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

Abstract

Background In-feed antibiotics are being phased out in livestock production worldwide. Alternatives to antibiotics are urgently needed to maintain animal health and production performance. Host defense peptides (HDPs) are known for their broad-spectrum antimicrobial and immunomodulatory capabilities. Enhancing the synthesis of endogenous HDPs represents a promising antibiotic alternative strategy to disease control and prevention. Methods To identify natural products with an ability to stimulate the synthesis of endogenous HDPs, we performed a high-throughput screening of 1261 natural products using a newly-established stable luciferase reporter cell line known as IPEC-J2/pBD3-luc. The ability of the hit compounds to induce HDP genes in porcine IPEC-J2 intestinal epithelial cells, 3D4/31 macrophages, and jejunal explants were verified using RT-qPCR. Augmentation of the antibacterial activity of porcine 3D4/31 macrophages against a Gram-negative bacterium (enterotoxigenic E. coli) and a Gram-positive bacterium (Staphylococcus aureus) were further confirmed with four selected HDP-inducing compounds. Results A total of 48 natural products with a minimum Z-score of 2.0 were identified after high-throughput screening, with 21 compounds giving at least 2-fold increase in luciferase activity in a follow-up dose-response experiment. Xanthohumol and deoxyshikonin were further found to be the most potent in inducing pBD3 mRNA expression, showing a minimum 10-fold increase in IPEC-J2, 3D4/31 cells, and jejunal explants. Other compounds such as isorhapontigenin and calycosin also enhanced pBD3 mRNA expression by at least 10-fold in both IPEC-J2 cells and jejunal explants, but not 3D4/31 cells. In addition to pBD3, other porcine HDP genes such as pBD2, PG1-5, and pEP2C were induced to different magnitudes by xanthohumol, deoxyshikonin, isorhapontigenin, and calycosin, although clear gene- and cell type-specific patterns of regulation were observed. Desirably, these four compounds had a minimum effect on the expression of several representative inflammatory cytokine genes. Furthermore, when used at HDP-inducing concentrations, these compounds showed no obvious direct antibacterial activity, but significantly augmented the antibacterial activity of 3D4/31 macrophages (P < 0.05) against both Gram-negative and Gram-positive bacteria. Conclusions Our results indicate that these newly-identified natural HDP-inducing compounds have the potential to be developed as novel alternatives to antibiotics for prophylactic and therapeutic treatment of infectious diseases in livestock production.

Deoxyshikonin-Induced Gene Expression Profile in Porcine Epithelial Cells

“…Maintenance of a histone hyperacetylation and hypomethylation status of target gene promoters largely contributes to transcript expression ( 16 , 17 ). Several nutrients and natural products upregulate HDP expression through histone modification ( 7 , 13 ). However, whether deoxyshikonin induces the expression of porcine HDP genes and other immunoregulatory genes through this way requires further investigation.…”

“…Deoxyshikonin, an active component of Lithospermum erythrorhizon , is a promising drug candidate for the treatment of wounds and cancers ( 5 , 6 ). In a screening of a library of 1,261 natural compounds using a newly established IPEC-J2/ pBD3 -luc cell line, we previously found that deoxyshikonin induces the expression of porcine host defense peptides (HDPs) ( 7 ). In addition, deoxyshikonin had a minimal effect on the expression of several representative inflammatory cytokine genes and obviously enhanced the antibacterial activity of 3D4/31 macrophages against both gram-negative and gram-positive bacteria.…”

High-Throughput Identification of Epigenetic Compounds to Enhance Chicken Host Defense Peptide Gene Expression

“…HTC/ AvBD10-luc cell clone D5 were seeded at 2 × 10 4 cells/well in 50 μL of complete RPMI 1640 medium in 96-well plates overnight prior to exposure to 20 µM of each of the 148 compounds in an Epigenetics Screening Library (Cayman Chemical) in individual wells for 24 h prior to luciferase assay. To assess potential cytotoxicity of each compound, an alamarBlue dye (Thermo Fisher Scientific) was added to a final concentration of 10% and incubated for 4 h as we previously described [ 23 , 37 , 50 ]. Cytotoxicity was assessed in live cells by measuring the fluorescence at 545 nm excitation and 590 nm emission on Fx80 Microplate Fluorescence Reader (BioTek Instruments, Winooski, VT, USA), followed by luciferase assay on L-Max II Luminescence Microplate Reader (Molecular Devices) using Steady-Glo ® Luciferase Assay System (Promega).…”

“…Cytotoxicity was assessed in live cells by measuring the fluorescence at 545 nm excitation and 590 nm emission on Fx80 Microplate Fluorescence Reader (BioTek Instruments, Winooski, VT, USA), followed by luciferase assay on L-Max II Luminescence Microplate Reader (Molecular Devices) using Steady-Glo ® Luciferase Assay System (Promega). The relative luciferase activity was normalized to the cell viability for each compound as we described [ 23 , 37 , 50 ]. To select hits, strictly standardized mean difference (SSMD) values were subsequently calculated as , where μ s and μ n are the mean luciferase activity of positive and negative controls, respectively, while σ is the standard deviation of the negative control [ 27 ].…”

Large-Scale Identification of Multiple Classes of Host Defense Peptide-Inducing Compounds for Antimicrobial Therapy

“…A high-throughput screening (HTS) assay based on a stable LL-37 promoter-driven luciferase reporter cell line (MN8CampLuc), has been developed and has led to the identification of multiple LL-37 inducers [ 22 , 23 ]. We also established different luciferase reporter cell lines through the stable integration of HDP promoter-driven luciferase reporter genes in macrophages or intestinal epithelial cell lines [ 24 , 25 ], which were subsequently employed to identify a number of HDP-inducing compounds [ 24 , 25 , 26 , 27 ]. Here, we report a large-scale screening of 5002 natural and synthetic small-molecule compounds with much greater structural and functional diversities using one such reporter cell line, termed HTC/ AvBD9-luc [ 25 ].…”