPDase2, NTPDase1/CD39 and ecto-5′-nucleotidase (eN/CD73) was analyzed in CD4+ T cells of a draining lymph node by fluorescence-activated cell sorting. The induction of EAE was associated with a transient decrease in a number of CD4+ NTPDase2+ T cells in a draining lymph node, whereas the recovery was characterized by an increase in NTPDase2+ cells in both CD4+ and CD4− cell populations. The opposite was found for NTPDase1/CD39+ and eN/CD73+ cells, which slightly increased in number with progression of the disease, particularly in CD4− cells, and then decreased in the recovery. Finally, CD4+ NTPDase2+ cells were never observed in the spinal cord parenchyma. Taken together, our results suggest that the process of neuroinflammation in EAE may be associated with altered ADP signaling.

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

“…A volume equivalent to 1 μg of RNA was used for reverse transcription with High Capacity cDNA Reverse transcription kit (Applied Biosystems, Foster City, CA, United States). cDNA was then diluted 10 times and these probes were used for real-time PCR standard protocol described previously (Jakovljevic et al, 2017) with QuantStudioTM 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, United States). For negative control, cDNA template was omitted from PCR mixture.…”

“…In rodent brain, NTPDase1 is constitutively expressed by microglia, endothelial and smooth muscle cells of the vasculature (Braun et al, 2000), thus being involved in microglial activation (Farber and Kettenmann, 2006), regulation of blood-brain barrier function (Bynoe et al, 2015), and control of cerebral blood flow (Sevigny et al, 2002). On the other hand, NTPDase2, mostly associated with white matter astrocytes and neural progenitors (Braun et al, 2003; Jakovljevic et al, 2017), produces a ligand for ADP-sensitive P2Y 1 , P2Y 12 , and P2Y 13 receptors present at astrocytes, microglial cells, oligodendrocytes and neurons (Abbracchio et al, 2009; Pérez-Sen et al, 2015). Finally, e-5NT/CD73, abundantly expressed by astrocytes, vascular endothelium, and choroid plexus epithelium (Jacobson and Gao, 2006; Mills et al, 2008; Lavrnja et al, 2015), generates adenosine (Zimmermann, 1992) which activates G-protein coupled adenosine receptor subtypes, A 1 , A 2A , A 2B , and A 3 (Fredholm et al, 2011).…”

“…Expression of NTPDase1 in dendritic cells supress EAE development through decreasing Th1 and Th17 activation, while vaccination with IL-27 treated dendritic cells reduce severity of chronic EAE due to their induction of NTPDase1 (Mascanfroni et al, 2013). Dynamic alterations in the activity of the whole ectonucleotidase enzyme chain have been reported in EAE (Lavrnja et al, 2009), due to significant induction of e-5NT and down-regulation of NTPDase2 at mRNA and protein level, mostly by reactive astrocytes in the spinal cord of affected animals (Lavrnja et al, 2015; Jakovljevic et al, 2017). Significant induction of NTPDase1 protein has been demonstrated in EAE (Jakovljevic et al, 2017) and traumatic brain injury model, where it was upregulated by reactive microglia (Nedeljkovic et al, 2006).…”

“…Dynamic alterations in the activity of the whole ectonucleotidase enzyme chain have been reported in EAE (Lavrnja et al, 2009), due to significant induction of e-5NT and down-regulation of NTPDase2 at mRNA and protein level, mostly by reactive astrocytes in the spinal cord of affected animals (Lavrnja et al, 2015; Jakovljevic et al, 2017). Significant induction of NTPDase1 protein has been demonstrated in EAE (Jakovljevic et al, 2017) and traumatic brain injury model, where it was upregulated by reactive microglia (Nedeljkovic et al, 2006). On the other hand, induction of NTPDase1 and e-5NT was shown in vitro in IL-4 stimulated macrophages exhibiting M2 phenotype (Zanin et al, 2012).…”

The Purinergic System as a Pharmacological Target for the Treatment of Immune-Mediated Inflammatory Diseases

“…CD39 on DCs was found to be important for limiting the onset and severity of EAE (Mascanfroni et al, 2013) and CD39-deficient CD4 T cells showed an enhanced capability to drive EAE progression (Wang et al, 2014b). Nucleoside, triphosphate diphosphohydrolase-2, which can also degrade ATP, was found to be downregulated in EAE, and this decrease was associated with the severity of disease symptoms (Jakovljevic et al, 2017).…”