quantification-global-histone-post-translational-modifications-using

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to ...

University of British Columbia

Microglia, the resident macrophages of the central nervous system, play important roles in maintaining brain homeostasis and facilitating the brain's innate immune responses. Following immune challenges microglia also retain immune memories, which can alter responses to secondary inflammatory challenges. Microglia have two main memory states, training and tolerance, which are associated with increased and attenuated expression of inflammatory cytokines, respectively. However, the mechanisms differentiating these two distinct states are not well understood. We investigated mechanisms underlying training versus tolerance memory paradigms in vitro in BV2 cells using B-cell-activating factor (BAFF) or bacterial lipopolysaccharide (LPS) as a priming stimulus followed by LPS as a second stimulus. BAFF followed by LPS showed enhanced responses indicative of priming, whereas LPS followed by LPS as the second stimulus caused reduced responses suggestive of tolerance. The main difference between the BAFF versus the LPS stimulus was the induction of aerobic glycolysis by LPS. Inhibiting aerobic glycolysis during the priming stimulus using sodium oxamate prevented the establishment of the tolerized memory state. In addition, tolerized microglia were unable to induce aerobic glycolysis upon LPS restimulus. Therefore, we conclude that aerobic glycolysis triggered by the first LPS stimulus was a critical step in the induction of innate immune tolerance.

Modelling Microglial Innate Immune Memory In Vitro: Understanding the Role of Aerobic Glycolysis in Innate Immune Memory.

As the primary innate immune cells of the brain, microglia respond to damage and disease through pro-inflammatory release of cytokines and neuroinflammatory molecules. Histone acetylation is an activating transcriptional mark that regulates inflammatory gene expression. Inhibition of histone deacetylase 3 (Hdac3) has been utilized in pre-clinical models of depression, stroke, and spinal cord injury to improve recovery following injury, but the molecular mechanisms underlying Hdac3's regulation of inflammatory gene expression in microglia is not well understood. To address this lack of knowledge, we examined how pharmacological inhibition of Hdac3 in an immortalized microglial cell line (BV2) impacted histone acetylation and gene expression of pro- and anti-inflammatory genes in response to immune challenge with lipopolysaccharide (LPS). Flow cytometry and cleavage under tags & release using nuclease (CUT & RUN) revealed that Hdac3 inhibition increases global and promoter-specific histone acetylation, resulting in the release of gene repression at baseline and enhanced responses to LPS. Hdac3 inhibition enhanced neuroprotective functions of microglia in response to LPS through reduced nitric oxide release and increased phagocytosis. The findings suggest Hdac3 serves as a regulator of microglial inflammation, and that inhibition of Hdac3 facilitates the microglial response to inflammation and its subsequent clearing of debris or damaged cells. Together, this work provides new mechanistic insights into therapeutic applications of Hdac3 inhibition which mediate reduced neuroinflammatory insults through microglial response.

Morgan Towriss

Department of Biochemistry and Molecular Biology,
Djavad Mowafaghian Centre for Brain Health,
Department of Biochemistry and Molecular Biology, Djavad Mowafaghian Centre for Brain Health

University of British Columbia

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Morgan Towriss

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry and Molecular Biology,Djavad Mowafaghian Centre for Brain Health,Department of Biochemistry and Molecular Biology, Djavad Mowafaghian Centre for Brain Health

University of British Columbia

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Department of Biochemistry and Molecular Biology,
Djavad Mowafaghian Centre for Brain Health,
Department of Biochemistry and Molecular Biology, Djavad Mowafaghian Centre for Brain Health