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. 2016 Jul 27;36(30):7946-56.
doi: 10.1523/JNEUROSCI.1161-16.2016.

The Alarmin HMGB1 Mediates Age-Induced Neuroinflammatory Priming

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Free PMC article

The Alarmin HMGB1 Mediates Age-Induced Neuroinflammatory Priming

Laura K Fonken et al. J Neurosci. .
Free PMC article

Abstract

Amplified neuroinflammatory responses following an immune challenge occur with normal aging and can elicit or exacerbate neuropathology. The mechanisms mediating this sensitized or "primed" immune response in the aged brain are not fully understood. The alarmin high mobility group box 1 (HMGB1) can be released under chronic pathological conditions and initiate inflammatory cascades. This led us to investigate whether HMGB1 regulates age-related priming of the neuroinflammatory response. Here, we show that HMGB1 protein and mRNA were elevated in the hippocampus of unmanipulated aged rats (24-month-old F344XBN rats). Furthermore, aged rats had increased HMGB1 in the CSF, suggesting increased HMGB1 release. We demonstrate that blocking HMGB1 signaling with an intracisterna magna (ICM) injection of the competitive antagonist to HMGB1, Box-A, downregulates basal expression of several inflammatory pathway genes in the hippocampus of aged rats. This indicates that blocking the actions of HMGB1 might reduce age-associated inflammatory priming. To test this hypothesis, we evaluated whether HMGB1 antagonism blocks the protracted neuroinflammatory and sickness response to peripheral Escherichia coli (E. coli) infection in aged rats. ICM pretreatment of aged rats with Box-A 24 h before E. coli infection prevented the extended hippocampal cytokine response and associated cognitive and affective behavioral changes. ICM pretreatment with Box-A also inhibited aging-induced potentiation of the microglial proinflammatory response to lipopolysaccharide ex vivo Together, these results suggest that HMGB1 mediates neuroinflammatory priming in the aged brain. Blocking the actions of HMGB1 appears to "desensitize" aged microglia to an immune challenge, thereby preventing exaggerated behavioral and neuroinflammatory responses following infection.

Significance statement: The world's population is aging, highlighting a need to develop treatments that promote quality of life in aged individuals. Normal aging is associated with precipitous drops in cognition, typically following events that induce peripheral inflammation (e.g., infection, surgery, heart attack). Peripheral immune stimuli cause exaggerated immune responses in the aged brain, which likely underlie these behavioral deficits. Here, we investigated whether the alarmin high mobility group box 1 (HMGB1) mediates age-associated "priming" of the neuroinflammatory response. HMGB1 is elevated in aged rodent brain and CSF. Blocking HMGB1 signaling downregulated expression of inflammatory pathway genes in aged rat brain. Further, HMGB1 antagonism prevented prolonged infection-induced neuroinflammatory and sickness responses in aged rats. Overall, blocking HMGB1 "desensitized" microglia in the aged brain, thereby preventing pathological infection-elicited neuroinflammatory responses.

Keywords: danger-associated molecular patterns; healthy aging; microglia; neuroimmunology; sickness behavior.

Figures

Figure 1.
Figure 1.
HMGB1 is elevated in aged rodent brain and CSF. A, B, Relative HMGB1 gene (A) and HMGB1 protein (B) expression were measured in the hippocampus of young (3-month-old, n = 5) and aged (24-month-old, n = 6) F344xBN rats. C, In a separate cohort of young and aged rats (n = 8/group) HMGB1 was measured in the CSF. Gene-expression data are presented relative to β-actin. All data are presented as mean ± SEM. *p < 0.05.
Figure 2.
Figure 2.
HMGB1 may be released from CD11b+ cells in the aged hippocampus. A–G, Nuclear colocalization of HMGB1 was quantified in the hippocampus of young and aged rats by triple-labeling DAPI and HMGB1 with either CD11b (microglia; A, B, E), GFAP (astrocytes; C, F), or NeuN (neurons; D, G; n = 8/group). Images were captured using a Nikon A1R Laser Scanning Confocal and TIRF Inverted Microscope and analyzed using Fiji (ImageJ) by a condition-blind observer. Arrows are highlighting triple-labeled cells and arrowheads represent DAPI+ cells lacking nuclear HMGB1. Data are presented as mean ± SEM. *p < 0.05.
Figure 3.
Figure 3.
Aged animals exhibit prolonged inflammation following infection. Hippocampal tissue was collected from young and aged rats 4 d following an E. coli or saline injection (n = 6/group). A–C, IL-1β protein and IL-1β gene-expression levels of inflammatory cytokines (B) and inflammatory receptors (C) were all evaluated. D–F, HMGB1 (D) and NLRP3 (E) mRNA expression were also evaluated and NLRP3 (F) was significantly associated with IL-1β protein. G, H, A separate cohort of rats (n = 8/group) underwent behavioral testing in a sucrose anhedonia (G) and juvenile social exploration task (H). Gene-expression data are presented relative to β actin. All data are presented as mean ± SEM. *, Main effect; , simple effect of E. coli; in all cases p < 0.05.
Figure 4.
Figure 4.
Blocking HMGB1 with Box-A prevents neuroinflammatory priming. A, Hippocampal tissue was collected 24 h after aged and young rats were treated with ICM Box-A and expression of inflammatory pathway genes was evaluated (n = 5–6/group). Microglia were isolated 24 h after aged and young rats were treated with ICM Box-A (n = 6/group). B–E, Microglia were stimulated with LPS ex vivo and relative gene expression for MHCII (B), TNFα (C), IL-1β (D), and IL-6 (E) was determined. Gene-expression data are presented relative to β actin. All data are presented as mean ± SEM. *p < 0.05.
Figure 5.
Figure 5.
Box-A pretreatment prevented prolonged inflammatory responses in aged E. coli-treated rats. A, Experimental schematic: aged and young rats received an ICM injection of Box-A or vehicle and an intraperitoneal injection of E. coli 24 h later. Tissue was collected at 0 h (no E. coli control), 24 h, or 4 d after E. coli injection. Relative expression of IL-1β protein (B), IL-1β mRNA (C), TNFα mRNA (D), and IL-6 mRNA (E) were quantified in hippocampal tissue (n = 5–6/group/time). Gene-expression data are presented relative to β actin. All data are presented as mean ± SEM. *, Main effect; , simple effect; in all cases p < 0.05.
Figure 6.
Figure 6.
Blocking HMGB1 prevents prolonged delirium-like behaviors in aged rats following infection. A, Experimental schematic: aged and young rats received an ICM injection of Box-A or vehicle followed by an intraperitoneal injection of saline or E. coli 24 h later (n = 7–8/group). Four days following E. coli injection, rats were trained in a pre-exposure fear-conditioning paradigm. B, Freezing behavior in the experimental context (context A) and control context (context B). C, Juvenile social exploration was evaluated in a separate cohort of young and aged rats (n = 6–8/group) that received an ICM injection of Box-A or vehicle followed 24 later by an intraperitoneal injection of saline or E. coli. Data are presented as mean ± SEM. *p < 0.05.
Figure 7.
Figure 7.
Proposed mechanism of microglia inflammatory priming in aged brain.

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