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In vitro cultured-cells from Homo sapiens control and treated with BHB and LPS raw sequence reads

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 transcriptionallyin 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE252513 | GEO | 2024/01/31

REPOSITORIES: GEO

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Project description:Acute cognitive impairment (i.e., delirium) is common in elderly emergency department patients and frequently results from infections that are unrelated to the central nervous system. Since activation of the peripheral innate immune system induces brain microglia to produce inflammatory cytokines that are responsible for behavioral deficits, we investigated if aging exacerbated neuroinflammation and sickness behavior after peripheral injection of lipopolysaccharide (LPS). Microarray analysis revealed a transcriptional profile indicating the presence of primed or activated microglia and increased inflammation in the aged brain. Furthermore, aged mice had a unique gene expression profile in the brain after an intraperitoneal injection of LPS, and the LPS-induced elevation in the brain inflammatory cytokines and oxidative stress was both exaggerated and prolonged compared with adults. Aged mice were anorectic longer and lost more weight than adults after peripheral LPS administration. Moreover, reductions in both locomotor and social behavior remained 24 h later in aged mice, when adults had fully recovered, and the exaggerated neuroinflammatory response in aged mice was not reliably paralleled by increased circulating cytokines in the periphery. Taken together these data establish that activation of the peripheral innate immune system leads to exacerbated neuroinflammation in the aged as compared with adult mice. This dysregulated link between the peripheral and central innate immune system is likely to be involved in the severe behavioral deficits that frequently occur in older adults with systemic infections. Experiment Overall Design: In this study, adult and aged mice were injected intraperitoneal with sterile saline or Escherichia coli LPS (0.33 mg/kg, ~10 µg/mouse; serotype 0127:B8, Sigma). This dosage of LPS was used because it induces a mild transient sickness behavior in young adults. Mice were killed 4 h after saline or LPS injection by CO2 asphyxiation. Blood samples were collected and brains were removed, separated in half at the longitudinal fissure, frozen in liquid nitrogen, and stored (-80°C) until assaying. Total RNA was later isolated from some brain samples for microarray analysis (n=3).

Project description:Microglia are innate immune cells of the brain that perform phagocytic and inflammatory functions in disease conditions. Transcriptomic studies of acutely-isolated microglia have provided novel insights into their molecular and functional diversity in homeostatic and neurodegenerative disease states. State-of-the-art mass spectrometric methods can comprehensively characterize proteomic alterations in microglia in neurodegenerative disorders, potentially providing novel functionally-relevant molecular insights that are not provided by transcriptomics. However, proteomic profiling of adult primary microglia in neurodegenerative disease conditions has not been performed. We performed quantitative proteomic analyses of purified CD11b+ acutely-isolated microglia adult mice in normal, acute neuroinflammatory (LPS-treatment) and chronic neurodegenerative states (5xFAD model of Alzheimer’s disease [AD]) using tandem mass tag mass spectrometry. Differential expression analyses were performed to characterize specific microglial proteomic changes in 5xFAD mice and identify overlap with LPS-induced pro-inflammatory changes. Our results were also contrasted with existing proteomic data from wild-type mouse microglia and from existing microglial transcriptomic data from wild-type and 5xFAD mice. Neuropathological validation studies of select proteins were performed in human AD and 5xFAD brains. Of 4,133 proteins identified, 187 microglial proteins were differentially expressed in the 5xFAD mouse model of AD pathology, including proteins with previously known (Apoe, Clu and Htra1) as well as previously unreported relevance to AD biology (Cotl1 and Hexb). Proteins upregulated in 5xFAD microglia shared significant overlap with pro-inflammatory changes observed in LPS-treated mice. Several proteins increased in human AD brain were also upregulated by 5xFAD microglia (Aβ peptide, Apoe, Htra1, Cotl1 and Clu). Cotl1 was identified as a novel microglia-specific marker with increased expression and strong association with AD neuropathology. Apoe protein was also detected within plaque-associated microglia in which Apoe and Aβ were highly co-localized suggesting a role for Apoe in phagocytic clearance of Aβ. We report the first comprehensive comparative proteomic study of adult mouse microglia derived from acute neuroinflammatory and AD models, representing a valuable resource to the neuroscience research community. We highlight shared and unique microglial proteomic changes in acute neuroinflammatory, aging and AD mouse models in addition to identifying novel roles for microglial proteins in human neurodegeneration.

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In vitro cultured-cells from Homo sapiens control and treated with BHB and LPS raw sequence reads

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