Metabolic Reprogramming in Response to Freund’s Adjuvants: Insights from Serum Metabolomics
Abstract
:1. Introduction
2. Materials and Methods
2.1. Mice
2.2. BCG Propagation and Enumeration
2.3. Immunization
2.4. Metabolite Extraction for Serum Sample
2.5. LC-MS Data Acquisition and Pre-Processing
2.6. Metabolomics Data Processing and Statistical Analysis
2.7. Metabolite Annotation
3. Results and Discussion
3.1. Serum Adjuvants Have Distinct Metabolic Phenotypes
3.2. BCG Reprograms Serum Metabolites Associated with Energy Metabolism
3.3. Minimal Metabolite Perturbation Between BCG and CFA
3.4. Multivariate Analysis of BCG and IFA Serum Metabolites
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AGC | Automatic gain control |
APCs | Antigen-presenting cells |
BCG | Bacillus Calmette–Guérin |
CFA | Complete Freund’s adjuvant |
CFU | Colony-forming units |
CMI | Cell-mediated immune |
HILIC | Hydrophilic interaction liquid chromatography |
HPLC | High-performance liquid chromatography |
IDO1 | Indoleamine 2,3-dioxygenase 1 |
IFA | Incomplete Freund’s adjuvant |
KEGG | Kyoto Encyclopedia of Genes and Genomes |
LC-MS | Liquid chromatography coupled with high-resolution mass spectrometry |
maxIT | Maximum injection time |
M. tb | Mycobacterium tuberculosis |
mTOR | Mammalian target of rapamycin |
NK | Natural killer |
NLRP3 | Nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein-3 |
OADC | Oleic Acid-Albumin-Dextrose-Catalase |
OD | Optical Density |
PCA | Principal component analysis |
PLS-DA | Partial least squares discriminant analysis |
PPM | Parts per million |
RT | Retention Time |
TCA | Tricarboxylic acid |
Th | T helper |
TID | Type I diabetes |
VIP | Variable importance in projection |
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Metabolite | Major Role | Ref. |
---|---|---|
Upregulated metabolites in the BCG group as compared to both saline and IFA groups | ||
Succinic acid | Intermediate of TCA cycle, metabolic reprogramming, epigenetic regulator and modulator of inflammatory response | [53,54,55] |
Aminomalonate | Amino acid synthesis | [101] |
2-hydroxybutanoic acid | Biomarker of preclampsia | [102] |
Methionine | Lipid metabolism and ferroptosis | [56,57,58] |
N-acetylmannosamine-6-phophate | Sialic acid synthesis | [103] |
Upregulated metabolites in the BCG group as compared to saline group | ||
4-hydroxybenzoic acid | NLRP3 inflammasome activation and oxidative stress | [59,60] |
Upregulated metabolites in the BCG group as compared to IFA group | ||
6-phosphogluconate | Reprogramming of Treg cells | [104] |
Kynurenine | Inflammation, oxidative stress, endothelial dysfunction and immune suppression | [61,62,63] |
Glutathione | Antioxidant, signaling and NK cell and T cell responses | [64,65,66,67,68,69,70] |
Acetylleucine | mTOR pathway | [105] |
Glycerophosphate | Adenosine triphosphate synthesis | [71] |
Glucose-6-phosphate | Central energy metabolism | [72] |
Pyruvate | Central energy metabolism, antioxidant and suppression of T cell responses | [73] |
Malate | TCA cycle intermediate | [74] |
Upregulated metabolites in the CFA group as compared to BCG group | ||
Phosphatidylcholine | Anti-inflammatory | [75] |
1-methyladenosine | Oncometabolite | [94,95] |
cGMP | Innate immune signaling | [106,107,108,109,110] |
Creatinine | Anti-inflammatory, immunomodulator and antioxidant | [76,77,78] |
Methylhistidine | Biomarker of skeletal muscle breakdown | [111] |
L-carnitine | Downregulate T cell responses and block inflammation | [79,80,81,82,83] |
2-hydroxyglutarte | Oncometabolite, promotes Treg cells, regulates T cell response, anti-inflammatory and blocks NF-AT activation | [84,85,86,87,88,89,90,91,92,93] |
Taurine | Oncometabolite, pro- and anti-inflammatory | [96,97,98,99,100] |
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Mone, K.; Garcia, E.J.T.; Abdullatif, F.; Rasquinha, M.T.; Sur, M.; Hanafy, M.; Zinniel, D.K.; Singh, S.; Thomas, R.; Barletta, R.G.; et al. Metabolic Reprogramming in Response to Freund’s Adjuvants: Insights from Serum Metabolomics. Microorganisms 2025, 13, 492. https://doi.org/10.3390/microorganisms13030492
Mone K, Garcia EJT, Abdullatif F, Rasquinha MT, Sur M, Hanafy M, Zinniel DK, Singh S, Thomas R, Barletta RG, et al. Metabolic Reprogramming in Response to Freund’s Adjuvants: Insights from Serum Metabolomics. Microorganisms. 2025; 13(3):492. https://doi.org/10.3390/microorganisms13030492
Chicago/Turabian StyleMone, Kiruthiga, Eloy Jose Torres Garcia, Fatema Abdullatif, Mahima T. Rasquinha, Meghna Sur, Mostafa Hanafy, Denise K. Zinniel, Shraddha Singh, Raymond Thomas, Raul G. Barletta, and et al. 2025. "Metabolic Reprogramming in Response to Freund’s Adjuvants: Insights from Serum Metabolomics" Microorganisms 13, no. 3: 492. https://doi.org/10.3390/microorganisms13030492
APA StyleMone, K., Garcia, E. J. T., Abdullatif, F., Rasquinha, M. T., Sur, M., Hanafy, M., Zinniel, D. K., Singh, S., Thomas, R., Barletta, R. G., Gebregiworgis, T., & Reddy, J. (2025). Metabolic Reprogramming in Response to Freund’s Adjuvants: Insights from Serum Metabolomics. Microorganisms, 13(3), 492. https://doi.org/10.3390/microorganisms13030492