Development of a Cell-Based High-Throughput Screening Assay to Identify Porcine Host Defense Peptide-Inducing Compounds

Abstract

Novel alternatives to antibiotics are needed for the swine industry, given increasing restrictions on subtherapeutic use of antibiotics. Augmenting the synthesis of endogenous host defense peptides (HDPs) has emerged as a promising antibiotic-alternative approach to disease control and prevention. To facilitate the identification of HDP inducers for swine use, we developed a stable luciferase reporter cell line, IPEC-J2/PBD3-luc, through permanent integration of a luciferase reporter gene driven by a 1.1 kb porcine β-defensin 3 (PBD3) gene promoter in porcine IPEC-J2 intestinal epithelial cells. Such a stable reporter cell line was employed in a high-throughput screening of 148 epigenetic compounds and 584 natural products, resulting in the identification of 41 unique hits with a minimum strictly standardized mean difference (SSMD) value of 3.0. Among them, 13 compounds were further confirmed to give at least a 5-fold increase in the luciferase activity in the stable reporter cell line, with 12 being histone deacetylase (HDAC) inhibitors. Eight compounds were subsequently observed to be comparable to sodium butyrate in inducing PBD3 mRNA expression in parental IPEC-J2 cells in the low micromolar range. Six HDAC inhibitors including suberoylanilide hydroxamine (SAHA), HC toxin, apicidin, panobinostat, SB939, and LAQ824 were additionally found to be highly effective HDP inducers in a porcine 3D4/31 macrophage cell line. Besides PBD3, other HDP genes such as PBD2 and cathelicidins (PG1–5) were concentration-dependently induced by those compounds in both IPEC-J2 and 3D4/31 cells. Furthermore, the antibacterial activities of 3D4/31 cells were augmented following 24 h exposure to HDAC inhibitors. In conclusion, a cell-based high-throughput screening assay was developed for the discovery of porcine HDP inducers, and newly identified HDP-inducing compounds may have potential to be developed as alternatives to antibiotics for applications in swine and possibly other animal species.

Discovery of natural products capable of inducing porcine host defense peptide gene expression using cell-based high throughput screening

“…We have screened a library of 584 natural products and identified a number of HDP-inducing compounds [ 17 , 18 ]. Using our newly-established IPEC-J2/ pBD3- luc cell line, which is the porcine IPEC-J2 intestinal epithelial cell line with stable integration of a luciferase reporter plasmid under the control of porcine pBD3 gene promoter [ 18 ], we screened in the present study a library of 1261 natural products, which was custom-made and non-redundant from the 584 compounds that were screened earlier [ 17 , 18 ]. The objective of this study was to discover additional and perhaps more potent, natural HDP inducers for livestock use.…”

“…To facilitate the identification of HDP-inducing compounds, we and other have developed cell-based highthroughput screening (HTS) assays for specific applications in humans, poultry, and pigs [16][17][18]. We have screened a library of 584 natural products and identified a number of HDP-inducing compounds [17,18].…”

“…To facilitate the identification of HDP-inducing compounds, we and other have developed cell-based highthroughput screening (HTS) assays for specific applications in humans, poultry, and pigs [16][17][18]. We have screened a library of 584 natural products and identified a number of HDP-inducing compounds [17,18]. Using our newly-established IPEC-J2/pBD3-luc cell line, which is the porcine IPEC-J2 intestinal epithelial cell line with stable integration of a luciferase reporter plasmid under the control of porcine pBD3 gene promoter [18], we screened in the present study a library of 1261 natural products, which was custom-made and non-redundant from the 584 compounds that were screened earlier [17,18].…”

Butyrate, Forskolin, and Lactose Synergistically Enhance Disease Resistance by Inducing the Expression of the Genes Involved in Innate Host Defense and Barrier Function

“…Butyrate is known to modulate HDP expression by acting as a histone deacetylase inhibitor (HDACi) and mitogen-activated protein kinases (MAPK) signaling pathways are also involved [20,33]. In fact, a number of natural and synthetic HDACi have been identified to be capable of inducing HDPs [10,34]. Forskolin is a natural agonist of adenylate cyclase and activator of cyclic adenosine monophosphate (cAMP) signaling [21,35] and triggers HDP expression by activating cAMP signaling [22].…”

Molecular characterization, mRNA gene expression, and antimicrobial activity of 2 new cathelicidin genes in goose

“…In addition, the immunomodulatory effects of avian cathelicidins exhibit the potential to be developed as adjuvants or components of an adjuvant complex ( Zhang and Sunkara, 2014 , van Dijk et al., 2016 , Kraaij et al., 2017 ). On the account of the cost and stability for the direct administration of HDPs, indirect instigation of synthesis of endogenous HDPs by dietary compounds seems to be a convenient and cost-effective strategy in animal production ( Deng et al., 2018 , Lyu et al., 2018 ). Because these HDP-inducing compounds have no direct interactions with pathogens, they can boost the synthesis of HDPs without triggering resistance and provoking inflammation ( Zhang and Sunkara, 2014 , Robinson et al., 2018 ).…”