NAD(P)H Fluorescence Lifetime Imaging for the Metabolic Analysis of the Murine Intestine and Parasites During Nematode Infection
Summary
The present protocol describes the NAD(P)H fluorescence lifetime imaging of an explanted murine intestine infected with the natural parasite Heligmosomoides polygyrus, which allows one to investigate metabolic processes both in host and parasite tissues in a spatially resolved manner.
Abstract
Parasites generally have a negative effect on the health of their host. They represent a huge health burden, as they globally affect the health of the infested human or animal in the long term and, thus, impact agricultural and socio-economic outcomes. However, parasite-driven immune-regulatory effects have been described, with potential therapeutic relevance for autoimmune diseases. While the metabolism in both the host and parasites contributes to their defense and is the basis for nematode survival in the intestine, it has remained largely understudied due to a lack of adequate technologies. We have developed and applied NAD(P)H fluorescence lifetime imaging to explanted murine intestinal tissue during infection with the natural nematode Heligmosomoides polygyrus to study the metabolic processes in both the host and parasites in a spatially resolved manner. The exploitation of the fluorescence lifetime of the co-enzymes nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), hereafter NAD(P)H, which are preserved across species, depends on their binding status and the binding site on the enzymes catalyzing metabolic processes. Focusing on the most abundantly expressed NAD(P)H-dependent enzymes, the metabolic pathways associated with anaerobic glycolysis, oxidative phosphorylation/aerobic glycolysis, and NOX-based oxidative burst, as a major defense mechanism, were distinguished, and the metabolic crosstalk between the host and parasite during infection was characterized.
Introduction
Parasitic infections impose a huge burden on human health1,2. A correlation between the rise in autoimmune diseases and the decline in parasitic infections has been observed in industrial countries. It is known that parasites can have beneficial effects by dampening excessive host immune responses. H. polygyrus is a natural parasite found in the intestine in rodents, and this parasite is known to induce immunoregulatory mechanisms that reduce the anti-parasitic immune response of the host via, among other mechanisms, the induction of regulatory T cells (Treg) in the infected host3,4,5,6,7,8,9,10,11. Those regulatory mechanisms are especially of interest in degenerative autoimmune diseases.
The analysis of the metabolic crosstalk between the host and intestinal nematodes remains widely neglected, although metabolism plays an important role in both the host and parasites for defense, survival, and function. We propose to adapt and apply NADH and NADPH fluorescence lifetime imaging upon two-photon excitation, a technology already widely used in different physiological and pathophysiological situations in mammalian cells and tissues12, to investigate host and nematode metabolism in living tissues correlatively.
NADH and NADPH, referred to as NAD(P)H, are ubiquitous molecules that are preserved in all cell-based lifeforms and play the role of co-enzymes in various metabolic pathways. For instance, they are involved in energy production, reductive biosynthesis, and NADPH oxidase-mediated reactive oxygen species (ROS) production, which are mainly linked to cell defense and cell communication13,14,15,16,17,20. Both co-enzymes emit fluorescence at ~450 nm upon two-photon excitation at 750 nm, thus allowing for marker-free metabolic imaging in cells and tissues19,21. Exciting both NADH and NADPH with only one wavelength is possible due to their similar and rather broad two-photon excitation spectra21.
The fluorescence lifetime of the co-enzyme NAD(P)H is directly dependent on the enzyme to which it binds18,21,22,23. Due to its chemical structure allowing for intramolecular energy transfer, the excited NADH or NADPH molecule loses energy through internal conversion processes, at a rate depending on its binding properties, to the enzymes (catalyst) before it relaxes and emits a fluorescence photon. This lifetime gives insight into the NAD(P)H binding site on the enzyme and, thus, the preferential biochemical reaction taking place19,21,22,23,24,25. The fluorescence lifetime of free NADH and NADPH molecules amounts to ~450 ps, whereas their fluorescence lifetime when bound to an enzyme is much longer (~2,000 ps) and depends on their binding site on the respective enzyme21.
There are more than 370 enzymes involved in NAD(P)H-linked processes; however, only the most abundant will be able to contribute to the resulting NAD(P)H fluorescence lifetime within the excitation range of the microscope. Using RNASeq data from mammal cells, we identified the most abundant NAD(P)H-dependent enzymes and generated a fluorescence lifetime reference to interpret the data generated in tissue and cell samples18. Thereby, this work distinguished for instance between the preferential activity of lactate dehydrogenase (LDH), which is associated with anaerobic glycolytic metabolic pathways, and isocitrate dehydrogenase (IDH) and pyruvate dehydrogenase (PDH) activity, which are mainly involved in aerobic glycolysis/oxidative phosphorylation metabolic pathways16,20. Additionally, NADPH binding to NADPH oxidases, which are the enzymes that are mainly responsible for oxidative burst, can be easily resolved due to the characteristic location of these enzymes in the cell (membrane-bound) and because of the particularly long NADPH fluorescence lifetime (3,650 ps)18,24,29,30,32. RNASeq data from H. polygyrus shows that the reference generated for mammalian cells also applies in adapted form to this nematode27.
Hence, in this work, by performing NAD(P)H fluorescence lifetime imaging (FLIM) in freshly explanted duodenum samples of mice infected with H. polygyrus, information on the ratio between free and enzyme-bound NAD(P)H was acquired, which depicted the general metabolic activity in all tissues, as well as the predominantly active enzyme in each pixel of the image (i.e., the enzyme to which NAD(P)H preferentially binds in that specific location). The success of these experiments relies on the accurate sample preparation of the explanted intestine, the reliable live imaging of the NAD(P)H fluorescence lifetime at subcellular resolution, and standardized data evaluation, as discussed in this protocol.
Protocol
Representative Results
Discussion
The critical steps within the protocol occur during the preparation and when finding the ROI. Fibers of partially digested food represent a challenge for imaging, mainly due to the endogenous luminescence of the fibers overlapping with the NAD(P)H fluorescence, but also due to their harmonic generation signal. It is of great importance to find ROIs that are free from feces. We aimed to avoid measuring areas containing feces. Washing was avoided because this affects the integrity of the fragile villi and influences the mu…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Robert Günther for their excellent technical support. Financial support from the German Research Council (DFG) under Grant SPP2332 HA2542/12-1 (S.H.), NI1167/7-1 (R.A.N.), HA5354/11-1 (A.E.H.), and RA2544/1-1 (S.R.), under Grant SFB1444, P14 (R.A.N., A.E.H.), under Grant HA5354/8-2 (A.E.H.), and under Grant GRK2046 B4 and B5 (S.H., S.R.) and HA2542/8-1 (S.H.) are greatly acknowledged. W.L. received a PhD fellowship from the Berliner Hochschule für Technik, School of Applied Sciences, Berlin in Medical Physics/Physical Engineering.
Materials
| Agarose | Thermo fisher | J32802.22 | ultra pure |
| Blunt scissors | FST fine science tools | 14108-09 | blund-blund 14 cm |
| Bodipy c12 | thermo fisher | D3822 | 1 mg solid |
| Control units, diode, TCSPC | LaVision Biontech | custom | TrimScope II |
| DMSO | Thermo fisher | D12345 | 3 mL |
| Filters | Chroma | 755 | 466 ± 20, 525 ± 25, 593 ± 20, 655 ± 20 nm |
| Foliodrape sheet | Hartmann | 277500 | |
| Gloves | Sigma-Aldrich | Z423262 | nitril |
| Halogen torch | Leica | This item has been phased out and is no longer available | KL 1500 LCD |
| hPMT | Hamamatsu, Germany | H7422 | GaAsP |
| Ilastik | Netlify | free Software | Java Backend |
| ImageJ | National Institutes of health | free Software | FIJI – standard plugins |
| Imspector | LaVision Biontech | – | Vers. 208 |
| Intravital stage | LaVision Biontech | custom | TrimScope II |
| Lens system 20x | Zeiss | custom | W-plan-apochom 20x Waterimmersion NA 1.05 |
| Mercury vapor torch | LaVision Biontech | custom | |
| microbrush | Fisher scientific | 22-020-002 | 85 mm |
| Microscope | LaVision Biontech | custom | TrimScope II |
| Oscilloscope | Rhode & Schwarz | 1326.2000.22 | |
| PBS | Sigma-Aldrich | AM9624 | 0.5 L |
| Petri dish | Sigma aldrich | P5606 | 40 x 15 mm |
| Pipette | thermo fisher | 4651280N | Einkanalpipette |
| Pipette tips | thermo fisher | 94056980 | Spitzen mit Filter |
| PMT | Hamamatsu, Japan | H7422 | GaAsP |
| Python | Python Software foundation | free Software | Anaconda 3.7 Spyder IDE, standard librarys with KYTE |
| Sterio microscope | Leica | This item has been phased out and is no longer available | M26, 6.3x zoom |
| Ti:Sa LASER CHAMELION ULTRA II | Coherent, APE | – | 690-1080 nm tunable, 80MHz |
| Tissueglue | 3M | 51115053603 | 3 mL |
| Tweezers | FST fine science tools | 11049-10 | blund, graefe, angeled |
| Tweezers | FST fine science tools | 91197-00 | Dumont, curved |
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