Innate Immune Tolerance in Microglia Does Not Impact on Central Nervous System Prion Disease

Abstract

Prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep, are infectious and chronic neurodegenerative diseases to which there are no cures. Infection with prions in the central nervous system (CNS) ultimately causes extensive neurodegeneration, and this is accompanied by prominent microglial and astrocytic activation in affected regions. The microglia are the CNS macrophages and help maintain neuronal homeostasis, clear dead or dying cells and provide defense against pathogens. The microglia also provide neuroprotection during CNS prion disease, but their pro-inflammatory activation may exacerbate the development of the neuropathology. Innate immune tolerance induced by consecutive systemic bacterial lipopolysaccharide (LPS) treatment can induce long-term epigenetic changes in the microglia in the brain that several months later can dampen their responsiveness to subsequent LPS treatment and impede the development of neuritic damage in a transgenic mouse model of Alzheimer's disease-like pathology. We therefore reasoned that innate immune tolerance in microglia might similarly impede the subsequent development of CNS prion disease. To test this hypothesis groups of mice were first infected with prions by intracerebral injection, and 35 days later given four consecutive systemic injections with LPS to induce innate immune tolerance. Our data show that consecutive systemic LPS treatment did not affect the subsequent development of CNS prion disease. Our data suggests innate immune tolerance in microglia does not influence the subsequent onset of prion disease-induced neuropathology in mice, despite previously published evidence of this effect in an Alzheimer's disease mouse model.

Keywords: central nervous system; innate immune tolerance; lipopolysaccharide; microglia; prion disease.

FIGURE 1

Multiple LPS exposure induces innate immune tolerance. (A) Groups of mice (n = 4/group) were given four daily IP injections with either LPS or PBS (control) and body weight measured at intervals afterward. (B,C) Mice were given a single (1x) or four (4x) daily IP injections with either LPS or PBS (control) and serum collected 3 h after the final injection. (B) Concentration of IL-1β in the serum of mice given a single or four daily IP injections with either LPS or PBS. (C) Concentration of IL-6 in the serum of mice given a single or four daily IP injections with either LPS or PBS. (D) Concentration of TNF-α in the serum of mice given a single or four daily IP injections with either LPS or PBS. (E) Concentration of IL-10 in the serum of mice given a single or four daily IP injections with either LPS or PBS. N = 3–4 mice/group; horizontal bar, median. *P < 0.05; **P < 0.01; ***P < 0.001.

FIGURE 2

Effect of systemic LPS treatment on expression of cytokine-encoding genes in the brain. Mice were given a single (1x) or four (4x) daily IP injections with either LPS or PBS (control). Brains were collected 3 h after the final injection and gene expression compared by RT-qPCR analysis. (A) Relative expression level of Il1b mRNA. (B) Relative expression level of Il6 mRNA. (C) Relative expression level of Tnf mRNA. (D) Relative expression level of Il10 mRNA. Gene expression data are normalized so that the mean level in the 1xPBS controls was 1.0. N = 4–8 mice/group; horizontal bar, median. *P < 0.05; **P < 0.01; ***P < 0.001.

FIGURE 3

Effect of systemic LPS treatment on microglia. Mice were given a single (1x) four (4x) daily IP injections with either LPS or PBS (control) and brains collected 3 h after the final injection. (A) Representative immunostaining of AIF1 + microglia (brown) in the CA1 region of the hippocampus striatum radiatum of each mouse. Scale bar 20 μm. (B) Relative abundance of AIF1+ microglia in 100,000 μm² images of the CA1 region of the hippocampus striatum radiatum of mice from each group. (C) Comparison of the magnitude of the AIF1+ immunostaining in the CA1 region of the hippocampus striatum radiatum of mice from each group. Assessment of AIF1+ microglia morphology in the CA1 region of the hippocampus striatum radiatum of mice from each group: (D), mean filament dendrite length; (E), mean number of dendrite segments; (F), mean number of dendrite branch points; (G), mean number of terminal points. (H–L) Relative expression level of Aif1, Csf1r, Cx3cr1, Itgam, and Tmem119 mRNA in half brains from each mouse from each group. Gene expression data are normalized so that the mean level in the 1xPBS controls was 1.0. N = 4–8 mice/group; horizontal bar in histograms, median. *P < 0.05; **P < 0.01; ***P < 0.001.

FIGURE 4

Effect of systemic LPS treatment on astrocytes. Mice were given a single (1x) or four (4x) daily IP injections with either LPS or PBS (control) and brains collected 3 h after the final injection. (A) Representative immunostaining of GFAP + astrocytes (brown) in the CA1 region of the hippocampus striatum radiatum of each mouse. Scale bar 20 μm. (B) Relative abundance of GFAP+ astrocytes in 100,000 μm² images of the CA1 region of the hippocampus striatum radiatum of mice from each group. (C) Comparison of the magnitude of the GFAP+ immunostaining in the CA1 region of the hippocampus striatum radiatum of mice from each group. (D) Relative expression level of Gfap mRNA in half brains from each mouse from each group. (E) Relative expression level of Gbp2, Iigp1, Psmb8, and Srgn mRNA in half brains from each mouse from each group. Gene expression data are normalized so that the mean level in the 1xPBS controls was 1.0. N = 3–8 mice/group; horizontal bar in histograms, median. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significantly different.

FIGURE 5

Effect of systemic LPS treatment on PrP^C expression in the brain. Mice were given a single (1x) or four (4x) daily IP injections with either LPS or PBS (control) and brains collected 3 h after the final injection. (A) Relative expression level of Prnp mRNA in half brains from each mouse from each group. Gene expression data are normalized so that the mean level in the 1xPBS controls was 1.0. N = 4–8 mice/group. Not significantly different. (B) Western blot analysis of PrP^C protein (upper panel) and β-actin (lower panel) in half brains from mice given 4 daily IP injections with either LPS or PBS (4xPBS, 4xLPS, respectively). (C) Quantitation of the relative abundance of PrP^C protein in half brains from each group. The abundance of PrP^C in each sample was normalized to β-actin and expressed as the% relative to the mean value in the 4xPBS-treated controls. N = 4 mice/group; horizontal bar in histogram, median. Not significantly different, student’s t-test.

FIGURE 6

Effect of systemic LPS treatment on CNS prion disease. (A) Cartoon describing the experimental design. Mice were first injected with ME7 scrapie prions directly into the brain by IC injection. Thirty-five days later, the mice were given either a single IP LPS injection (prions + 1xLPS) or four consecutive daily IP LPS injections (prions + 4xLPS) to induce innate immune training or tolerance, respectively. A parallel group of mice were given four consecutive daily IP PBS injections (prions + 4xLPS) as a control. (B) A single IP LPS injection (prions + 1xLPS) or four consecutive daily IP LPS injections (prions + 4xLPS) caused a transient reduction in body weight compared to prions + 4xPBS-treated controls. Open circles, P < 0.05 compared to prions + 4xPBS-treated controls. Closed circles, not significantly different compared to prions + 4xPBS-treated controls. (C) Survival curve for prion infected mice in each treatment group. N = 5–6 mice/group. Not significantly different, Log-rank (Mantel-Cox) test.

FIGURE 7

Effect of systemic LPS treatment on the histopathological signs of prion disease in the brain. Mice were first injected with ME7 scrapie prions directly into the brain by IC injection. Thirty five days later the mice were given a either single IP LPS injection (prions + 1xLPS) or four consecutive daily IP LPS injections (prions + 4xLPS) to induce innate immune training or tolerance, respectively. A parallel group of mice were given four consecutive daily IP PBS injections (prions + 4xLPS) as a control. Brains were collected at the terminal stage. (A) The severity and distribution of the spongiform pathology (vacuolation) within each clinically affected brain from each treatment group was scored on H&E sections using a scale of 1–5 in nine gray matter regions and three white matter regions: G1, dorsal medulla; G2, cerebellar cortex; G3, superior colliculus; G4, hypothalamus; G5, thalamus; G6,hippocampus; G7, septum; G8, retrosplenial and adjacent motor cortex; G9, cingulate and adjacent motor cortex; W1, inferior and middle cerebellar peduncles; W2, decussation of superior cerebellar peduncles; and W3, cerebellar peduncles. (B) Representative images showing high levels of spongiform pathology (H&E, upper row), microgliosis (AIF1+ cells, brown, 2nd row), reactive astrocytes (GFAP+ cells, brown, 3rd row) and heavy prion disease-specific PrPd accumulation (brown, bottom row) in the hippocampus of all prion infected mice from each treatment group at the terminal clinical stage. Sections counterstained with hematoxylin to detect cell nuclei (blue). Scale bar, 200 μm. (C,D) Comparison of the magnitude of the AIF1+ immunostaining and abundance of AIF1 + microglia, respectively, in the CA1 region of the hippocampus of mice from each group. (E,F) Comparison of the magnitude of the GFAP+ immunostaining and abundance of GFAP+ reactive astrocytes, respectively, in the CA1 region of the hippocampus of mice from each group. (G) Comparison of the magnitude of the PrP^d + immunostaining in the CA1 region of the hippocampus of mice from each group. (H) Relative expression level of Il1b mRNA. (I) Relative expression level of Il6 mRNA. (J) Relative expression level of Tnf mRNA. (K) Relative expression level of Il10 mRNA. Gene expression data are normalized so that the mean level in the 1xPBS controls was 1.0. N = 4–6 mice/group; horizontal bar, median. *P < 0.05; **P < 0.01; ***P < 0.001.

FIGURE 8

Effect of systemic LPS treatment on prion disease-specific PrP^Sc accumulation in the brain. Mice were first injected with ME7 scrapie prions directly into the brain by IC injection. Thirty five days later the mice were given a either single IP LPS injection (prions + 1xLPS) or four consecutive daily IP LPS injections (prions + 4xLPS) to induce innate immune training or tolerance, respectively. A parallel group of mice were given four consecutive daily IP PBS injections (prions + 4xLPS) as a control. Brains were collected at the terminal stage. (A) Western blot analysis of prion disease specific PrP^Sc accumulation in half brains from each group. PK, proteinase-K. Upper panel, total PrP; middle panel, relatively PK-resistant prion disease-specific PrP^Sc; lower panel β-actin. (B) Quantitation of the relative abundance of PrP^Sc in half brains from each group. The abundance of PrP^Sc in each sample was expressed as the% relative to the mean value in the 1xPBS-treated controls. N = 5–6 mice/group; horizontal bar in histograms, median.