A ketogenic diet reduces age-induced chronic neuroinflammation in mice Running title: ketogenic diet and brain inflammaging

Abstract

Beta-hydroxybutyrate (BHB) is a ketone body synthesized during fasting or strenuous exercise. Our previous study demonstrated that a cyclic ketogenic diet (KD), which induces BHB levels similar to fasting every other week, reduces midlife mortality and improves memory in aging mice. BHB actively regulates gene expression and inflammatory activation through non-energetic signaling pathways. Neither of these activities has been well-characterized in the brain and they may represent mechanisms by which BHB affects brain function during aging. First, we analyzed hepatic gene expression in an aging KD-treated mouse cohort using bulk RNA-seq. In addition to the downregulation of TOR pathway activity, cyclic KD reduces inflammatory gene expression in the liver. We observed via flow cytometry that KD also modulates age-related systemic T cell functions. Next, we investigated whether BHB affects brain cells transcriptionally in vitro. Gene expression analysis in primary human brain cells (microglia, astrocytes, neurons) using RNA-seq shows that BHB causes a mild level of inflammation in all three cell types. However, BHB inhibits the more pronounced LPS-induced inflammatory gene activation in microglia. Furthermore, we confirmed that BHB similarly reduces LPS-induced inflammation in primary mouse microglia and bone marrow-derived macrophages (BMDMs). BHB is recognized as an inhibitor of histone deacetylase (HDAC), an inhibitor of NLRP3 inflammasome, and an agonist of the GPCR Hcar2. Nevertheless, in microglia, BHB's anti-inflammatory effects are independent of these known mechanisms. Finally, we examined the brain gene expression of 12-month-old male mice fed with one-week and one-year cyclic KD. While a one-week KD increases inflammatory signaling, a one-year cyclic KD reduces neuroinflammation induced by aging. In summary, our findings demonstrate that BHB mitigates the microglial response to inflammatory stimuli, like LPS, possibly leading to decreased chronic inflammation in the brain after long-term KD treatment in aging mice.

Keywords: Beta-hydroxybutyrate (BHB); brain; inflammaging; ketogenic diet.

Figure 1.. One-year KD reduces hepatic chronic inflammation

(A) Diet composition and experimental timeline with mouse numbers. An analysis of RNA-seq after the one-week KD was published before. (B) GSEA (hallmark) analysis (KD vs. CD) of 26-month liver on diets for 14 months, collected at a dark cycle during CD-fed week (n=8 mice per group). (C) Heatmap of gene expressions in the liver.

Figure 2.. R-BHB reduces LPS-induced inflammation in human primary microglia

(A) Experimental timeline. (B, C) GSEA (hallmark) analysis (R-BHB vs. Ctrl) without (B) or with (C) LPS treatment in human primary neurons/astrocytes/microglia (n=3 per group). Gray squares: not significant.

Figure 3.. BHB acids reduce LPS-induced inflammation in mouse primary microglia

(A) Experimental timeline. (B) mRNA expression in mouse primary microglia (n=3 per group). All data are presented as mean ± SD. One-way ANOVA with Dunnet’s correction for multiple comparisons. Compare the mean of each sample with the mean of Ctrl. (C) Protein expression in mouse primary microglia (n=2 per group). All data are representative of two independent experiments. Abbreviations: R-BHB, R-BHB acid; S-BHB, S-BHB acid; Na-R-BHB, R-BHB salt; Na-S-BHB, S-BHB salt; Bu, butyric acid; Na-Bu, butyrate (salt).

Figure 4.. One-year KD reduces neuroinflammation distinct from one-week KD

(A) Experimental timeline with mouse numbers. (B) GSEA (hallmark) analysis (KD vs. CD) of 26-month brain on diets for 14 months, collected at night during CD-fed week (n=8 mice per group). (C) Heatmap of gene expressions in the brain. (D) Graphical abstract of the study.