Quantitative 1H NMR Metabolomics Reveal Distinct Metabolic Adaptations in Human Macrophages Following Differential Activation

Abstract

Macrophages (MΦs) are phagocytic immune cells that are found in nearly all human tissues, where they modulate innate and adaptive immune responses, thereby maintaining cellular homeostasis. MΦs display a spectrum of functional phenotypes as a result of microenvironmental and stress-induced stimuli. Evidence has emerged demonstrating that metabolism is not only crucial for the generation of energy and biomolecular precursors, but also contributes to the function and plasticity of MΦs. Here, 1D 1H NMR-based metabolomics was employed to identify metabolic pathways that are differentially modulated following primary human monocyte-derived MΦ activation with pro-inflammatory (M1) or anti-inflammatory (M2a) stimuli relative to resting (M0) MΦs. The metabolic profiling of M1 MΦs indicated a substantial increase in oxidative stress as well as a decrease in mitochondrial respiration. These metabolic profiles also provide compelling evidence that M1 MΦs divert metabolites from de novo glycerophospholipid synthesis to inhibit oxidative phosphorylation. Furthermore, glycolysis and lactic acid fermentation were significantly increased in both M1 and M2a MΦs. These metabolic patterns highlight robust metabolic activation markers of MΦ phenotypes. Overall, our study generates additional support to previous observations, presents novel findings regarding the metabolic modulation of human MΦs following activation, and contributes new knowledge to the rapidly evolving field of immunometabolism.

Pseudomonas aeruginosa Planktonic- and Biofilm-Conditioned Media Elicit Discrete Metabolic Responses in Human Macrophages

“…In addition, our data indicated that BCM-exposed MΦs exhibit significantly decreased levels of intracellular lactate, relative to PCM-exposed MΦs ( Figure 3 ; Table 1 ). This finding differs from what was previously demonstrated [ 39 ], where a significant increase in both intra- and extracellular lactate was observed in MΦs activated with a combination of lipopolysaccharide (LPS) and interferon-γ relative to resting MΦs. However, MΦs in our current study were only stimulated with bacterial-conditioned media for 1 h prior to metabolite extraction, whereas MΦs in our prior study were activated with purified recombinant human cytokines over a period of 72 h, which may account for this difference.…”

“…Furthermore, Cheng et al demonstrated that leukocytes isolated from patients with sepsis exhibit a shift to aerobic glycolysis, which dissipates following patient recovery [ 77 ]. Our findings provide further evidence that pro-inflammatory stimulation of MΦs induces Warburg-like metabolic traits, as previously reported in M1 MΦs [ 39 , 57 , 78 ]. Accordingly, we postulate that PCM- and BCM-exposed MΦs upregulate glycolysis, a well-known and established metabolic marker of M1 MΦ activation, relative to control MΦs in order to promote M1-like MΦ effector functions.…”

“…A total of 5 donors that ranged in age from 19 to 35 were included in this particular study, of whom 40% of these volunteers were female. Peripheral blood mononuclear cells were isolated by differential centrifugation in lymphocyte separation (Ficoll) media (Corning Inc., Corning, NY, USA) at 800× g for 25 min at room temperature, following protocols described previously [ 39 ]. CD14 + monocytes were isolated from peripheral blood mononuclear cells by MACS using CD14 human microbeads (Miltenyi Biotec, San Diego, CA, USA), which amounted to an average purity of 97.1 ± 1.3% ( Supplementary Materials , Figure S1 ) when analyzed on an LSR Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA).…”

“…Spectral phase correction and chemical shift referencing of DSS were performed using Topspin software (Bruker Scientific LLC, Billerica, MA, USA; version 3.6). Metabolite assignments, annotated using Chenomx NMR Suite software (version 8.1; Chenomx Inc., Edmonton, Alberta, Canada) as detailed in the “NMR Data Analysis” section below, were further validated using 2D 1 H- 1 H total correlation spectroscopy (TOCSY) NMR as previously described [ 39 ] or by spiking metabolite reference standards into the samples, as needed.…”

Triacylglycerol synthesis enhances macrophage inflammatory function

“…Altered mitochondrial function, in which FAO and OXPHOS are inhibited and the TCA cycle is fragmented to support the use of citrate and aconitate for fatty acid and itaconate production respectively, are core metabolic features of inflammatory macrophage activation 3,6,7,[29][30][31] . These events, alongside increased FA uptake and increased DGAT expression, create an environment in which TG synthesis is promoted.…”