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 health^1,2. 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 host

Protocol

All the experiments were performed in accordance with the National Animal Protection Guidelines and approved by the German Animal Ethics Committee for the protection of animals (G0176/16 and G0207/19). The protocol describes NAD(P)H fluorescence lifetime imaging data acquisition and data evaluation, which allow one to assess the general metabolic activity and specific metabolic pathways in both the host intestine and the parasites upon infection with the natural murine intestinal nematode, H. polygyrus. For this.......

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.......

Materials

Name	Company	Catalog Number	Comments
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