A Comparison of the Immunometabolic Effect of Antibiotics and Plant Extracts in a Chicken Macrophage-like Cell Line during a Salmonella Enteritidis Challenge

Abstract

Immunometabolic modulation of macrophages can play an important role in the innate immune response of chickens triggered with a multiplicity of insults. In this study, the immunometabolic role of two antibiotics (oxytetracycline and gentamicin) and four plant extracts (thyme essential oil, grape seed extract, garlic oil, and capsicum oleoresin) were investigated on a chicken macrophage-like cell line (HD11) during a Salmonella Enteritidis infection. To study the effect of these substances, kinome peptide array analysis, Seahorse metabolic assay, and gene expression techniques were employed. Oxytetracycline, to which the bacterial strain was resistant, thyme essential oil, and capsicum oleoresin did not show any noteworthy immunometabolic effect. Garlic oil affected glycolysis, but this change was not detected by the kinome analysis. Gentamicin and grape seed extract showed the best immunometabolic profile among treatments, being able to both help the host with the activation of immune response pathways and with maintaining a less inflammatory status from a metabolic point of view.

Keywords: HD11; Salmonella Enteritidis; antibiotic alternatives; cellular metabolism; foodborne diseases; innate immunity; plant extracts.

Figure 1

Summary of the main phosphorylation changes of significant metabolic peptides (p ≤ 0.05) of HD11 cells infected with S. Enteritidis and treated with gentamicin. ↑ protein, significantly activated; ↓ protein, significantly deactivated; protein, significantly phosphorylated but with no site information; ↓↑ protein, more than one site significantly phosphorylated with both activation and deactivation of the protein; inhibitor arrow indicates that phosphorylation of the protein leads to the inhibition of the linked pathway; normal arrow indicates that phosphorylation of the protein leads to the activation of the linked pathway; dashed arrow indicates that a product of a cellular metabolic pathway enters in a different metabolic pathway. Created with BioRender.com.

Figure 2

Section of Salmonella infection KEGG reference pathway (map05132) indicating the main phosphorylation changes of significant immune peptides (p ≤ 0.05) of HD11 cells infected with S. Enteritidis and treated with gentamicin. Green squares indicate significantly activated protein; red squares indicate significantly deactivated protein; pink squares indicate either no site information was available or more sites of the same protein were significantly affected with both activation/deactivation of the protein. Adapted with permission from [33]. 2023, Kanehisa Laboratories.

Figure 3

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of HD11 cells infected with Salmonella Enteritidis for 6 h. The reads start from 60 min since, during the first hour, the Seahorse XFp Analyzer requires an internal calibration. Data are expressed as the mean of all the experiments ± SEM (n = 18).

Figure 4

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of HD11 cells infected with Salmonella Enteritidis and treated with antibiotics ((a)—oxytetracycline (OXY) or (b)—gentamicin (GEN)) for 6 h. The reads start from 60 min since, during the first hour, the Seahorse XFp Analyzer requires an internal calibration. Data are expressed as the mean ± SEM (n = 3). Asterisks * indicate statistical significance with p ≤ 0.05.

Figure 5

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of HD11 cells infected with Salmonella Enteritidis and treated with plant extracts ((a)—thyme essential oil (TEO), (b)—grape seed extract (GSE), (c)—garlic oil (GAR), or (d)—capsicum oleoresin (CAP)) for 6 h. The reads start from 60 min since, during the first hour, the Seahorse XFp Analyzer requires an internal calibration. Data are expressed as the mean ± SEM (n = 3). Asterisks * indicate statistical significance with p ≤ 0.05.

Figure 6

mRNA expression of IL-1β, IL-8, IL-22, iNOS, TNFα, IFNγ, IL-6, IL-10, and TGFβ of HD11 cells infected with S. Enteritidis and treated with antibiotics (oxytetracycline (OXY), gentamicin (GEN)) or plant extracts (thyme essential oil (TEO), grape seed extract (GSE), garlic oil (GAR), capsicum oleoresin (CAP)). Dashed lines represent the CTR—threshold, set as 1. Data are presented as mean ± SEM. Different letters indicate statistical significance with p < 0.05.