Systems Immunology Approaches to Metabolism.

Over the last decade, immunometabolism has emerged as a novel interdisciplinary field of research and yielded significant fundamental insights into the regulation of immune responses. Multiple classical approaches to interrogate immunometabolism, including bulk metabolic profiling and analysis of metabolic regulator expression, paved the way to appreciating the physiological complexity of immunometabolic regulation in vivo. Studying immunometabolism at the systems level raised the need to transition towards the next-generation technology for metabolic profiling and analysis. Spatially resolved metabolic imaging and computational algorithms for multi-modal data integration are new approaches to connecting metabolism and immunity. In this review, we discuss recent studies that highlight the complex physiological interplay between immune responses and metabolism and give an overview of technological developments that bear the promise of capturing this complexity most directly and comprehensively.

Hyperpolarized [5-13C,4,4-2H2,5-15N]-L-glutamine provides a means of annotating in vivo metabolic utilization of glutamine.

Glutamine is consumed by rapidly proliferating cells and can provide the carbon and nitrogen required for growth through various metabolic pathways. However, delineating the metabolic fate of glutamine is challenging to interrogate in vivo. Hyperpolarized magnetic resonance, by providing high transient nuclear magnetic resonance signals, provides an approach to measure fast biochemical processes in vivo. Aminohydrolysis of glutamine at carbon-5 plays an important role in providing nitrogen and carbon for multiple pathways. Here, we provide a synthetic strategy for isotope-enriched forms of glutamine that prolongs glutamine-C5 relaxation times and thereby reveals in vivo reactions involving carbon-5. We investigate multiple enrichment states, finding [5-13C,4,4-2H2,5-15N]-L-glutamine to be optimal for hyperpolarized measurement of glutamine conversion to glutamate in vivo. Leveraging this compound, we explore pancreatic cancer glutamine metabolism in vivo. Taken together, this work provides a means for studying glutamine metabolic flux in vivo and demonstrates on-target effects of metabolic enzyme inhibitors.

Whole-brain deuterium metabolic imaging via concentric ring trajectory readout enables assessment of regional variations in neuronal glucose metabolism.

Deuterium metabolic imaging (DMI) is an emerging magnetic resonance technique, for non-invasive mapping of human brain glucose metabolism following oral or intravenous administration of deuterium-labeled glucose. Regional differences in glucose metabolism can be observed in various brain pathologies, such as Alzheimer's disease, cancer, epilepsy or schizophrenia, but the achievable spatial resolution of conventional phase-encoded DMI methods is limited due to prolonged acquisition times rendering submilliliter isotropic spatial resolution for dynamic whole brain DMI not feasible. The purpose of this study was to implement non-Cartesian spatial-spectral sampling schemes for whole-brain 2H FID-MR Spectroscopic Imaging to assess time-resolved metabolic maps with sufficient spatial resolution to reliably detect metabolic differences between healthy gray and white matter regions. Results were compared with lower-resolution DMI maps, conventionally acquired within the same session. Six healthy volunteers (4 m/2 f) were scanned for ~90 min after administration of 0.8 g/kg oral [6,6']-2H glucose. Time-resolved whole brain 2H FID-DMI maps of glucose (Glc) and glutamate + glutamine (Glx) were acquired with 0.75 and 2 mL isotropic spatial resolution using density-weighted concentric ring trajectory (CRT) and conventional phase encoding (PE) readout, respectively, at 7 T. To minimize the effect of decreased signal-to-noise ratios associated with smaller voxels, low-rank denoising of the spatiotemporal data was performed during reconstruction. Sixty-three minutes after oral tracer uptake three-dimensional (3D) CRT-DMI maps featured 19% higher (p = .006) deuterium-labeled Glc concentrations in GM (1.98 ± 0.43 mM) compared with WM (1.66 ± 0.36 mM) dominated regions, across all volunteers. Similarly, 48% higher (p = .01) 2H-Glx concentrations were observed in GM (2.21 ± 0.44 mM) compared with WM (1.49 ± 0.20 mM). Low-resolution PE-DMI maps acquired 70 min after tracer uptake featured smaller regional differences between GM- and WM-dominated areas for 2H-Glc concentrations with 2.00 ± 0.35 mM and 1.71 ± 0.31 mM, respectively (+16%; p = .045), while no regional differences were observed for 2H-Glx concentrations. In this study, we successfully implemented 3D FID-MRSI with fast CRT encoding for dynamic whole-brain DMI at 7 T with 2.5-fold increased spatial resolution compared with conventional whole-brain phase encoded (PE) DMI to visualize regional metabolic differences. The faster metabolic activity represented by 48% higher Glx concentrations was observed in GM- compared with WM-dominated regions, which could not be reproduced using whole-brain DMI with the low spatial resolution protocol. Improved assessment of regional pathologic alterations using a fully non-invasive imaging method is of high clinical relevance and could push DMI one step toward clinical applications.

Viewing early life without labels: optical approaches for imaging the early embryo†.

Embryo quality is an important determinant of successful implantation and a resultant live birth. Current clinical approaches for evaluating embryo quality rely on subjective morphology assessments or an invasive biopsy for genetic testing. However, both approaches can be inherently inaccurate and crucially, fail to improve the live birth rate following the transfer of in vitro produced embryos. Optical imaging offers a potential non-invasive and accurate avenue for assessing embryo viability. Recent advances in various label-free optical imaging approaches have garnered increased interest in the field of reproductive biology due to their ability to rapidly capture images at high resolution, delivering both morphological and molecular information. This burgeoning field holds immense potential for further development, with profound implications for clinical translation. Here, our review aims to: (1) describe the principles of various imaging systems, distinguishing between approaches that capture morphological and molecular information, (2) highlight the recent application of these technologies in the field of reproductive biology, and (3) assess their respective merits and limitations concerning the capacity to evaluate embryo quality. Additionally, the review summarizes challenges in the translation of optical imaging systems into routine clinical practice, providing recommendations for their future development. Finally, we identify suitable imaging approaches for interrogating the mechanisms underpinning successful embryo development.

A vendor-neutral EPI sequence for hyperpolarized 13C MRI.

PURPOSE: To develop a flexible, vendor-neutral EPI sequence for hyperpolarized 13C metabolic imaging. METHODS: An open-source EPI sequence consisting of a metabolite-specific spectral-spatial RF excitation pulse and a customizable EPI readout was created using the Pulseq framework. To explore the flexibility of our sequence, we tested several versions of the sequence including a symmetric 3D readout with different spatial resolutions for each metabolite (1.0 cm3 and 1.5 cm3). A multichamber phantom constructed with a Shepp-Logan geometry, containing two chambers filled with either natural abundance 13C compounds or hyperpolarized (HP) [1-13C]pyruvate, was used to test each sequence. For experiments involving HP [1-13C]pyruvate, a single chamber was prefilled with nicotinamide adenine dinucleotide hydride and lactate dehydrogenase to facilitate the conversion of [1-13C]pyruvate to [1-13C]lactate. All experiments were performed on a Siemens Prisma 3T scanner. RESULTS: All the sequence variations localized natural-abundance 13C ethylene glycol and methanol to the appropriate compartment of the multichamber phantom. [1-13C]pyruvate was detectable in both chambers following the injection of HP [1-13C]pyruvate, whereas [1-13C]lactate was only found in the chamber containing nicotinamide adenine dinucleotide hydride and lactate dehydrogenase. The conversion rate from [1-13C]pyruvate to [1-13C]lactate (kPL) was 0.01 s-1 (95% confidence interval [0.00, 0.02]). CONCLUSION: We have developed and tested a vendor-neutral EPI sequence for imaging HP 13C agents. We have made all of our sequence creation and image reconstruction code freely available online for other investigators to use.

Current methods for hyperpolarized [1-13C]pyruvate MRI human studies.

MRI with hyperpolarized (HP) 13C agents, also known as HP 13C MRI, can measure processes such as localized metabolism that is altered in numerous cancers, liver, heart, kidney diseases, and more. It has been translated into human studies during the past 10 years, with recent rapid growth in studies largely based on increasing availability of HP agent preparation methods suitable for use in humans. This paper aims to capture the current successful practices for HP MRI human studies with [1-13C]pyruvate-by far the most commonly used agent, which sits at a key metabolic junction in glycolysis. The paper is divided into four major topic areas: (1) HP 13C-pyruvate preparation; (2) MRI system setup and calibrations; (3) data acquisition and image reconstruction; and (4) data analysis and quantification. In each area, we identified the key components for a successful study, summarized both published studies and current practices, and discuss evidence gaps, strengths, and limitations. This paper is the output of the "HP 13C MRI Consensus Group" as well as the ISMRM Hyperpolarized Media MR and Hyperpolarized Methods and Equipment study groups. It further aims to provide a comprehensive reference for future consensus, building as the field continues to advance human studies with this metabolic imaging modality.

Dynamic T 2 * relaxometry of hyperpolarized [1-13 C]pyruvate MRI in the human brain and kidneys.

PURPOSE: This study aimed to quantify T 2 * $$ {T}_2^{\ast } $$ for hyperpolarized [1-13 C]pyruvate and metabolites in the healthy human brain and renal cell carcinoma (RCC) patients at 3 T. METHODS: Dynamic T 2 * $$ {T}_2^{\ast } $$ values were measured with a metabolite-specific multi-echo spiral sequence. The dynamic T 2 * $$ {T}_2^{\ast } $$ of [1-13 C]pyruvate, [1-13 C]lactate, and 13 C-bicarbonate was estimated in regions of interest in the whole brain, sinus vein, gray matter, and white matter in healthy volunteers, as well as in kidney tumors and the contralateral healthy kidneys in a separate group of RCC patients. T 2 * $$ {T}_2^{\ast } $$ was fit using a mono-exponential function; and metabolism was quantified using pyruvate-to-lactate conversion rate maps and lactate-to-pyruvate ratio maps, which were compared with and without an estimated T 2 * $$ {T}_2^{\ast } $$ correction. RESULTS: The T 2 * $$ {T}_2^{\ast } $$ of pyruvate was shown to vary during the acquisition, whereas the T 2 * $$ {T}_2^{\ast } $$ of lactate and bicarbonate were relatively constant through time and across the organs studied. The T 2 * $$ {T}_2^{\ast } $$ of lactate was similar in gray matter (29.75 ± 1.04 ms), white matter (32.89 ± 0.9 ms), healthy kidney (34.61 ± 4.07 ms), and kidney tumor (33.01 ± 2.31 ms); and the T 2 * $$ {T}_2^{\ast } $$ of bicarbonate was different between whole-brain (108.17 ± 14.05 ms) and healthy kidney (58.45 ± 6.63 ms). The T 2 * $$ {T}_2^{\ast } $$ of pyruvate had similar trends in both brain and RCC studies, reducing from 75.56 ± 2.23 ms to 22.24 ± 1.24 ms in the brain and reducing from 122.72 ± 9.86 ms to 57.38 ± 7.65 ms in the kidneys. CONCLUSION: Multi-echo dynamic imaging can quantify T 2 * $$ {T}_2^{\ast } $$ and metabolism in a single integrated acquisition. Clear differences were observed in the T 2 * $$ {T}_2^{\ast } $$ of metabolites and in their behavior throughout the timecourse.

Deuterium metabolic imaging for 3D mapping of glucose metabolism in humans with central nervous system lesions at 3T.

PURPOSE: To explore the potential of 3T deuterium metabolic imaging (DMI) using a birdcage 2 H radiofrequency (RF) coil in both healthy volunteers and patients with central nervous system (CNS) lesions. METHODS: A modified gradient filter, home-built 2 H volume RF coil, and spherical k-space sampling were employed in a three-dimensional chemical shift imaging acquisition to obtain high-quality whole-brain metabolic images of 2 H-labeled water and glucose metabolic products. These images were acquired in a healthy volunteer and three subjects with CNS lesions of varying pathologies. Hardware and pulse sequence experiments were also conducted to improve the signal-to-noise ratio of DMI at 3T. RESULTS: The ability to quantify local glucose metabolism in correspondence to anatomical landmarks across patients with varying CNS lesions is demonstrated, and increased lactate is observed in one patient with the most active disease. CONCLUSION: DMI offers the potential to examine metabolic activity in human subjects with CNS lesions with DMI at 3T, promising for the potential of the future clinical translation of this metabolic imaging technique.

Label-Free Optical Metabolic Imaging in Cells and Tissues.

Over the last half century, the autofluorescence of the metabolic cofactors NADH (reduced nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) has been quantified in a variety of cell types and disease states. With the spread of nonlinear optical microscopy techniques in biomedical research, NADH and FAD imaging has offered an attractive solution to noninvasively monitor cell and tissue status and elucidate dynamic changes in cell or tissue metabolism. Various tools and methods to measure the temporal, spectral, and spatial properties of NADH and FAD autofluorescence have been developed. Specifically, an optical redox ratio of cofactor fluorescence intensities and NADH fluorescence lifetime parameters have been used in numerous applications, but significant work remains to mature this technology for understanding dynamic changes in metabolism. This article describes the current understanding of our optical sensitivity to different metabolic pathways and highlights current challenges in the field. Recent progress in addressing these challenges and acquiring more quantitative information in faster and more metabolically relevant formats is also discussed.

Metabolic imaging of human embryos is predictive of ploidy status but is not associated with clinical pregnancy outcomes: a pilot trial.

STUDY QUESTION: Does fluorescence lifetime imaging microscopy (FLIM)-based metabolic imaging assessment of human blastocysts prior to frozen transfer correlate with pregnancy outcomes? SUMMARY ANSWER: FLIM failed to distinguish consistent patterns in mitochondrial metabolism between blastocysts leading to pregnancy compared to those that did not. WHAT IS KNOWN ALREADY: FLIM measurements provide quantitative information on NAD(P)H and flavin adenine dinucleotide (FAD+) concentrations. The metabolism of embryos has long been linked to their viability, suggesting the potential utility of metabolic measurements to aid in selection. STUDY DESIGN, SIZE, DURATION: This was a pilot trial enrolling 121 IVF couples who consented to have their frozen blastocyst measured using non-invasive metabolic imaging. After being warmed, 105 couples' good-quality blastocysts underwent a 6-min scan in a controlled temperature and gas environment. FLIM-assessed blastocysts were then transferred without any intervention in management. PARTICIPANTS/MATERIALS, SETTING, METHODS: Eight metabolic parameters were obtained from each blastocyst (4 for NAD(P)H and 4 for FAD): short and long fluorescence lifetime, fluorescence intensity, and fraction of the molecule engaged with enzyme. The redox ratio (intensity of NAD(P)H)/(intensity of FAD) was also calculated. FLIM data were combined with known metadata and analyzed to quantify the ability of metabolic imaging to differentiate embryos that resulted in pregnancy from embryos that did not. De-identified discarded aneuploid human embryos (n = 158) were also measured to quantify correlations with ploidy status and other factors. Statistical comparisons were performed using logistic regression and receiver operating characteristic (ROC) curves with 5-fold cross-validation averaged over 100 repeats with random sampling. AUC values were used to quantify the ability to distinguish between classes. MAIN RESULTS AND THE ROLE OF CHANCE: No metabolic imaging parameters showed significant differences between good-quality blastocysts resulting in pregnancy versus those that did not. A logistic regression using metabolic data and metadata produced an ROC AUC of 0.58. In contrast, robust AUCs were obtained when classifying other factors such as comparison of Day 5 (n = 64) versus Day 6 (n = 41) blastocysts (AUC = 0.78), inner cell mass versus trophectoderm (n = 105: AUC = 0.88) and aneuploid (n = 158) versus euploid and positive pregnancy embryos (n = 108) (AUC = 0.82). LIMITATIONS, REASONS FOR CAUTION: The study protocol did not select which embryo to transfer and the cohort of 105 included blastocysts were all high quality. The study was also limited in number of participants and study sites. Increased power and performing the trial in more sites may have provided a stronger conclusion regarding the merits of the use of FLIM clinically. WIDER IMPLICATIONS OF THE FINDINGS: FLIM failed to distinguish consistent patterns in mitochondrial metabolism between good-quality blastocysts leading to pregnancy compared to those that did not. Blastocyst ploidy status was, however, highly distinguishable. In addition, embryo regions and embryo day were consistently revealed by FLIM. While metabolic imaging detects mitochondrial metabolic features in human blastocysts, this pilot trial indicates it does not have the potential to serve as an effective embryo viability detection tool. This may be because mitochondrial metabolism plays an alternative role post-implantation. STUDY FUNDING/COMPETING INTEREST(S): This study was sponsored by Optiva Fertility, Inc. Boston IVF contributed to the clinical site and services. Becker Hickl, GmbH, provided the FLIM system on loan. T.S. was the founder and held stock in Optiva Fertility, Inc., and D.S. and E.S. had options with Optiva Fertility, Inc., during this study. TRIAL REGISTRATION NUMBER: The study was approved by WCG Connexus IRB (Study Number 1298156).