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Macrophages represent an important line of defense of the organism against external insults and their immune and metabolic profiles are strictly interconnected. The aim of the study was to evaluate the immunometabolic role of a thymol-based blend of botanicals (TBB) on a chicken macrophage-like cell line during a Salmonella Enteritidis challenge in vitro. In particular, the blend was used at a sub-inhibitory concentration against the bacterial strain, in order to exclude its antimicrobial effect and focus on its role on cells.
A chicken macrophage-like cell line (HD11 cells) was infected with a S. Enteritidis strain for 6 hours and, at the same time, cells were treated with TBB at 150 ppm. A group infected but not treated (CTR) was also included. Three different experiments were performed: (1) the gene expression of a panel of inflammatory markers was carried out through qPCR analysis; (2) the energetic metabolic profile of cells was investigated using the Seahorse metabolic assay, by measuring mitochondrial respiration and glycolysis over the time of infection; (3) the kinomic profile of macrophages was assessed with the kinome array analysis. A t-test was performed for the gene expression analysis and the kinome peptide array, whereas the Seahorse assay results were analysed with an ordinary two-way ANOVA followed by Šidák’s multiple comparison test.
qPCR results showed that, when compared to CTR, TBB tended to increase the expression of IL-10 (p = 0.072) and significantly decreased IL-6 expression (p = 0.015).
At the end of the infection, the Seahorse assay highlighted a lower extracellular acidification rate (p < 0.0001), that is proportional to cellular glycolysis, for TBB group compared to CTR. At the same time, a little but not significant increase was observed for the oxygen consumption rate, that is proportional to mitochondrial respiration. These two data indicate that macrophages treated with TBB are in aerobic status, that is usually correlated with the M2 phenotype. Based on different stimuli, macrophages can differentiate into M2 anti-inflammatory profile, that is associated with production of regulatory cytokines and the shift from a glycolytic metabolism to TCA cycle and oxidative phosphorylation.
The kinome array showed an increased phosphorylation of RAF1 that could suggest the IL-10 production through the ERK pathway. Moreover, the observed activation of AMPK and SIRT1 could lead to increased mitochondrial functionality, further confirming the M2 phenotype.
To conclude, the thymol-based blend of botanicals is shown to switch the phenotype of macrophages to a less-inflammatory status based on the immunometabolic findings of the study.
Keywords: chicken macrophages; thymol; immunometabolic modulation; inflammation.
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