The molecular mechanism and the functional role of pyruvate dehydrogenase complex regulation in macrophages

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PROJECT SUMMARY: Precise control of innate immunity is critical for human health. Both insufficient or excess inflammation can have detrimental effects and both are related to a variety of common and costly human pathologies including sepsis, arthritis, heart disease, and diabetes. Macrophages are crucial players in the coordination of this balance. In response to extracellular signals, macrophages can adopt diverse phenotypes that act in both the mounting and resolution of the immune response. Therefore, detailed understanding of the mechanisms regulating macrophage function is crucial for understanding immune-mediated disease pathology. Increasing evidence has shown that metabolism is important in controlling macrophage function. When stimulated, macrophages dramatically and dynamically alter their metabolism. However, in many cases, the mechanisms controlling and functional relevance of these metabolic alterations are unknown. In response to signals associated with infection, lipopolysaccharide and interferon-γ (LPS and IFN-γ), macrophages rapidly develop a pro-inflammatory phenotype. Following this initial activation, the cells eventually transition into a more immuno-suppressive state. Coupled to the dynamic change in function is a dynamic change in metabolism. In particular, TCA cycle metabolism is substantially rewired, and this rewiring is largely driven by inhibition of pyruvate dehydrogenase complex (PDHC) activity. Altering PDHC activity affects the function of LPS and IFN-γ stimulated macrophages. However, the detailed mechanism controlling PDHC activity and the mechanisms dictating the functional importance of PDHC are unknown. Aim 1 will elucidate the molecular mechanism controlling PDHC inhibition. In response to LPS and IFN-γ stimulation, the activity of PDHC’s E2 subunit decreases. Data shows that this is due to increased covalent modification of the E2 cofactor lipoic acid, on its reactive thiol group. We will identify the modification using a targeted mass spectrometry technique and will assess its role in controlling PHDC activity using genetic or chemical perturbation of the processes required for modification. Aim 2 will test the hypothesis that PDHC inhibition, via control of its product acetyl-CoA, influences functionally relevant histone acetylation and gene expression patterns. To test this model, the impact of genetic and chemical manipulation of acetyl-CoA levels and PDHC activity on histone acetylation will be assessed. To identify the consequences of PDHC-regulated histone acetylation, ChIP-seq and qPCR analyses will assess the impact of PDHC modulation on the histone acetylation and transcriptional landscape. The proposed work will illuminate novel mechanisms directing the metabolic and epigenetic reprogramming in macrophages. It will provide a broader understanding of the control of inflammatory state in macrophages and lay the groundwork for developing metabolic interventions to modulate immunity and treat disease.