Abstract
M1 (LPS) macrophages are characterized by a high expression of pro-inflammatory mediators, and distinct metabolic features that comprise increased glycolysis, a broken TCA cycle, or impaired OXPHOS with augmented mitochondrial ROS production. This study investigated whether the phytochemical sulforaphane (Sfn) influences mitochondrial reprogramming during M1 polarization, as well as to what extent this can contribute to Sfn-mediated inhibition of M1 marker expression in murine macrophages. The use of extracellular flux-, metabolite-, and immunoblot analyses as well as fluorescent dyes indicative for mitochondrial morphology, membrane potential or superoxide production, demonstrated that M1 (LPS/Sfn) macrophages maintain an unbroken TCA cycle, higher OXPHOS rate, boosted fusion dynamics, lower membrane potential, and less superoxide production in their mitochondria when compared to control M1 (LPS) cells. Sustained OXPHOS and TCA activity but not the concomitantly observed high dependency on fatty acids as fuel appeared necessary for M1 (LPS/Sfn) macrophages to reduce expression of nos2, il1β, il6 and tnfα. M1 (LPS/Sfn) macrophages also displayed lower nucleo/cytosolic acetyl-CoA levels in association with lower global and site-specific histone acetylation at selected pro-inflammatory gene promoters than M1 (LPS), evident in colorimetric coupled enzyme assays, immunoblot and ChIP-qPCR analyses, respectively. Supplementation with acetate or citrate was able to rescue both histone acetylation and mRNA expression of the investigated M1 marker genes in Sfn-treated cells. Overall, Sfn preserves mitochondrial functionality and restricts indispensable nuclear acetyl-CoA for histone acetylation and M1 marker expression in LPS-stimulated macrophages.
| Original language | English |
|---|---|
| Pages (from-to) | 443-456 |
| Number of pages | 14 |
| Journal | Free Radical Biology and Medicine |
| Volume | 213 |
| DOIs | |
| Publication status | Published - Mar 2024 |
Funding
The FWF (Austrian Science Fund; P32600 to EHH) and the University of Vienna provided financial support for this study. Macrophages possess exceptional versatility and plasticity, making them crucial constituents of the innate immune system. They exhibit distinct activation (polarization) states and metabolic alterations in response to various environmental stimuli, enabling them to fulfill diverse functions ranging from bacterial killing over tissue clearance to antigen presentation [1,2]. Pro-inflammatory M1 macrophages can be induced by lipopolysaccharide (LPS) and/or interferon-gamma (IFNγ) [3,4]. One of the well-accepted characteristics of M1 macrophages is the elevated production of various inflammatory mediators, including nitric oxide (NO), reactive oxygen species (ROS), and pro-inflammatory cytokines like interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor (TNFα) [5,6]. Metabolic reprogramming is an additional essential feature of activated macrophages. In fact, metabolic pathways do not only serve as mere source of energy but also are decisive for the functional state of macrophages and other immune cells (immunometabolism) [ 7–9]. M1 macrophages depend on the rapid initiation of glycolysis to facilitate effective bacterial removal and the release of inflammatory mediators [10]. Their tricarboxylic acid (TCA) cycle shows two breaks that result in accumulation of citrate and succinate [11,12]. Built-up citrate and its subsequent cytosolic conversion into acetyl-CoA facilitate cholesterol or fatty acid synthesis (FAS) and histone acetylation, which support inflammatory signaling in M1 cells and the relaxation of chromatin structure around inflammatory genes, respectively [ 13–15]. Piled up succinate drives the pro-inflammatory M1 phenotype by increased activity of succinate dehydrogenase (complex II) within the electron transport chain (ETC), promoting mitochondrial superoxide production via reverse electron transport (RET) at complex I, and enhanced hypoxia-inducible factor1-α (HIF1-α) stabilization [ 16–19]. Overall, M1 macrophages are characterized by elevated glycolysis, a broken TCA cycle, repressed oxidative phosphorylation (OXPHOS) and mitochondrial metabolism repurposed from ATP generation to production of ROS and precursors for biomolecule synthesis or signaling [20,21].Inhibitors of OXPHOS activity not only consistently prevented Sfn from maximally reducing LPS-induced nos2, ilβ, il6 and tnfα expression (Fig. 2) but also distinctly affected expression of different M1 (LPS) markers on their own (Supplementary Fig. 4): The extent of nos2 and tnfα induction was not affected whereas ilβ and il6 were rather boosted, supporting the notions that (i) an impaired mitochondrial OXPHOS does not interfere with pro-inflammatory (M1) macrophage polarization [58,59], but rather boosts it (e.g. by increased glycolysis as compensatory response to inhibited OXPHOS) [60], and that (ii) individual genes differ in their susceptibility to control by bioenergetics [61].The FWF (Austrian Science Fund; P32600 to EHH) and the University of Vienna provided financial support for this study.
Austrian Fields of Science 2012
- 301209 Pharmacy
- 301204 Pharmacognosy
Keywords
- Fatty acid synthesis
- Histone acetylation
- Immunometabolism
- M1 macrophages
- Mitochondria
- Sulforaphane
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