Beta-Defensin-2 and Beta-Defensin-3 Reduce Intestinal Damage Caused by Salmonella typhimurium Modulating the Expression of Cytokines and Enhancing the Probiotic Activity of Enterococcus faecium

Abstract

The intestinal microbiota is a major factor in human health and disease. This microbial community includes autochthonous (permanent inhabitants) and allochthonous (transient inhabitants) microorganisms that contribute to maintaining the integrity of the intestinal wall, modulating responses to pathogenic noxae and representing a key factor in the maturation of the immune system. If this healthy microbiota is disrupted by antibiotics, chemotherapy, or a change in diet, intestinal colonization by pathogenic bacteria or viruses may occur, leading to disease. To manage substantial microbial exposure, epithelial surfaces of the intestinal tract produce a diverse arsenal of antimicrobial peptides (AMPs), including, of considerable importance, the β-defensins, which directly kill or inhibit the growth of microorganisms. Based on the literature data, the purpose of this work was to create a line of intestinal epithelial cells able to stably express gene encoding human β-defensin-2 (hBD-2) and human β-defensin-3 (hBD-3), in order to test their role in S. typhimurium infections and their interaction with the bacteria of the gut microbiota.

Figure 1

(a) hBD-2 mRNA expression in untransfected cells (lane 1), 24 hours (lane 2), and 48 hours (lane 3) after transfection; hBD-3 mRNA expression in untransfected cells (lane 4), 24 hours (lane 5), and 48 hours (lane 6) after transfection. (b) hBD-2 concentration in cell supernatants 48 hours after transfection. (c) hBD-3 concentration in cell supernatants 48 hours after transfection.

Figure 2

Comparison between relative gene expression (a) and protein concentration (b) in Caco-2 cells infected with S. typhimurium and Caco-2 cells infected with E. faecium. Data are mean ± SD and are expressed as the percentage of increment compared to uninfected controls.

Figure 3

Comparison between relative gene expression (a) and protein concentration (b) in Caco-2 cells infected with S. typhimurium alone and Caco-2 cells coinfected with S. typhimurium and E. faecium. Data are mean ± SD and are expressed as the percentage of increment compared to uninfected controls.

Figure 4

S. typhimurium internalization assay. Untransfected Caco-2 cells were infected with S. typhimurium alone or coinfected with live or heat-killed E. faecium for 4 hours. The number of internalized bacteria was determined by host cell lysis, plating, and counting CFU/well. The data shown are representative of three different experiments ± SD. Error bars represent standard deviations.