Pleiotrophin regulates microglia-mediated neuroinflammation

Abstract

Background: Pleiotrophin (PTN) is a cytokine found highly upregulated in the brain in different disorders characterized by overt neuroinflammation such as neurodegenerative diseases, drug addiction, traumatic injury, and ischemia. In the present work, we have explored whether PTN modulates neuroinflammation and if Toll-like receptor 4 (TLR4), crucial in the initiation of an immune response, is involved.

Methods: In immunohistochemistry assays, we studied lipopolysaccharide (LPS, 7.5 mg/kg i.p.)-induced changes in glial fibrillary acidic protein (GFAP, astrocyte marker) and ionized calcium-binding adaptor molecule 1 (Iba1, microglia marker) expression in the prefrontal cortex (PFC) and striatum of mice with transgenic PTN overexpression in the brain (PTN-Tg) and in wild-type (WT) mice. Cytokine protein levels were assessed in the PFC by X-MAP technology. The influence of TLR4 signaling in LPS effects in both genotypes was assessed by pretreatment with the TLR4 antagonist (TAK-242, 3.0 mg/kg i.p.). Murine BV2 microglial cells were treated with PTN (0.5 μg/ml) and LPS (1.0 μg/ml) and assessed for the release of nitric oxide (NO).

Results: We found that LPS-induced microglial activation is significantly increased in the PFC of PTN-Tg mice compared to that of WT mice. The levels of TNF-α, IL-6, and MCP-1 in response to LPS were significantly increased in the PFC of PTN-Tg mice compared to that of WT mice. Pretreatment with TAK-242 efficiently blocked increases in cytokine contents in a similar manner in both genotypes. Concomitant incubation of BV2 cells with LPS and PTN significantly potentiated the production of NO compared to cells only treated with LPS.

Conclusions: Our findings identify for the first time that PTN is a novel and potent regulator of neuroinflammation. Pleiotrophin potentiates LPS-stimulated microglia activation. Our results suggest that regulation of the PTN signaling pathways may constitute new therapeutic opportunities particularly in those neurological disorders characterized by increased PTN cerebral levels and neuroinflammation.

Keywords: Microglia activation; Microgliosis; Midkine; Neuroimmune response; Neuroinflammation; Pleiotrophin; TLR4.

Fig. 1

LPS effects on astrocytosis in the PFC of WT and PTN-Tg mice. Photomicrographs are from GFAP-immunostained PFC sections of saline + saline (Sal)- or saline + LPS (LPS)-treated animals (n = 4–5/group). Higher magnification images in the lower right corner of every representative picture show that astrocytes were hypertrophic and densely stained in WT mice treated with LPS. The graph represents quantification of data obtained from the counts of GFAP-positive cells in PFC whole sections. ^## P < 0.01 vs. WT-LPS. Scale bar = 500 μm, (high magnification, scale bar = 50 μm). SAL saline, LPS lipopolysaccharide, WT wild-type mice, PTN-Tg mice with transgenic pleiotrophin overexpression in the brain, GFAP glial fibrillary acidic protein

Fig. 2

Effects of TAK-242 and LPS on microglia activation in the PFC of PTN-Tg mice. Photomicrographs are from Iba-1-immunostained PFC sections of saline (Sal)- or TAK-242 (TAK)-pretreated and saline (Sal)- or LPS-treated animals (n = 4–5/group) (a). Graphs represent quantification of data (mean ± SEM) obtained from the counts of Iba-1-positive cells (b), total marked area (c), cell area (d), soma perimeter (e), and circularity index (f) in PFC whole sections. *P < 0.05, ***P < 0.001, ****P < 0.0001 vs. Sal + Sal within the same genotype. ^# P < 0.05, ^## P < 0.01 vs. WT within the same treatment. Scale bar = 100 μm. SAL saline, LPS lipopolysaccharide, TAK TAK-242, WT wild-type mice, PTN-Tg mice with transgenic pleiotrophin overexpression in the brain, Iba1 ionized calcium-binding adaptor molecule 1

Fig. 3

Effects of TAK-242 and LPS on cytokine expression in the PFC of PTN-Tg mice. TNF-α, Il-1β, IL-6, MCP-1, IL-4, and IL-10 protein levels measured using a Milliplex system in PFC of mice pretreated with saline (Sal) or TAK-242 (TAK) and treated with saline (Sal) or LPS (n = 5/group). *P < 0.05, ***P < 0.001 vs. Sal + Sal within the same genotype. ^## P < 0.01, ^### P < 0.001 vs. WT within the same treatment. &&P < 0.01, &&&P < 0.001 vs. Sal + LPS within the same genotype. SAL saline, LPS lipopolysaccharide, TAK TAK-242, WT wild-type mice, PTN-Tg mice with transgenic pleiotrophin overexpression in the brain, TNF Tumor necrosis factor-α, IL-1β interleukin 1β, IL-6 interleukin 6, MCP-1 monocyte chemoattractant protein-1, IL-4 interleukin 4, IL-10 interleukin 10

Fig. 4

LPS effects on astrocytosis in the striatum of WT and PTN-Tg mice. Photomicrographs are from GFAP-immunostained striatal sections of saline + saline (Sal)- or saline + LPS (LPS)-treated animals (n = 4–5/group). The graph represents quantification of data obtained from the counts of GFAP-positive cells in the striatum. Scale bar = 200 μm. SAL saline, LPS lipopolysaccharide, WT wild-type mice, PTN-Tg mice with transgenic pleiotrophin overexpression in the brain, GFAP glial fibrillary acidic protein

Fig. 5

Effects of TAK-242 and LPS on microglia activation in the striatum of PTN-Tg mice. Photomicrographs are from Iba-1-immunostained striatal sections of saline (Sal)- or TAK-242 (TAK)-pretreated and saline (Sal)- or LPS-treated animals (n = 4–5/group) (a). Graphs represent quantification of data (mean ± SEM) obtained from the counts of Iba-1-positive cells (b), total marked area (c), cell area (d), soma perimeter (e), and circularity index (f) in the striatum. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Sal + Sal within the same genotype. Scale bar = 100 μm. SAL saline, LPS lipopolysaccharide, TAK TAK-242, WT wild-type mice, PTN-Tg mice with transgenic pleiotrophin overexpression in the brain, Iba1 ionized calcium-binding adaptor molecule 1

Fig. 6

Effects of PTN on LPS-induced NO production in BV2 microglial cells. Cells were treated with the indicated concentrations of PTN (0.05 or 0.5 μg/ml) and/or with LPS (1.0 μg/ml) for 24 h. The data of the levels of NO in the media are expressed as the mean ± SEM. *P < 0.05 vs. cells treated with LPS. NO nitric oxide, LPS lipopolysaccharide, PTN pleiotrophin