Astrocytes Are Primed by Chronic Neurodegeneration to Produce Exaggerated Chemokine and Cell Infiltration Responses to Acute Stimulation with the Cytokines IL-1β and TNF-α - PubMed (original) (raw)

Astrocytes Are Primed by Chronic Neurodegeneration to Produce Exaggerated Chemokine and Cell Infiltration Responses to Acute Stimulation with the Cytokines IL-1β and TNF-α

Edel Hennessy et al. J Neurosci. 2015.

Abstract

Microgliosis and astrogliosis are standard pathological features of neurodegenerative disease. Microglia are primed by chronic neurodegeneration such that toll-like receptor agonists, such as LPS, drive exaggerated cytokine responses on this background. However, sterile inflammatory insults are more common than direct CNS infection in the degenerating brain and these insults drive robust IL-1β and TNF-α responses. It is unclear whether these pro-inflammatory cytokines can directly induce exaggerated responses in the degenerating brain. We hypothesized that glial cells in the hippocampus of animals with chronic neurodegenerative disease (ME7 prion disease) would display exaggerated responses to central cytokine challenges. TNF-α or IL-1β were administered intrahippocampally to ME7-inoculated mice and normal brain homogenate-injected (NBH) controls. Both IL-1β and TNF-α produced much more robust IL-1β synthesis in ME7 than in NBH animals and this occurred exclusively in microglia. However, there was strong nuclear localization of the NFκB subunit p65 in the astrocyte population, associated with marked astrocytic synthesis of the chemokines CXCL1 and CCL2 in response to both cytokine challenges in ME7 animals. Conversely, very limited expression of these chemokines was apparent in NBH animals similarly challenged. Thus, astrocytes are primed in the degenerating brain to produce exaggerated chemokine responses to acute stimulation with pro-inflammatory cytokines. Furthermore, this results in markedly increased neutrophil, T-cell, and monocyte infiltration in the diseased brain. These data have significant implications for acute sterile inflammatory insults such as stroke and traumatic brain injury occurring on a background of aging or neurodegeneration.

Keywords: astrocyte; chemokine; cytokine; microglia; neurodegeneration; neuroinflammation.

Copyright © 2015 Hennessy et al.

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Figures

Figure 1.

Figure 1.

Microglial priming, activation, and IL-1β synthesis. Microglial activation status was examined at 2 h post-intrahippocampal IL-1β (10 ng) or TNF-α (300 ng) challenge in NBH and ME7 animals. Hippocampal CA1 IBA-1 microglial labeling ×20 (a–f) and ×100 (g–i). Hippocampal TNF-α labeling ×5 (j–o). n, Inset shows TNF-α labeling, post-intrahippocampal LPS, with and without pre-absorption of the antibody with excess TNF-α. Hippocampal IL-1β labeling is shown at ×20 (p–u). u, Inset shows IL-1β-positive microglia adjacent to unlabeled astrocytic-like nucleus. Fluorescent double labeling of IBA-1 (594 nm) with IL-1β (488 nm) and Hoechst 33258 (blue) in ME7 animals 2 h post IL-1β (I–III) or TNF-α (IV–VI).

Figure 2.

Figure 2.

Astrocyte priming, activation, and chemokine synthesis. NFκB activation and chemokine expression 2 h post intrahippocampal IL-1β (10 ng) or TNF-α (300 ng) challenge of NBH and ME7 animals. NFκB p65 labeling (×100) in the hippocampus (a–f) with astrocytic-like nucleus indicated by white arrow and microglial-like nucleus indicated by black arrow in f. Hippocampal CCL2 labeling at ×100 magnification (g–l). CXCL1 labeling at ×100 magnification (m–r). l, r, Insets show CCL2 and CXCL1 labeling, respectively, associated with astrocyte-like nucleus adjacent to microglia-like nucleus devoid of labeling. Hippocampal CA1 GFAP astrocyte labeling in NBH and ME7 animals is shown at ×20 (s–x). Fluorescent labeling of GFAP (488 nm), p65 (633 nm), CCL2 (594 nm), and CXCL1 (594 nm) 2 h post TNF-α (i–ix) or IL-1β challenge (x–xiii).

Figure 3.

Figure 3.

Analysis of cytokine, chemokine, and astrocytosis transcripts. Hippocampal expression of transcripts for inflammatory genes was analyzed 2 h post intrahippocampal IL-1β (10 ng) or TNF-α (300 ng). a, IL-1β; b, TNF-α; c, CCL2; d, CXCL1; e, RANTES; f, CXCL10; g, GFAP; h, PTX3; i, STEAP4. Data were analyzed using two-way ANOVA and main effects and interactions are described in the main text. Statistically significant differences between NBH and ME7 by Bonferroni post hoc tests are denoted by ***p < 0.001, **p < 0.01, and *p < 0.05. All data are represented as the mean ± SEM and n = 4 for all groups except for the analysis of TNF-α, CCL2, and CXCL10, in which both ME7 + saline and ME7 + IL-1β had n = 6.

Figure 4.

Figure 4.

Cytokine-induced neutrophil infiltration. Hippocampal neutrophil infiltration at 24 h post intrahippocampal IL-1β (10 ng) or TNF-α (300 ng) challenge. MBS-1 neutrophil labeling at ×20 magnification (a–f) at 24 h post challenge. Quantification of neutrophils in an area of 4.64 mm2 centered on the injected dorsal hippocampus following IL-1β (g) and TNF-α (h) injection. Data were analyzed using a two-way ANOVA followed by Bonferroni post hoc test. Interactions between treatment and disease are denoted by #p < 0.05 and ##p < 0.01. All data are represented as the mean ± SEM, n = 3–5 for all groups.

Figure 5.

Figure 5.

Chemokine expression and immune cell recruitment at brain barriers. Chemokine expression at ventricular and vascular surfaces at 2 h post intrahippocampal cytokine challenge. Representative images of CCL2 at the ventricular (a) and vascular (b) surfaces of ME7 animals ×100 at 2 h. b, Inset shows CCL2 labeling with and without pre-absorption of the antibody with excess CCL2. CXCL-1 at the ventricular (c) and vascular (d) surfaces ×100 at 2 h. d, Inset shows CXCL1 labeling with and without pre-absorption of the antibody with excess CXCL1. GFAP and CCL2 colocalization at a hippocampal blood vessel at 2 h post TNF-α (e). MBS-1 neutrophil labeling at the ventricular membrane at 24 h post challenge with IL-1β (10 ng) or TNF-α (300 ng) challenge (×40, f–k). CD68 macrophage labeling (×40, l–q) at the ventricular surface (glia limitans) at 24 h post challenge with IL-1β (10 ng) or TNF-α (300 ng).

Figure 6.

Figure 6.

Immune cell infiltration at 72 h. Cellular infiltration at 72 h post intrahippocampal TNF-α (300 ng) challenge. Hippocampal MBS-1 neutrophil labeling ×40 (a–d), CD3 T-cell labeling ×40 (e–h), and Pu.1 macrophage/microglial labeling ×20 (i–l). Quantification of MBS-1-positive neutrophils (m) and CD3-positive T-cells (n) in the injected hippocampus at 72 h. Pu.1-positive macrophages and microglia were quantified per 0.29 mm2 of the injected hippocampus (o). The latter were quantified in the presence and absence of peripherally administered Mc21 to deplete circulating monocytes. Data were analyzed using two-way ANOVA (full analysis in main text) followed by Bonferroni post hoc test. Selected statistically significant pairwise comparisons are denoted by *p < 0.05, **p < 0.01, ***p < 0.001. All data are represented as the mean ± SEM, n = 3–7.

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