P2x7 receptors control demyelination and inflammation in the cuprizone model

Abstract

The contribution of P2x7 receptors to multiple sclerosis remains controversial, as both detrimental and beneficial effects resulting from P2x7 receptor loss-of-function have been reported in autoimmune models of the disease. Here we investigated the relevance of P2x7 receptors to de- and remyelination in the cuprizone model of T cell-independent myelin degeneration. Primary demyelination was induced by administration of 0.3% cuprizone in the diet for 3 and 6 weeks. Remyelination was studied in mice treated with the P2x7 receptor antagonists Brilliant Blue G (BBG, 50 ​mg/Kg) and JNJ-47965567 (30 ​mg/Kg) for 2 weeks following 6 weeks of cuprizone challenge. Toxic demyelination induced a robust up-regulation of P2x7 receptors mainly localized on microglial cells. In parallel, we measured increased expression of several NLPR3-inflammasome and M1 polarization-associated genes in demyelinated tissue. Notably, mice deficient in P2x7 receptors exhibited attenuated demyelination, reduced presence of M1 microglia and reactive astrocytes as well as blunted expression of pro-inflammatory genes in response to cuprizone feeding. Nevertheless, blockade of P2x7 receptors during the remyelination phase did not improve the extent of myelin recovery nor attenuated glial reaction and inflammation in damaged white matter. These findings suggest that P2x7 receptors drive T cell-independent inflammation and demyelination, but are not relevant to regenerative responses involved in myelin repair.

Keywords: Cuprizone; De- and remyelination; Inflammation; Microglia; Multiple sclerosis; P2x7 receptors.

Fig. 1

Up-regulated P2x7 receptor expression and signalling during cuprizone intoxication. (A) RT-qPCR analyses showed significant increases in P2rx7 expression during the time-course of cuprizone administration (n ​= ​7 mice per group; normalized to Gapdh and Hprt1. (B) Up-regulated P2x7 receptor levels were detected following 6 weeks of cuprizone feeding (n ​= ​5 mice per group). At each treatment point, brain slices from cuprizone-treated mice were compared with tissue from mice fed a control diet. (C) Representative images of P2x7 receptor immunostaining in the corpus callosum of mice treated with a cuprizone containing diet for 6 weeks and untreated controls. Quantification of immunolabelled tissue sections revealed significant up-regulation of P2x7 receptors in demyelinated corpus callosum (n ​= ​4 mice per group). (D) Increased expression of inflammasome-related molecules following 6 weeks of cuprizone intoxication. Transcript levels of indicated genes were assessed by RT-qPCR. Data represent relative fold change compared with controls normalized to Gapdh, Hprt1 and Ppia (n ​= ​4-5 mice per group). CPZ, cuprizone. ∗p ​< ​0.05, ∗∗p ​< ​0.01 and ∗∗∗p ​< ​0.001 referred to the control groups, Student’s t tests. Scale bars: 20 ​μm.

Fig. 2

P2x7 receptor deficient mice are resistant to cuprizone-induced demyelination. (A) P2x7^+/+ and P2x7^-/- mice were fed 0.3% cuprizone in the diet for 6 weeks. Analysis of tissue sections stained for LFB and MBP showed attenuated myelin loss in P2x7 receptor deficient mice (n ​= ​3-5 mice). Representative images depict the loss of MBP immunolabelling after cuprizone administration in P2x7^+/+ and P2x7^-/- mice. (B-D) The corpus callosum was immunostained for CD11b, GFAP, NG2, CD3, MAC387 and myeloperoxidase (MPO) as markers of microglia/macrophages, astrocytes, OPCs, T cells, recently infiltrating monocytes/macrophages and neutrophils, respectively. Quantification and representative images show a reduced accumulation of astrocytes and microglia in P2x7^-/- mice at 6 weeks of cuprizone diet (B), but no differences regarding the recruitment of OPCs (C) or peripheral immune cells (D) to demyelinated tissue (n ​= ​4-5 mice). (E) Transcript levels of inflammasome-associated genes were assessed by RT-qPCR in brain tissue from P2x7^+/+ and P2x7^-/- mice at 6 weeks of cuprizone intoxication. Data represent relative fold change compared with controls normalized to Gapdh, Hprt1 and Ppia (n ​= ​4-5 mice per group). Scale bars: 200 ​μm (A) and 50 ​μm (B). CPZ, cuprizone; N.D., not detected. ∗p ​< ​0.05, ∗∗p ​< ​0.01 and ∗∗∗p ​< ​0.001 referred to the control groups, Student’s t tests.

Fig. 3

Cell-specific analysis of P2x7 receptor expression in cuprizone-treated mice. Confocal images corresponding to double immunostaining of P2x7 receptors and the microglia/macrophage marker CD11b (A) and the astrocytic marker GFAP (B) in the corpus callosum of mice fed a cuprizone containing diet for 6 weeks. (C-D) Analysis of confocal fluorescent images indicated higher colocalization of P2x7 receptor immunopositive puncta with CD11b⁺ microglia than with GFAP⁺ astrocytic profiles, both in control and in cuprizone-treated mice (n ​= ​4 mice per group). CPZ, cuprizone. ∗p ​< ​0.05 and ∗p ​< ​0.01 versus P2x7/GFAP colocalization; ^###p ​< ​0.001 versus untreated controls; Student’s t tests. Scale bar: 10 ​μm.

Fig. 4

P2x7 receptors promote the accumulation of pro-inflammatory microglia during cuprizone-induced demyelination. (A) RT-qPCR analysis of M1 and M2 signature genes in brain tissue from mice treated with a cuprizone-containing diet for 6 weeks. Data represent relative fold change compared with controls normalized to Gapdh, Hprt1 and Ppia (n ​= ​4-5 mice per group). (B) P2x7 knockout mice displayed down-regulated induction of M1 phenotype markers at 6 weeks of cuprizone intoxication. Results are expressed as relative fold change relative to controls normalized to Gapdh, Hprt1 and Ppia (n ​= ​6 mice per group). (C-D) Quantification and representative images depicting iNOS and Arg-1 colocalization with CD11b in mice treated with cuprizone for 6 weeks. P2x7^-/- mice exhibited a significant reduction in the number of CD11b⁺ cells expressing iNOS in demyelinated corpus callosum. Scale bars: 20 ​μm. CPZ, cuprizone. ∗p ​< ​0.05, ∗∗p ​< ​0.01 and ∗∗∗p ​< ​0.001 referred to the control groups, Student’s t tests.

Fig. 5

P2x7 receptors are not master regulators of demyelination-associated microglia phenotype. Wild-type and P2x7 knockout mice were treated with cuprizone for 6 weeks and microglial cells purified from the forebrain using flow cytometry. RT-qPCR analysis of inflammasome activationmarkers (A) and M1/M2 phenotype genes (C) in cell sorted from control and cuprizone-treated mice showed selective up-regulation of several pro-inflammatory genes in demyelination-associated microglia. Data represent relative fold change compared with microglial cells purified from control mice normalized to B2m and Ppia (n ​= ​3-6 mice per group). (B, D) Relative expression of indicate genes in microglia sorted from cuprizone-treated P2x7 knockout mice (n ​= ​3-6 mice per group; fold change compared with expression levels in cells purified from cuprizone-treated wild-type mice; normalized to B2m and Ppia). CPZ, cuprizone. ∗p ​< ​0.05, ∗∗p ​< ​0.01 and ∗∗∗p ​< ​0.001 referred to the control groups, Student’s t tests.

Fig. 6

Pharmacological blockade of P2x7 receptors does not improve spontaneous remyelination in the cuprizone model. Mice received cuprizone in the diet for 6 weeks and BBG (50 ​mg/Kg), JNJ-47965567 (JNJ; 30 ​mg/Kg) or vehicle solutions during a 2 week remyelination phase. (A, B) LFB and MBP immunostaining revealed extensive demyelination at 6 weeks of cuprizone administration and partial recovery 2 weeks after toxin withdrawal. Scoring of LFB and MBP staining showed no differences between mice treated with BBG (A), JNJ-47965567 (B) or vehicle during the recovery phase (n ​= ​4-6 mice per group). ∗∗∗p ​< ​0.0001 control mice; ^#p ​< ​0.05 and ^###p ​< ​0.001 versus mice treated with cuprizone for 6 weeks; One-way ANOVA followed by Newman-Keuls multiple comparison tests. Scale bars: 25 ​μm. Scale bars: 500 ​μm (LFB) and 50 ​μm (MBP).

Fig. 7

Effect of P2x7 receptor antagonists on glial cells and inflammatory responses during remyelination. (A) Quantification of CD11b⁺ and Iba1⁺ cells and GFAP immunoreactivity following treatment with P2x7 receptor antagonists. Treatment with BBG or JNJ-47965567 during recovery from cuprizone intoxication did not modulate the presence of microglia/macrophages or astrocytes in remyelinating corpus callosum (n ​= ​5-6 mice per group). (B, C) Expression of inflammasome- and polarization-related molecules assessed by RT-qPCR in brain tissue from mice treated with BBG (B) or JNJ-47965567 (C). Data represent fold change relative to vehicle-treated mice normalized to Gapdh, Hprt1 and Ppia (n ​= ​5-6 mice per group). ∗p ​< ​0.05; Student’s t tests.