NAD(P)H fluorescence lifetime imaging of live intestinal nematodes reveals metabolic crosstalk between parasite and host

Abstract

Infections with intestinal nematodes have an equivocal impact: they represent a burden for human health and animal husbandry, but, at the same time, may ameliorate auto-immune diseases due to the immunomodulatory effect of the parasites. Thus, it is key to understand how intestinal nematodes arrive and persist in their luminal niche and interact with the host over long periods of time. One basic mechanism governing parasite and host cellular and tissue functions, metabolism, has largely been neglected in the study of intestinal nematode infections. Here we use NADH (nicotinamide adenine dinucleotide) and NADPH (nicotinamide adenine dinucleotide phosphate) fluorescence lifetime imaging of explanted murine duodenum infected with the natural nematode Heligmosomoides polygyrus and define the link between general metabolic activity and possible metabolic pathways in parasite and host tissue, during acute infection. In both healthy and infected host intestine, energy is effectively produced, mainly via metabolic pathways resembling oxidative phosphorylation/aerobic glycolysis features. In contrast, the nematodes shift their energy production from balanced fast anaerobic glycolysis-like and effective oxidative phosphorylation-like metabolic pathways, towards mainly anaerobic glycolysis-like pathways, back to oxidative phosphorylation/aerobic glycolysis-like pathways during their different life cycle phases in the submucosa versus the intestinal lumen. Additionally, we found an increased NADPH oxidase (NOX) enzymes-dependent oxidative burst in infected intestinal host tissue as compared to healthy tissue, which was mirrored by a similar defense reaction in the parasites. We expect that, the here presented application of NAD(P)H-FLIM in live tissues constitutes a unique tool to study possible shifts between metabolic pathways in host-parasite crosstalk, in various parasitic intestinal infections.

Th2 and metabolic responses to nematodes are independent of prolonged host microbiota abrogation

“…Along this line, we therefore investigated whether prolonged antibiotic treatment affects general NAD(P)H‐dependent metabolic activity and enzyme activity associated with specific metabolic pathways, both in the mouse intestine and in H. polygyrus . Using NAD(P)H fluorescence lifetime imaging (FLIM) in freshly explanted duodenum of infected mice, with and without antibiotic treatment, we confirmed a slightly higher general metabolic activity in host villi as compared to the parasites 12 . The energy production both in host and nematodes mainly follows metabolic pathways resembling aerobic glycolysis, rather than anaerobic glycolysis or β‐oxidation of fatty acids (preferential activity of hydroxyacyl‐coenzyme‐A dehydrogenase—HADH), independent of antibiotic treatment.…”

“…We imbedded the samples in low‐melting agarose (0.7%–1%) to preserve villi orientation. The Petri dish was kept at 37°C using a heating plate, to ensure in vivo‐like NAD(P)H‐dependent metabolic activity both in host tissue and in nematodes 12 . Keeping the Petri dish on ice, at 4°C, resulted in lack of fluorescence signal upon excitation at 760 nm, detected at 466 ± 30 nm, in both intestinal tissue of the host and in nematode tissue, confirming the NAD(P)H specificity of the fluorescence used for FLIM evaluation.…”

“…In order to distinguish between host and parasite tissues, we performed image segmentation of the NAD(P)H‐fluorescence intensity images using a UNet‐based algorithm (Python 3.1), as previously described 12 . The algorithm was trained to identify image areas with high fluorescence signal resembling the structure of villi, classified as host tissue, regions belonging the nematodes, identified based on their coiled structure, and in these areas, regions with high fluorescence signal, classified as nematode tissue with high enzymatic activity.…”

“…Yet, how the metabolism of the host immune system changes during infection with H. polygyrus and upon prolonged interference with the host microbiota, remains poorly studied. By using fluorescence lifetime imaging (FLIM) of the co‐enzymes NADH and NADPH—conserved across species—in live infected duodenal tissue, 12 we are able to explore the activity of specific metabolic pathways related to energy production and to defence using NADPH oxidases activation, both in host and in parasite, as an additional parameter to the characterization of immune responses.…”

“…Here, we therefore investigated how antibiotics potentially affect the formation, maintenance and recall responses of host memory Th2 cells. In parallel, we evaluated possible changes in the activity of different metabolic pathways in the parasite and host gut tissue via FLIM of the co‐enzymes NADH and NADPH, present in all cells and involved in key metabolic processes 12 …”