Molecular detection of inflammation in cell models using hyperpolarized 13C-pyruvate - PubMed (original) (raw)

. 2018 May 23;8(12):3400-3407.

doi: 10.7150/thno.24322. eCollection 2018.

Affiliations

Molecular detection of inflammation in cell models using hyperpolarized 13C-pyruvate

Renuka Sriram et al. Theranostics. 2018.

Abstract

The detection and treatment monitoring of inflammatory states remain challenging in part due to the multifactorial mechanisms of immune activation and spectrum of clinical manifestations. Currently, diagnostic strategies tend to be subjective and limited quantitative tools exist to monitor optimal treatment strategies. Pro-inflammatory M1 polarized macrophages exhibit a distinct metabolic glycolytic phenotype compared to the continuum of M2 polarization states. In the present study, the distinct metabolic phenotypes of resting and activated macrophages were successfully characterized and quantified using hyperpolarized carbon-13 (13C) labeled pyruvate and its metabolic products, i.e. lactate, as a biomarker of resting, disease and treated states. Methods: Mouse macrophage J774A.1 cells were used as a model system in an NMR compatible bioreactor to facilitate dynamic hyperpolarized 13C measurements. The glycolytic metabolism of the cells in the quiescent or resting state were compared with macrophages stimulated by lipopolysaccharide, a classical M1 activator using hyperpolarized 13C labeled pyruvate. Additionally, the activated macrophages were also treated with a non-steroidal anti-inflammatory drug to assess the changes in hyperpolarized lactate signal. The hyperpolarized lactate signals were then correlated using biochemical and molecular assays. Results: We first validated our model system of inflammatory cells by the hallmarks of M1 polarization using steady state metabolic profiling with high resolution NMR in conjunction with nitric oxide Greiss assay, enzyme activity, and mRNA expression. Subsequently, we clearly showed that the cutting edge technology of hyperpolarized 13C NMR can be used to detect elevated lactate levels in M1 polarized macrophages in comparison to control and non-steroidal anti-inflammatory drug treated M2 states. Conclusion: Hyperpolarized 13C lactate has the potential to serve as a biomarker to non-invasively detect and quantify pro-inflammatory state of immune regulatory cells and its response to therapy.

Keywords: M1 polarization; dynamic nuclear polarization (DNP); hyperpolarized 13C nuclear magnetic resonance (MR); inflammation; lactate; macrophages; pyruvate.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1

Figure 1

1H spectra of cell extracts. 800 MHz spectra of cell extracts of control macrophages (lower spectrum) and that of LPS-treated macrophages (top spectrum). The labeled peaks, namely itaconate, arginine and lactate, indicate the significantly elevated metabolites in LPS-stimulated macrophages that are indicative of the M1 polarization state.

Figure 2

Figure 2

Three-fold elevation in the production of hyperpolarized 13C-lactate after LPS stimulation in a macrophage cell model of inflammation. (A) 31P spectrum shows viable cells with normal physiology. (B) 13C-NMR spectrum of hyperpolarized signals normalized to pyruvate signal shows elevation of lactate production in LPS-activated macrophages in comparison to control (inset is 5x magnification of the lactate spectra). (C) Bar graph shows the 3.5-fold increase in hyperpolarized 13C-lactate after LPS stimulation in the macrophages and its subsequent reduction to baseline (control cells) with indomethacin treatment.

Figure 3

Figure 3

Molecular underpinnings of increased hyperpolarized lactate signal. (A) Schematic representation of hyperpolarized pyruvate metabolism in activated macrophages. (B) NADH content in the stimulated and control cells measured using a calorimetric assay. (C) RNA expression of stimulated macrophages relative to control cells (normalized to the housekeeping gene L19). Increased LDHA expression with a concomitant increase in the monocarboxylate transporters (MCT1 and MCT4) synergistically promote increased lactate production as well as efflux. LDHb/a: isoenzymes A and B of lactate dehydrogenase; MCT1/4: monocarboxylate transporters 1 and 4. (D) The LDH activity measured in stimulated macrophages reflects the increased LDHA expression compared to the control cells. (* denotes significant change from control, p<0.05)

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