Abstract
A rise in antimicrobial resistance demands novel alternatives to antimicrobials for disease control and prevention. As an important component of innate immunity, host defense peptides (HDPs) are capable of killing a broad spectrum of pathogens and modulating a range of host immune responses. Enhancing the synthesis of endogenous HDPs has emerged as a novel host-directed antimicrobial therapeutic strategy. To facilitate the identification of natural products with a strong capacity to induce HDP synthesis, a stable macrophage cell line expressing a luciferase reporter gene driven by a 2-Kb avian β-defensin 9 (AvBD9) gene promoter was constructed through lentiviral transduction and puromycin selection. A high throughput screening assay was subsequently developed using the stable reporter cell line to screen a library of 584 natural products. A total of 21 compounds with a minimum Z-score of 2.0 were identified. Secondary screening in chicken HTC macrophages and jejunal explants further validated most compounds with a potent HDP-inducing activity in a dose-dependent manner. A follow-up oral administration of a lead natural compound, wortmannin, confirmed its capacity to enhance the AvBD9 gene expression in the duodenum of chickens. Besides AvBD9, most other chicken HDP genes were also induced by wortmannin. Additionally, butyrate was also found to synergize with wortmannin and several other newly-identified compounds in AvBD9 induction in HTC cells. Furthermore, wortmannin acted synergistically with butyrate in augmenting the antibacterial activity of chicken monocytes. Therefore, these natural HDP-inducing products may have the potential to be developed individually or in combinations as novel antibiotic alternatives for disease control and prevention in poultry and possibly other animal species including humans.
Highlights
Antimicrobial resistance is posing a major threat to public health
Increased resistance to conventional antibiotics necessitates the development of novel antimicrobial strategies
A cellbased high-throughput screening (HTS) assay was recently developed by employing human HT-29 intestinal epithelial cells transfected with a fusion of a 4Kb human cathelicidin LL-37 gene promoter and its entire open reading frame with a luciferase reporter gene (Nylen et al, 2014)
Summary
While it is necessary to continue the development of antibiotics with direct antimicrobial activities, host-directed therapies have emerged as attractive alternative strategies to combating infectious and non-communicable diseases (Zumla et al, 2016). Host defense peptides (HDPs), known as antimicrobial peptides, are represented by a large diverse group of small peptides that are synthesized primarily by phagocytic cells and epithelial cells lining the gastrointestinal, respiratory, and urogenital tracts (Zasloff, 2002). Immunomodulatory and barrier protective activities, HDPs constitute an important phylogenetically conserved first line of defense in virtually all species of life (Hilchie et al, 2013; Mansour et al, 2014; Robinson et al, 2015). One cathelicidin known as LL-37, six α-defensins, and a minimum of 39 β-defensins exist in humans (Wang, 2014), whereas four cathelicidins (CATH1-3 and CATH-B1) and 14 avian β-defensins (AvBD1-14) are present in chickens (Cuperus et al, 2013; Zhang and Sunkara, 2014)
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