Protocol for identifying immune mediators during Leishmania infection in mice using metabolomic analysis

TL;DR

This paper outlines a protocol to identify immune mediators during Leishmania infection in mice using metabolomic analysis.

Contribution

The study introduces a detailed protocol for metabolomic analysis of immune responses in Leishmania-infected mice.

Findings

Steps for Leishmania infection and metabolite extraction from infected tissues are described.

LC-MS is used to identify enriched metabolic pathways and altered metabolites.

PCR and ELISA are employed to verify the role of identified metabolites in immune responses.

Abstract

Here, we present a protocol for identifying immune mediators during Leishmania infection in mice using metabolomic analysis. We describe steps for Leishmania infection, harvesting infected tissues, and performing liquid chromatography-mass spectrometry (LC-MS) to identify enriched metabolic pathways and altered metabolites. We then detail procedures for verifying the metabolite role in immune response via PCR and ELISA. This protocol focuses on cutaneous leishmaniasis models, but the analyses here are applicable to other infection models.

For complete details on the use and execution of this protocol, please refer to Volpedo et al.1 and Oljuskin et al.2

•Development of a murine model of infection with two dermotropic Leishmania species•Metabolite extraction from infection site and downstream characterization via HPLC•Metabolic pathway analysis using MetaboAnalyst and MetScape•In vitro validation via orthogonal methods, such as PCR and ELISA

Development of a murine model of infection with two dermotropic Leishmania species

Metabolite extraction from infection site and downstream characterization via HPLC

Metabolic pathway analysis using MetaboAnalyst and MetScape

In vitro validation via orthogonal methods, such as PCR and ELISA

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol for identifying immune mediators during Leishmania infection in mice using metabolomic analysis. We describe steps for Leishmania infection, harvesting infected tissues, and performing liquid chromatography-mass spectrometry (LC-MS) to identify enriched metabolic pathways and altered metabolites. We then detail procedures for verifying the metabolite role in immune response via PCR and ELISA. This protocol focuses on cutaneous leishmaniasis models, but the analyses here are applicable to other infection models.

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