CENCAT enables immunometabolic profiling by measuring protein synthesis via bioorthogonal noncanonical amino acid tagging

Abstract

Cellular energy metabolism significantly contributes to immune cell function. To further advance immunometabolic research, novel methods to study the metabolism of immune cells in complex samples are required. Here, we introduce CENCAT (cellular energetics through noncanonical amino acid tagging). This technique utilizes click labeling of alkyne-bearing noncanonical amino acids to measure protein synthesis inhibition as a proxy for metabolic activity. CENCAT successfully reproduced known metabolic signatures of lipopolysaccharide (LPS)/interferon (IFN)γ and interleukin (IL)-4 activation in human primary macrophages. Application of CENCAT in peripheral blood mononuclear cells revealed diverse metabolic rewiring upon stimulation with different activators. Finally, CENCAT was used to analyze the cellular metabolism of murine tissue-resident immune cells from various organs. Tissue-specific clustering was observed based on metabolic profiles, likely driven by microenvironmental priming. In conclusion, CENCAT offers valuable insights into immune cell metabolic responses, presenting a powerful platform for studying cellular metabolism in complex samples and tissues in both humans and mice.

Keywords: CP: Immunology; CP: Metabolism; OXPHOS; SCENITH; energy metabolism; glycolysis; immunometabolism.

Graphical abstract

Figure 1

Overview of performed experiments

Figure 2

Comparison of CENCAT with the original SCENITH protocol (A) Structures of L-methionine and HPG. (B and C) Primary human macrophages (M-CSF) were treated with HPG and harvested at different time points for click labeling. (B) Representative flow cytometry histograms of HPG-AZ488 staining at 0, 0.5, 1, 2, 4, and 6 h post-treatment. (C) HPG incorporation kinetics of macrophages treated with homoharringtonine (red) or DMSO control (blue). Data are displayed as means ± SD of the geometric MFI (gMFI; n = 3). (D and E) Representative histograms of protein synthesis for DMSO, 2-DG, oligomycin, and 2-DG + oligomycin conditions as measured using puromycin (Puro) (D) or HPG (E) in GM-CSF macrophages. (F and G) Primary human macrophages (GM-CSF and M-CSF) were stimulated for 24 h with culture medium (green), LPS + IFNγ (red), or IL-4 (blue) before SCENITH analysis using either HPG or Puro as substrates. (F) Mitochondrial dependence (%) and glycolytic capacity (%) of GM-CSF macrophages. (G) Mitochondrial dependence (%) and glycolytic capacity (%) of M-CSF macrophages. Data are displayed as mean percentages ±SD (n = 6). Significance was tested by two-way ANOVA with Sidak correction for multiple testing. Individual donors are displayed by different symbols. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 3

CENCAT analysis of PBMC metabolic profiles after different stimulations (A) PBMCs isolated from healthy blood donors (n = 6) were stimulated for 2 h with Medium control (green), LPS (red), ODN2006 (orange), TransAct (blue), or all stimuli combined (Mix, purple). (B) Representative uniform manifold approximation and projection (UMAP) plot of PBMCs stained with 9-marker flow cytometry panel. Colors and numbers indicate different cell populations. (C) Mean fold changes of HPG incorporation compared to Medium control of classical monocytes, B cells, CD4 T cells, and CD8 T cells. Significance was tested by repeated one-way ANOVA with Dunnett’s multiple comparisons test. Individual donors are displayed by different symbols. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. (D) Structures of L-threonine and βES. (E) Representative histograms of protein synthesis for DMSO, 2-DG, oligomycin, and 2-DG + oligomycin conditions as measured using HPG or βES in naive CD4 T cells. (F) Mean glucose dependence (%) and mitochondrial dependence (%) of classical monocytes, effector CD4 T cells, effector CD8 T cells, naive CD4 T cells, and naive CD8 T cells. Significance was tested by paired t test. Individual donors are displayed by different symbols. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. CENCAT was performed using HPG (C and E) or βES (E and F).

Figure 4

CENCAT analysis of murine tissue-resident immune cell populations The following tissues were isolated from male C57BL/6J mice and subjected to CENCAT analysis: eWAT (red, n = 4), kidney (yellow, n = 6), liver (green, n = 6), lung (cyan, n = 6), PECs (blue, n = 6), and spleen (pink, n = 6). (A) βES incorporation (MFI) of immune cell populations from all six tissues. Data are displayed as means ± SD. (B) PCA score plot based on metabolic dependencies of immune cell populations in all six tissues. (C and D) Top loadings on PC1 (C) and PC2 (D) of the PCA score plot. Measures of glucose dependence are represented by blue bars and mitochondrial dependence by red bars. (E and F) Boxplots of mitochondrial and glucose dependence (%) of cDC2s (E) and macrophages (F) from all six tissues.