2-AA tolerization decreases crucial metabolites of cellular energy and affects mitochondrial respiration in mouse BMDM.

(A) Schematic representation showing experimental design: naïve cells were exposed to 2-AA for 1, 6, or 48 h (black). Cells exposed to 2-AA (400 µM) for 48 h were washed, rested for 24 or 106 h, and re-exposed (200 µM) for 1 or 6 h (red), respectively. The same color code, black cells after 1st exposure and red cells after 2nd exposure, was kept throughout the manuscript with corresponding controls in grey and pink, respectively. The levels of (B) ATP and (C) acetyl-CoA in BMDM cells after 1st and 2nd 2-AA exposure. (D) Real-time oxygen consumption rate (OCR) traces were recorded using a Seahorse XF analyzer and normalized to protein content. Cells were exposed to 2-AA for 1 or 24 h (black), washed and rested for 24 h and re-exposed for 1 h (red). Mitochondrial respiratory parameters, (E) basal respiration, and (F) maximal respiration. Data are presented as mean ± SD, n ≥ 4, *p < 0.05, **p < 0.01, ***p < 0.001, and ns indicates no significant difference. One-way ANOVA followed by Tukey’s post hoc test was applied.

2-AA perturbs the mitochondrial MPC1-mediated import and metabolism of pyruvate.

(A) Cytosolic and mitochondrial pyruvate levels following 2-AA exposure (black) or re-exposure (red) and corresponding controls in grey and pink, respectively, for indicated time points. (B) Representative Western blot and results of densitometric analysis of MPC1 protein levels following 2-AA exposure or re-exposure for indicated time points. β-actin was used as a control. Corresponding controls are shown in grey or pink, respectively. Mean ± SD is shown, n = 3, ***p < 0.001, ****p < 0.0001, and ns indicates no significant difference. One-way ANOVA followed by Tukey’s post hoc test was applied.

2-AA-mediated macrophage tolerization deranges PGC-1α/ERRα-dependent metabolic programing.

(A) Representative Western blot and results of densitometric analysis of ERRα protein levels following 2-AA exposure or re-exposure for indicated time points. β-actin was used as a control. (B) Western blots and its corresponding densitometric analysis of ERRα in cytoplasmic or nuclear lysates of tolerized macrophages exposed to 2-AA for 24 h or not exposed to 2-AA. (C) ChIP-qPCR assay of ERRα binding at the MPC1 promoter in RAW 264.7 tolerized macrophages exposed to 2-AA (200 μM) for 24 h (black) compared to untreated control macrophages (grey). IgG served as a negative control (D) Representative Western blot of co-immunoprecipitation (co-IP) studies of ERRα and PGC-1α in nuclear extracts of 2-AA tolerized (24 h) and control RAW 264.7 cells. Pull down with IgG served as a negative control. 2-AA tolerized macrophages are shown in black, and untreated control macrophages in grey. Mean ± SD is shown, n ≥ 3, *p < 0.05, **p < 0.01, ****p < 0.0001, and ns indicates no significant difference. One-way ANOVA followed by Tukey’s post hoc test was applied.

Increased intracellular burden in macrophages is associated with decreased expression of MPC1, ERR-α, and TNF-α genes.

Real-time PCR analysis of MPC1 (A), ERR-α (B), and PGC-1α (C) expression in RAW 246.7 macrophages infected with PA14 or ΔmvfR in the presence or absence of exogenous addition of 2-AA or ATP for 6 h as indicated. Transcript levels were normalized to 18srRNA. PA14-infected cells served as controls. (D) The intracellular burden of PA14 or ΔmvfR of infected macrophages in the presence or absence of exogenous addition of 2-AA, UK5099, or ATP. Untreated cells infected with PA14 were set as 100%. (E) Real-time PCR analysis of TNF-α expression in RAW 246.7 macrophages infected with PA14 or ΔmvfR in the presence or absence of exogenous addition of 2-AA, ATP, or UK5099. Transcript levels were normalized to 18srRNA. PA14-infected cells served as controls. The compound concentration used for UK5099 was 10 µM and ATP 20 µM. Mean ± SD is shown, n = 3, * p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001***, and ns indicates no significant difference. One-way ANOVA followed by Tukey’s post hoc test was applied.

2-AA promotes a long-lasting decrease in ATP, acetyl-CoA levels, and bacterial persistence in PA-infected mice.

(A) ATP and (B) acetyl-CoA concentrations in the spleens of mice infected with PA wild type (PA14), the isogenic mutant ΔmvfR, ΔmvfR injected with 2-AA at the time of infection (ΔmvfR + 2-AA), or non-infected but injected with 2-AA (6.75 mg/kg), (C) Bacterial burden in muscles expressed as CFU count was analyzed using the Kruskal–Wallis non-parametric test with Dunn’s post-test; ***p < 0.001, and ns indicates no significant difference. Control mice groups: naïve were not given 2-AA; mice receiving 2-AA were given a single intraperitoneal injection of 2-AA; sham represents a burn/PBS group since the burn and infection model was used. Results of three independent replicates with 4 mice per group for 1, 5, and 10 d are shown. Means ± SD are shown, *p < 0.05, **p < 0.01, ***p < 0.001, and ns indicate no significant difference. One-way ANOVA followed by Tukey’s post hoc test was applied.

Proposed mechanism by which 2-AA impairs bioenergetics through the inhibition of MPC1-mediated pyruvate transport into mitochondria and its impact on the PGC-1α/ERRα axis.

2-AA tolerized macrophages exhibit diminished pyruvate levels in mitochondria due to the decreased expression of MPC1, a consequence of the 2-AA impact on the interaction of ERRα with the transcriptional coactivator PGC-1α for the effective transcription of ERRα since ERRα controls its own transcription and that of MPC1. In the presence of 2-AA, the weakened interaction between ERRα and PGC-1α results in reduced expression of MPC1 and ERRα. The reduction in mitochondrial pyruvate levels leads to decreased acetyl-CoA and ATP levels, which modulate HDAC1- and HAT-catalyzed remodeling of H3K18 acetylation. The diminished levels of this epigenetic mark have previously been associated with an increased intracellular presence of bacteria in macrophages, as demonstrated by our group [18]. Pathways, proteins, and metabolites that are negatively affected are indicated in red, while positively affected are denoted in black. The figure was created using BioRender.com.

© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.

2-AA tolerization decreases metabolites in murine RAW 264.7 (A-B) and human THP-1 (C-D) cells. (A and C) ATP, (B and D) acetyl-CoA, and (E) cytosolic and (F) mitochondrial pyruvate levels were quantified in 2-AA (400 μM) exposed and re-exposed cells for the hours indicated using the same condition as shown in Fig. 1A. Mean ± SD is shown (n = 3); *** p < 0.001, and ns indicates no significant difference. One-way ANOVA followed by Tukey’s post hoc test was applied. The same color code, black cells after 1st exposure and red cells after 2nd exposure, was kept throughout the main manuscript, with corresponding controls shown in grey and pink, respectively.

ATP levels in murine BMDM (A) cells with and without 2-AA (400 μM) or LPS stimulation (100 μg/mL). Each dot represents 1 experimental replicate of 4 independent experiments (n = 4). Data are presented as mean ± SD, **p < 0.01, ***p < 0.001, and ns indicates no significant difference. One-way ANOVA followed by Tukey’s post hoc test was applied.

Effect of 2-AA on mitochondrial spare respiratory capacity in BMDM non-tolerized (black) and tolerized (red) macrophages. Controls are shown in grey and pink, respectively. Mitochondrial spare respiratory capacity data extrapolated from the OCR profiles shown in Figure 1D. Unstimulated BMDM macrophages were used as a control (c). Means ± SDs are shown, n = 6, * p < 0.05, *** p < 0.001, and ns indicates no significant difference. One-way ANOVA followed by Tukey’s post hoc test was applied.

Transmission electron microscopy (TEM) images showing structural alterations of mitochondria in 2-AA exposed macrophages for 48 h. High-magnification (30,000X) TEM images showing mitochondrial abundance and structural changes in 2-AA exposed macrophages as compared to the non-exposed macrophages.

Exposure and re-exposure to 2-AA promotes a long-lasting decrease in ATP and acetyl-CoA levels and sustains bacterial presence in mice receiving 1st exposure to 2-AA by injecting 2-AA and 2nd exposure through infection with PA14 or ΔmvfR four days post-2-AA injection. (A) ATP and (B) acetyl-CoA concentrations in the spleens of mice. (C) Bacterial burden in muscles expressed as CFU counts was analyzed using the Kruskal–Wallis non-parametric test with Dunn’s post-test; ***p < 0.001, and ns indicates no significant difference. Control mice groups: naïve were not given 2-AA; mice receiving 2-AA were given a single intraperitoneal injection of 2-AA four days prior to infection; sham represents a burn/PBS group since the burn and infection model was used. N = 4 mice per group, and 10 days post-infection is shown. Means ± SD are shown, *p < 0.05, **p < 0.01, ***p < 0.001, and ns indicate no significant difference. One-way ANOVA followed by Tukey’s post hoc test was applied.