Dysregulation of C-X-C motif ligand 10 during aging and association with cognitive performance

Abstract

Chronic low-grade inflammation during aging (inflammaging) is associated with cognitive decline and neurodegeneration; however, the mechanisms underlying inflammaging are unclear. We studied a population (n = 361) of healthy young and old adults from the MyoAge cohort. Peripheral levels of C-X-C motif chemokine ligand 10 (CXCL10) was found to be higher in older adults, compared with young, and negatively associated with working memory performance. This coincided with an age-related reduction in blood DNA methylation at specific CpGs within the CXCL10 gene promoter. In vitro analysis supported the role of DNA methylation in regulating CXCL10 transcription. A polymorphism (rs56061981) that altered methylation at one of these CpG sites further associated with working memory performance in 2 independent aging cohorts. Studying prefrontal cortex samples, we found higher CXCL10 protein levels in those with Alzheimer's disease, compared with aged controls. These findings support the association of peripheral inflammation, as demonstrated by CXCL10, in aging and cognitive decline. We reveal age-related epigenetic and genetic factors which contribute to the dysregulation of CXCL10.

Keywords: Alzheimer's disease; Cognitive aging; DNA methylation; Epigenetics; Inflammaging; Neurodegeneration.

Fig. 1

Plasma cytokines which were significantly different between the young and old adult group. (A) Plasma cytokines which were significantly higher in the older adult group compared to the younger adult group. (B) Plasma cytokines which were significantly lower in the older adult group compared to the younger adult group. Young n = 44–135; old n = 64–225. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Fig. 2

DNA methylation differences between age groups for the CXCL10 proximal promoter and associations with plasma protein concentrations. (A) Full-length human CXCL10 promoter region (negative strand shown). Transcription factor binding sites were predicted using MatInspector (core: >0.9; matrix similarity: optimized). CpG sites are represented as a red beacon with the positions relative to the transcriptional start site (extended arrow) in base pairs. There are 4 and 3 CpG sites located in the distal and proximal regions respectively. (B) Differences in CXCL10 proximal promoter methylation between young and old adults. Young n = 69; old n = 82. ^∗∗p < 0.01; ^∗∗∗p < 0.001. Association between plasma CXCL10 levels (ln transformed) with CpG-168 (C), CpG-136 (D), and CpG-8 (E) methylation levels. n = 122. Abbreviations: AP-1, activating protein 1; CCAAT, CAT box; CEBP-α, CCAAT/enhancer binding protein alpha; CEBP-β, CCAAT/enhancer binding protein beta; IRF, interferon regulatory factor; CXCL10, C-X-C motif chemokine ligand 10; ISRE, IFN-stimulatory element; NF-κB, nuclear factor-kappa B; STAT, signal transducers and activators of transcription. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Analysis of CXCL10 regulation, DNA methylation, and promoter activity. (A) CXCL10 mRNA expression in U937 cells following stimulation with 10 ng/mL TNF-α and IFN-γ for 90 minutes. Values are normalized to GAPDH and BACTIN expression. n = 3. (B) DNA methylation from U937 cells for the 3 CpG sites following stimulation with TNF-α and IFN-γ. n = 3. (C) The half inhibitory concentration of U937 cells exposed to 5-azacytidine (5-Aza). Cell viability was determined by the trypan blue exclusion method. Briefly, U937 cells were seeded at 1 × 10⁵ in a 24-well plate overnight. The next day cells were incubated with various concentrations (0.1, 0.2, 1, 2, 5, and 10 μM) of 5-Aza for 72 hours. Fresh 5-Aza was added every 24 hours. Red line indicates a cell viability of 50%. n = 3. (D) DNA methylation from U937 cells treated with and without 5-Aza for the 3 CpG sites. Cells were incubated with 0.65 μM 5-Aza for 72 hours, with fresh 5-Aza added every 24 hours, before stimulation. n = 3. (E) CXCL10 mRNA expression in U937 cells pre-treated with 5-Aza before and after the addition of TNF-α and IFN-γ. Values are normalized to GAPDH and BACTIN expression. Simulated levels of CXCL10 mRNA gene expression are relative to their unstimulated counterparts. n = 3. (A–E) ^∗∗p < 0.01; ^∗∗∗p < 0.001. (F) Promoter activity of the CXCL10 promoter following in vitro methylation. Schematics denote either plasmid methylation or patch methylation techniques. Beacons represent methylated (red) or unmethylated (white) CpG's. Luciferase values were normalized via the β-galactosidase signal to account for transfection efficiency, and results are expressed as the promoter percentage activity relative to the unmethylated constructs (100% activity; red dotted line). n = 4–5. ^∗p < 0.05; ^∗∗p < 0.01 relative to corresponding unmethylated constructs. Abbreviations: CXCL10, C-X-C motif chemokine ligand 10; mRNA, messenger RNA; TNF, tumor necrosis factor. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

DNA methylation differences between rs56061981 genotypes in the healthy young and old adults. (A) Schematic representation of CXCL10 proximal promoter showing the rs56061981 polymorphism and the resulting change in the CpG-136 site (negative strand shown). (B) Difference between CpG-136 methylation in GG (n = 65) and GA (n = 14) carriers in the old group. ^∗p < 0.05. Abbreviation: CXCL10, C-X-C motif chemokine ligand 10.

Fig. 5

Comparison of CXCL10 methylation and protein levels in the prefrontal cortex. (A) Difference between CpG-136 methylation in GG (n = 61) and GA (n = 5) carriers in the prefrontal cortex samples. (B) CXCL10 protein levels from the prefrontal cortex samples of those showing intermediate pathological signs of AD and those with no neuropathologies. Aged n = 17; intermediate AD n = 16. ^∗p < 0.05; ^∗∗p < 0.01. Abbreviations: AD, Alzheimer's disease; CXCL10, C-X-C motif chemokine ligand 10.