Figures and data
CoA reverses tolerance and enhances TLR-dependent proinflammatory responses in vitro.
A. Steady-state levels of (left) and relative 13C6-glucose fractional contribution (FC) to (right) select TCA cycle metabolites in recombinant murine macrophage colony stimulating factor (rMCSF)-differentiated bone morrow-derived macrophages (rBMDMs) stimulated with LPS for 2h. n=3 biological replicates per condition, numbers above bars are Student’s t-test P values (planned comparisons). B. Quantitative realtime PCR (qPCR) for LPS-induced proinflammatory gene expression in naïve rBMDMs, RAW 264.7s, and THP-1s with CoA supplementation (250μM). Numbers denote fold-induction over the untreated condition. C. qPCR for LPS-induced proinflammatory gene expression in naïve or tolerized bone morrow-derived macrophages (BMDMs) with CoA supplementation (2.5mM). Numbers denote fold-induction over the tolerized, untreated condition. D. left – RNAseq heatmap denoting relative expression (RPKM) of select genes in untreated or LPS-stimulated (6h) rBMDMs from ref. 34. top right - qPCR for LPS-induced proinflammatory gene expression with CoA supplementation as described in B. bottom – qPCR for gene expression in cells supplemented with 500μM (lo) or 2.5mM (high) CoA for 2h. For all qPCRs, n=2 or 3 biological replicates per condition, mean ± s.e.m is shown, numbers above bars are Student’s t-test P values (planned comparisons).
Macrophages directly take up exogenous CoA.
A. Model for proinflammatory effects of CoA, and testable hypotheses from the model. Image created with Biorender.com. B. SILEC strategy for isotopic labeling of the endogenous CoA pool, and tracing of unlabeled CoA into macrophages. Image created with Biorender.com. C. Abundance of unlabeled CoA over time in SILEC RAW264.7 macrophages treated with unlabeled CoA, PPanSH, or Pan (all 500μM). n=4, mean ± s.e.m. is shown. D. For CoA-treated cells from C, unlabeled CoA as a percentage of the total CoA pool over time (set to 100%). E. For cells from C, the total amount of CoA (labeled + unlabeled) at 60 minutes following CoA, PPanSH, or Pan treatment. Mean is shown, numbers above data are Student’s t-test P values (planned comparison with untreated condition). F. Abundance of unlabeled CoA in SILEC rBMDMs 60 minutes after treatment with unlabeled CoA, PPanSH, or Pan all 500μM). n=4, mean is shown, numbers above data are Student’s t-test P values (planned comparison with the untreated condition). G. ForCoA-treated cells from F, unlabeled CoA as a percentage of the total CoA pool over time (set to 100%). H. For cells from F, the total amount of CoA (labeled + unlabeled) at 60 minutes following CoA, PPanSH, or Pan treatment. Mean is shown, numbers above data are Student’s t-test P values (planned comparison with untreated condition).
CoA taken up by macrophages enhances mitochondrial glucose oxidation and directly supports acetyl-CoA production.
A. Top – experimental design for 13C6-glucose isotope tracing in rBMDMs. Competitive labeling of metabolites with 13C6-glucose is performed from the final 30 minutes of a 1.5h LPS stimulation to determine how CoA supplementation (250μM) influences glucose labeling of metabolites, serving as a proxy for metabolic pathway activity. Bottom – Heatmap depicting 13C6-glucose Fractional Contribution (FC) to glycolytic and TCA cycle metabolites, represented as a Z-score for each metabolite. Metabolites with significant FC differences between conditions were identified by One-Way ANOVA. *P<0.05. *P<0.01,***P<0.001. n=3 biological replicates per condition. B. Relative 13C6-glucose FC to acetyl-CoA and citrate from A. Numbers above bars are P values from Dunnett’s post-hoc test correcting for multiple comparisons. C. Heatmap depicting Z-score-normalized 13C6-glucose FC to indicated metabolites in naïve BMDMs treated with CoA (250μM) or LPS for 1.75h. D. qPCR for CoA-induced I1lb expression (2h) in cells pretreated with ACLY inhibitors and 2-DG (left, n=3, mean ± s.e.m), or 4-OHE1 for 1h (right, n=2, mean). Numbers above data are Student’s t-test P values (planned comparison). E. left – schematic depicting production of unlabeled acetyl-CoA from unlabeled CoA taken up by SILEC macrophages. right - unlabeled acetyl-CoA levels in SILEC rBMDMs 60 minutes following unlabeled CoA or Pan treatment (both 500μM). n=4, mean is shown, numbers above data are Student’s t-test P values (planned comparison with the untreated condition).
Exogenous CoA increases histone acetylation at cis-regulatory regions of proinflammatory genes.
A. Model depicting how exogenous CoA uptake expands the CoA pool and supports increased acetyl-CoA-dependent histone acetylation at proinflammatory gene promoters/enhancers. Image created with Biorender.com. B. H3K27ac levels measured by intracellular staining (ICS)-flow cytometry in untreated or CoA-treated (500μM) rBMDMs. Treatment with histone deacetylase (HDAC) inhibitor Trichostatin A (TSA, 1μM 18h) serves as a positive control. C. H3K27ac levels at Il1b and Il1a cis-regulatory regions assessed by ChIP-qPCR in control, CoA, or LPS-treated supBMDMs. Mean is shown, each data point represents an independent ChIP experiment/biological replicate (n=2 per condition). IgG control ChIP-qPCR shows specificity of H3K27ac signal. D. Venn diagram depicting total number of H3K27ac peaks identified by Cut- and-Tag (CnT) in rBMDMs, relative to the total numbers of peaks increased in read density versus untreated control rBMDMs by either CoA (500μM) or LPS treatment. E. Overlap between CoA- and LPS-induced H3K27ac CnT peaks. F. Gene ontology (GO) analysis of 2432 genes near CoA- and LPS-induced H3K27ac CnT peaks. G. HOMER de novo motif finding results for enriched transcription factor motifs in CoA- and LPS-induced H3K27ac CnT peaks. H. Cumulative read density at the 2432 H3K27ac peaks in cells treated with either CoA or LPS alone, or CoA/LPS co-treatment. I. Response of putative H3K27ac-marked cis-regulatory regions in the Ccl5 locus to treatment with CoA, LPS, or CoA/LPS co-treatment.
Tonic TLR signaling regulates basal macrophage glucose uptake, metabolic homeostasis, histone acetylation, and gene expression.
A. Model depicting predicted regulation of macrophage biology by tonic TLR signaling. Image created with Biorender.com. B. 2-NDBG uptake in WT vs. TLR KO rBMDMs. n=3, mean is shown, numbers above bars are Student’s t-test P values (planned comparisons). C. For approximately 130 metabolites detected, Z score-normalized steady-state levels of 26 metabolites displaying significant differences (all decreased, p<0.05, One-Way ANOVA) between WT and TLR KO rBMDMs. n=3 biological replicates per genotype. D. Left – H3K27ac ICS-flow cytometry in rBMDMs cultured in DMEM with 25mM versus 2.5mM glucose. Top histograms representative of data quantified below. Right - H3K27ac ICS-flow cytometry in WT vs. TLR KO supBMDMs cultured in RPMI with 10mM glucose. Top histograms representative of data quantified below. n=3, mean is shown, and numbers above bars are P values from Student’s t-test (planned comparisons). TSA-treated cells represent H3K27ac positive control. E. H3K27ac levels at cis-regulatory regions in Il1b and Il1a loci (left) and non-TLR4 target gene promoters (right) assessed by ChIP-qPCR in WT versus TLR KO supBMDMs. Mean is shown, each data point represents an independent ChIP experiment/biological replicate (n=2 per condition). IgG control ChIP-qPCR shows specificity of H3K27ac signal. F. Basal Il1b expression in WT versus TLR KO rBMDMs. n=3, mean ± s.e.m. is shown. G. Cell-surface expression of the indicated markers measured by flow cytometry on WT versus TLR KO supBMDMs. n=3, mean is shown, numbers above bars are Student’s t-test P values (planned comparisons).
Exogenous CoA’s effects on histone acetylation and proinflammatory gene transcription are dependent on tonic TLR signaling.
A. Il1b qPCR in rBMDMs treated with LPS alone, CoA alone (250μM), or CoA/LPS co-treatment, without or with 4-OHE1 (5μM) pretreatment. n=2, mean is shown. B. Il1b qPCR in WT and TLR KO BMDMs treated with CoA or TLR ligands for 4h. n=3, mean ± s.e.m. is shown. C. Relative abundance of unlabeled CoA (top) and unlabeled acetyl-CoA (bottom) in WT versus TLR KO SILEC rBMDMs treated with unlabeled CoA (500μM) for 60 minutes. n=3, mean is shown, numbers above bars are Student’s t-test P values (planned comparisons). D. H3K27ac levels at Il1b and Il1a cis-regulatory regions as assessed by ChIP-qPCR in control or CoA-treated (500μM, 1.5h) TLR KO supBMDMs. Mean is shown, each data point represents an independent ChIP experiment/biological replicate (n=2 per condition). IgG control ChIP-qPCR shows specificity of H3K27ac signal. E. Il1b qPCR in WT and TLR KO BMDMs pretreated with 500μM CoA for 2h before stimulation with 50ng/mL recombinant mouse IL-1β for 4h. F. Model depicting how exogenous CoA enhances macrophage mitochondrial glucose oxidation and acetyl-CoA production capacity, cooperating with TLR signaling to provide metabolic-epigenetic support of proinflammatory gene transcription. Image created with Biorender.com.
CoA enhances TLR agonist anti-tumor activity.
A. Serum levels of select proteins in male C57BL/6 mice intraperitoneally (i.p.) injected with CoA (250 mg/kg), LPS (3 mg/kg), or CoA/LPS. Violin plots for select serum proteins (left), and heatmap showing relative induction of all cytokines, chemokines, and growth factors quantified (right), with induction in “LPS” group set to 100%. For violin plots, numbers above bars are P values from Student’s t-test for the indicated conditions (planned comparisons). For heatmap, *P<0.05. *P<0.01, ***P<0.001, and ****P<0.0001 between “LPS” and “CoA/LPS” groups in each row (Student’s t-test, planned comparisons). “x” denotes protein for which at least one sample in the “CoA/LPS” group exceeded the linear range of the standard curve, underestimating induction in this group. n=5 mice per group except “CoA/LPS”, n=4. B. Orthotopic MMTV-PyMT breast tumor volume in female C57BL/6 mice. Left – volume in control tumors versus tumors treated intratumorally (i.t.) with MPLA, CoA, or MPLA+CoA. Right – volume in control, MPLA+CoA-treated, and MPLA+IFNγ-treated tumors. Numbers represent Student’s t-test P values at Day 27 (planned comparisons). n=8 mice per group. C. Intratumoral gene expression (left) and cytokine levels (right) from mice in A. 16 hours after final i.t. treatment. n=7 or 8 mice per group. Numbers represent Student’s t-test P values (planned comparisons). D. Orthotopic MMTV-PyMT breast tumor volume (left) and survival (right) in female C57BL/6 mice treated for 25 days with controls, CoA, αCTLA-4, or CoA/αCTLA-4 combination therapy. n=10 mice per group.
CoA enhances macrophage anti-microbial activity against Legionella pneumophila via CoA-supported itaconate biosynthesis.
A. Luminescent ΔflaA Legionella pneumophila intracellular growth in infected supBMDMs. Left – intracellular bacterial replication over time in control or CoA-treated supBMDMs. Right - intracellular bacterial replication over time in supBMDMs treated with CoA or IFNγ (6ng/mL). Numbers represent Student’s t-test P values (planned comparisons). n=3 biological replicates per condition. B. Model depicting how CoA could support itaconate production via 1) increasing metabolic-epigenetic support of Irg1 expression, and 2) supporting increased flux of glucose carbon into the TCA cycle. C. Irg1 qPCR in supBMDMs treated with CoA (250μM), LPS, or CoA/LPS for 2h. D. Total steady-state itaconate levels (left), and relative 13C6-glucose fractional contribution (FC) to itaconate (right), in rBMDMs treated with CoA (250μM), LPS, or CoA/LPS for 2h. E. Luminescent ΔflaA Legionella pneumophila intracellular growth over time in control or CoA-treated supBMDMs from WT or Irg1−/− macrophages. F. L. pneumophila colony forming units (CFUs) from infected WT and Irg1−/− supBMDMs in E at 96h.
A. Top – Illustrated summary of 4-OHE1’s disruption of CoA and acetyl-CoA homeostasis and LPS-induced histone acetylation. Bottom - qPCR for LPS-induced Il1b and Nos2 in BMDMs (1.5h) pretreated for 1h with 4-hydroxyestrone (5μM) and/or CoA (250μM). B. qPCR for LPS-induced Il1b (2h) with CoA supplementation (250μM) in naïve BMDMs differentiated with MCSF-containing supernatant from 3T3-L1 cells (supBMDMs). C. qPCR for proinflammatory gene expression in primary human monocytes from 3 independent donors with CoA supplementation at two doses (lo = 500μM CoA, hi = 5mM CoA) either alone or with LPS. D. Endotoxin levels in CoAs and CoA precursors/degradation products utilized in this study, and estimated levels of contaminating endotoxin in a 500μM in vitro CoA treatment. E. qPCR for Il1b in RAW264.7 macrophages treated with CoA (250μM), LPS, or simultaneously cotreated with CoA/LPS, for 6h. F. qPCR for LPS-induced Tnf expression in naïve or tolerized rBMDMs with CoA supplementation (2.5mM). For qPCRs in A, B, E, and F, n=2 or 3 biological replicates per condition, mean ± s.e.m is shown, numbers above bars are Student’s t-test P values (planned comparisons).
A. Schematic depicting known pathways of extracellular CoA degradation product entry into intracellular CoA biosynthesis pathway. B. Il1b qPCR in rBMDMs treated with CoA or CoA degradation products (500μM). Percentages represent Il1b induction relative to CoA. C. Il1b qPCR in rBMDMs treated with CoA (500μM) for 2h. Prior to addition to rBMDMs, CoA was preincubated at 37°C for 3h in PBS, media, non-heat inactivated FBS, of heat-inactivated (HIA) FBS. For qPCRs in B and C, n=4, and mean ± s.e.m. is shown, and numbers above data are Student’s t-test P values. D. Unlabeled CoA as a percentage of the total CoA pool (set to 100%) pre- and post-SILEC labeling of RAW264.7 cells. n=1. E. Unlabeled and labeled CoA levels in SILEC RAW264.7 cells treated with unlabeled CoA for 5 or 60 minutes and subjected to the indicated numbers of 0.9% NaCl washes. n=1, mean is shown. F. Unlabeled CoA as a percentage of the total CoA pool (set to 100%) in SILEC RAW264.7 cells treated with unlabeled CoA for the indicated times and subjected to 5 0.9% NaCl washes. n=1. G. Unlabeled CoA as a percentage of the total CoA pool (set to 100%) pre- and post-SILEC labeling of rBMDMs. n=1.
A. Unlabeled acetyl-CoA as a percentage of the total CoA pool (set to 100%) pre- and post-SILEC labeling of RAW264.7 cells (left) and rBMDMs (right). n=4. B. Unlabeled acetyl-CoA levels in SILEC RAW264.7 macrophages 60 minutes following unlabeled CoA or Pan treatment (both 500μM). n=4, mean is shown, numbers above data are Student’s t-test P values (planned comparison with the untreated condition). C. Total lactate levels (left), and relative 13C6-glucose fractional contribution (FC) to lactate (right), in LPS-tolerized BMDMs treated with CoA (2.5mM), LPS, or CoA/LPS. n=3 biological replicates per condition, mean ± s.e.m is shown, numbers above bars are Student’s t-test P values (planned comparisons).
A. Quantification of Fig.4B H3K27ac ICS-flow cytometry experiment assessing global H3K27ac upon CoA treatment. n=3, mean is shown. B. Luciferase activity in RAW264.7 (left) and THP-1 cells (right) with stably-integrated NFκB luciferase reporters following 2 hour treatment with either CoA (500μM), LPS, or CoA/LPS co-treatment. n=3, mean ± s.e.m. is shown, and numbers above bars are Student’s t-test P values (planned comparisons). C. H3K27ac levels at Nrh1h2 and Cox5a promoters assessed by ChIP-qPCR in cells treated with CoA or LPS. D. Comparison of Il1b locus H3K27ac peaks captured in previously published ChIP-seq experiment from reference 49, and by our H3K27ac CnT experiments. E. CnT read density at putative cis-regulatory region H3K27ac peaks in the indicated genes in rBMDMs treated with either CoA (500μM), LPS, or CoA/LPS co-treatment.
A. 2-NDBG uptake in WT vs. TLR KO supBMDMs. n=3, mean is shown, numbers above bars are Student’s t-test P values (planned comparisons). B. Steady-state levels of acetyl-CoA in WT versus TLR KO rBMDMs measured as part of Fig.5C experiment.
A. Il1b, Il1a, and Nos2 expression measured by qPCR in rBMDMs treated with CoA (top), or TLR ligands (bottom). n=2, mean is shown. B. Flow cytometry measurement of FITC-Dextran uptake over time in WT versus TLR KO rBMDMs. n=2, mean is shown.
A, B. Day 30 tumor weights (A) and intratumoral lactate level (B) for tumors from mice in Fig. 7B experiment. C. Top - Experimental strategy to test the contribution of tumor-associated macrophages (TAMs) to the anti-tumor effect of MPLA+CoA therapy. Bottom - Effects of TAM depletion on MPLA+CoA therapy efficacy, represented by comparing tumor size among all animals (left), or by comparing tumor growth rates (right), on experimental Days 13 and 27 between IgG and anti-CSFR1 cohorts receiving vehicle or MPLA+CoA therapy. n=8 or 7 mice per group. D. Left – flow cytometry of CD45+ tumor cells depicting F4/80 hi CD11b hi TAM populations in representative mice from IgG control antibody-treated or anti-CSF1R antibody-treated cohorts. Right – quantification of flow cytometry results confirming TAM depletion in anti-CSF1R-treated mice. 6 mice were chosen at random from the IgG control or anti-CSF1R cohorts. Black indicates mice receiving vehicle control treatment, red indicates mice receiving MPLA+CoA therapy. All numbers above bars in figure represent Student’s t-test P values (planned comparisons).
A. qPCR for L. pneumophila restriction genes in rBMDMs treated with CoA alone (250μM), or CoA in combination with LPS, for 2h. n=3, mean ± s.e.m is shown, numbers represent Student’s t-test P values (planned comparisons). B. L. pneumophila colony forming units (CFUs) from infected BMDMs in Fig. 8A 96h after infection. n=3, mean ± s.e.m is shown, numbers represent Student’s t-test P values (planned comparisons). C. L. pneumophila growth (left) and luminescence (right) in broth supplemented with 500μM CoA for 24 hours. n=3, mean ± s.e.m is shown. D. supBMDM viability assessed by flow cytometry after 72h of continuous 500μM CoA supplementation. n=3, mean ± s.e.m is shown. E. Total steady-state itaconate levels (left), and relative 13C6-glucose fractional contribution (FC) to itaconate (right), in rBMDMs treated with IFNψ (xxx), LPS, or IFNψ/LPS for 2h. F. Model depicting how a pathogen-driven mitoOXPHOS to aerobic glycolysis shift reduces production of proinflammatory/anti-microbial metabolites to create a replicative niche for pathogens. Image created with Biorender.com.
