Abstract
Lung macrophages (LM) are in the first line of defense against inhaled pathogens and can undergo phenotypic polarization to the proinflammatory M1 after stimulation with Toll-like receptor agonists. The objective of the present work was to characterize the metabolic alterations occurring during the experimental M1 LM polarization. Human LM were obtained from resected lungs and cultured for 24 hrs in medium alone or with 10 ng.mL-1 lipopolysaccharide. Cells and culture supernatants were subjected to extraction for metabolomic analysis with high-resolution LC-MS (HILIC and reverse phase -RP- chromatography in both negative and positive ionization modes) and GC-MS. The data were analyzed with R and the Worklow4Metabolomics and MetaboAnalyst online infrastructures. A total of 8,741 and 4,356 features were detected in the intracellular and extracellular content, respectively, after the filtering steps. Pathway analysis showed involvement of arachidonic acid metabolism, tryptophan metabolism and Krebs cycle in the response of LM to LPS, which was confirmed by the specific quantitation of selected compounds. This refined analysis highlighted a regulation of the kynurenin pathway as well as the serotonin biosynthesis pathway, and an involvement of aspartate-arginosuccinate shunt in the malate production. Macrophages M1 polarization is accompanied by changes in the cell metabolome, with the differential expression of metabolites involved in the promotion and regulation of inflammation and antimicrobial activity. The analysis of this macrophage immunometabolome may be of interest for the understanding of the pathophysiology of lung inflammatory disesases.
Highlights
Lung macrophages are in the first line of defense against inhaled pathogens
hydrophilic interaction liquid chromatography (HILIC) was the most contributive, followed with reverse phase (RP) chromatography and GC-MS and a total of 8,741 features were remaining after the filtering steps
RP chromatography allowed the detection of the highest number of features, followed with HILIC and GC-MS
Summary
Lung macrophages are in the first line of defense against inhaled pathogens. They are major effectors of the immune response as they express membrane receptors, including Toll-like receptors (TLRs), able to recognize conserved microbial ligands [1]. Like allergens (e.g. ragweed pollen, house dust extract, and cat dander) or air pollutants, LPS binds to and activates TLR4, the first subtype among the TLR family to be identified in humans Beyond this role in cell signaling, TLRs play a role in the primary function of macrophages, consisting in the phagocytosis and killing of pathogens [3]. With respect to lung pathogenesis, macrophages play a role in infections and inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD), where they can undergo a phenotypic differentiation [14,15,16,17] In these cases, TLRs are of prime importance for their role in the recognition of pathogens during infections and in microbe-induced acute exacerbations of asthma and COPD [18, 19]. Since metabolic changes associated with the stimulation of TLRs in human lung macrophages were not yet described, the objective of the present study was to perform an extensive intra- and extracellular metabolomic characterization of LPS-induced alterations in human lung macrophages using a combined untargeted liquid chromatography high-resolution mass spectrometry and gas chromatography mass spectrometry approach [23]
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