Deciphering the Mechanism of Action of Antibiotics and Botanical Compounds Using Advanced Immunometabolic Techniques

The immunometabolic regulation of macrophages may significantly influence the response of chickens exposed to various challenges. 

In this study, the immunometabolic role of two antibiotics (oxytetracycline and gentamicin) and a botanical compound (thymol) were investigated on a chicken macrophage-like cell line (HD11) during a Salmonella Enteritidis infection. 

Seahorse metabolic assay was used to characterize the immunometabolic profile of macrophages during the experiment and results were then validated with gene expression and kinome peptide array analysis. 

Macrophages under exposure to Salmonella Enteritidis induced a shift from quiescent to an energetic state. The treatment with oxytetracycline, to which the bacterium was resistant, showed no detectable effects during the challenge, as indicated by the Seahorse assay. Gentamicin, to which the bacterium was sensitive, was shown to keep the cells in a quiescent status even at sub-inhibitory dosages with the Seahorse metabolic assay, with a significant upregulation of IL1B and IL10 gene expression. Thymol was able to shift the metabolism of macrophages to an anaerobic status, associated with the anti-inflammatory phenotype M2. 

Gene expression analysis revealed a significant increase in IL1B and a numerical upregulation of IL10.

Using the kinome array analysis we investigated the main immunometabolic KEGG pathways with changes in phosphorylation. One of the main pathways influenced by the treatments was the C-type lectin receptor signaling pathway. These receptors are expressed on macrophages and are involved in numerous patterns that include pathogen recognition, phagocytosis, and activation of immune signaling. Differences in protein phosphorylation between thymol and gentamicin treatments in this pathway may explain the observed upregulation of IL1B and IL10, and more in general lead to activation of pathways involved in Th17 cell differentiation. 

Carefully selected antibiotics and botanical compounds can support macrophages during a challenge with Salmonella Enteritidis, suggesting that advanced in vitro techniques can help unveil different mechanisms of action of compounds prior to their use in vivo.