Abstract
The immune system defends the body from infection and plays a vital role in a wide range of health conditions. Metabolism affects a series of physiological processes, including those linked to the function of human immune system. Cellular metabolism modulates immune cell activation and cytokine production. Understanding the relationship between metabolism and immune response has important implications for the development of immune-based therapeutics. However, the deployment of large-scale functional assays to investigate the metabolic regulation of immune response has been limited by the lack of standardized procedures. Here, we present a protocol for the analysis of immune response using standardized whole-blood stimulation with metabolism modulation. Diverse immune stimuli including pattern recognition receptor (PRR) ligands and microbial stimuli were incubated with fresh human whole blood. The metabolic inhibitors were used to modulate metabolic status in the immune cells. The variable immune responses after metabolic interventions were evaluated. We described in detail the main steps involved in the whole-blood stimulation and cytokines quantification, namely, collection and treatment of whole blood, preparation of samples and controls, cytokines detection, and stimulation with metabolic interventions. The metabolic inhibitors for anabolic pathways and catabolic pathways exert selective effects on the production of cytokines from immune cells. In addition to a robust and accurate assessment of immune response in cohort studies, the standardized whole-blood stimulation with metabolic regulation might provide new insights for modulating immunity.
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Data Availability
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
Abbreviations
- 2-DG:
-
2-Deoxyglucose
- ELISA:
-
Enzyme linked immunosorbent assay
- FAO:
-
Fatty acid oxidation
- FBS:
-
Fetal bovine serum
- FLA:
-
Flagellin
- GVHD:
-
Graft-versus-host disease
- HKMT:
-
Heat killed mycobacterium tuberculosis
- HKSA:
-
Heat killed S. aureus
- HRP:
-
Horseradish peroxidase
- IDO1:
-
Indoleamine-2,3-dioxygenase 1
- IL-1β:
-
Interleukin-1 beta
- IL-6:
-
Interleukin-6
- IMPDH:
-
Inosine monophosphate dehydrogenase
- LPS:
-
Lipopolysaccharide
- MPA:
-
Mycophenolic acid
- NADPH:
-
Nicotinamide adenine dinucleotide phosphate
- NLRP3:
-
Nucleotide-binding oligomerization domain-like receptor family pyrin domain–containing 3
- NOX4:
-
Nicotinamide adenine dinucleotide phosphate oxidase 4
- OD:
-
Optical density
- Pam3CSK4:
-
Pam3CysSerLys4
- PBMC:
-
Peripheral blood mononuclear cell
- PBS:
-
Phosphate-buffered saline
- PRR:
-
Pattern recognition receptor
- RT:
-
Room temperature
- TLR:
-
Toll-like receptor
- TNF-α:
-
Tumor necrosis factor-alpha
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Acknowledgements
This project was supported by National Key Research and Development Program of China (2021YFA1301000), Shanghai Municipal Technology Standards Project (23DZ2202600), Shanghai Municipal Science and Technology Major Project (Grant No. 2017SHZDZX01).
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JLZ, XLH and FQ designed and coordinated the study. JLZ, XLH, HLL, YLL, YF, YW, JG, YRZ and JXH performed experiments. JLZ, XLH and FQ contributed to scientific discussion. JLZ and FQ wrote the first draft of the manuscript. All authors reviewed, revised, and approved the final manuscript.
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Yiran Zhao is the editorial operation team member of Phenomics, and she was not involved in reviewing this paper.
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This study was approved by the Institutional Review Board at School of Life Science, Fudan University.
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Zhao, J., Han, X., Li, H. et al. Analysis of the Immune Response by Standardized Whole-Blood Stimulation with Metabolism Modulation. Phenomics 4, 81–89 (2024). https://doi.org/10.1007/s43657-023-00114-0
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DOI: https://doi.org/10.1007/s43657-023-00114-0