Natural molecules induce and synergize to boost expression of the human antimicrobial peptide β-defensin-3 Emmanuel Secheta,b,1, Erica Telforda,b,1, Clément Bonamya,b, Philippe J. Sansonettia,b,c,2, and Brice Sperandioa,b,2 aUnité de Pathogénie Microbienne Moléculaire, Institut Pasteur, 75015 Paris, France; bUnité INSERM U1202, Institut Pasteur, 75015 Paris, France; and cChaire de Microbiologie et Maladies Infectieuses, Collège de France, 75005 Paris, France Contributed by Philippe J. Sansonetti, September 7, 2018 (sent for review March 28, 2018; reviewed by Richard L. Gallo and Mathias Hornef) Antimicrobial peptides (AMPs) are mucosal defense effectors of In humans, defensins include two families: α- and β-defensins. the human innate immune response. In the intestine, AMPs are Human α-defensins are stored in granules of polymorphonuclear produced and secreted by epithelial cells to protect the host leukocytes (HNP 1–4) or Paneth cells (HD-5 and HD-6) local- against pathogens and to support homeostasis with commensals. ized at the bottom of intestinal crypts. In contrast, β-defensins The inducible nature of AMPs suggests that potent inducers could are produced by epithelial cells along the entirety of the intes- be used to increase their endogenous expression for the pre- tinal tract, and their synthesis is regulated at the gene-expression vention or treatment of diseases. Here we aimed at identifying level. Expression of the best-known β-defensins, HBD1–4, by molecules from the natural pharmacopoeia that induce expression epithelial cells, is either constitutive or inducible in response to of human β-defensin-3 (HBD3), one of the most efficient AMPs, proinflammatory stimuli. Expression of the HBD1 gene is es- without modifying the production of proinflammatory cytokines. sentially constitutive, whereas expression of HBD2–4 genes is By screening, we identified three molecules isolated from medici- inducible in response to various stimuli, including bacteria, nal plants, andrographolide, oridonin, and isoliquiritigenin, which PAMPs, and proinflammatory cytokines (5). induced HBD3 production in human colonic epithelial cells. This β-Defensins have a broad spectrum of antimicrobial efficacy and effect was observed without activation of the NF-κB pathway or act on Gram-positive and Gram-negative bacteria as well as on INFLAMMATION the expression of associated proinflammatory cytokines. We iden- fungi and enveloped viruses (9). They exert their action by inter- IMMUNOLOGY AND tified the EGF receptor as the target of these compounds and acting with the surface of microorganisms. In Gram-negative bac- characterized the downstream-activated MAPK pathways. At the teria they bind the anionic portion of the LPS molecule; in Gram- chromatin level, molecules increased phosphorylation of histone positive bacteria they interact with teichoic acids or with anionic H3 on serine S10 and recruitment of the c-Fos, c-Jun, and Elk1 or c- groups present in the peptidoglycan molecule. The interaction of Myc transcription factors at the HBD3 promoter. Interestingly, β-defensins with the different structures of the bacterial cell enve- stimulating cells with a combination of andrographolide and iso- liquiritigenin synergistically enhanced HBD3 induction 10-fold lope permeabilizes the membrane either through a detergent effect more than observed with each molecule alone. Finally, we investi- resulting in the leakage of cytoplasmic components or through the gated the molecular basis governing the synergistic effect, confirmed formation of pores subsequent to peptide aggregation (9). our findings in human colonic primary cells, and demonstrated that The growing threat of antibiotic resistance in pathogenic synergism increased cellular antimicrobial activity. This work shows bacteria and the need for new antibiotics stimulate interest in the β the capability of small molecules to achieve induction of epithelial use of -defensins as candidate therapeutic molecules due to antimicrobial defenses while simultaneously avoiding the deleterious their significant antimicrobial activities and their additional risks of an inflammatory response. protective properties, such as in wound healing, modulation of epithelial cell | β-defensin | gene regulation | natural inducer | Significance synergistic effect Defensins are antimicrobial peptides exhibiting antibacterial, ntimicrobial defenses from the human intestinal tract rely antifungal, and antiviral activity. They are expressed by epi- Aon the ability of the mucosal immune system to recognize thelial cells at the intestinal mucosal surface where they play a and neutralize pathogenic microbes. In this context of innate crucial role in the host intestinal homeostasis. Therefore, ap- immunity, sensing of bacteria occurs mainly through the en- proaches aiming to boost their expression represent a prom- gagement of host cell Toll-like receptors (TLRs), nucleotide ising therapeutic strategy to treat infections and dysbiosis- oligomerization domain (NOD) proteins, and Nod-like receptors driven diseases in humans at a time of increasing incidence of (NLRs) by pathogen-associated molecular patterns (PAMPs) (1– antibiotic resistance. 3). After recognition of the pathogen, the antimicrobial response is achieved by immediate secretion of epithelial antimicrobial Author contributions: P.J.S. and B.S. designed research; E.S., E.T., C.B., and B.S. performed research; E.S., E.T., C.B., P.J.S., and B.S. analyzed data; and B.S. wrote the paper. peptides (AMPs), such as defensins and lectins, and subsequent – Reviewers: R.L.G., University of California, San Diego; and M.H., Rheinisch-Westfälische recruitment of immune cells (4 6). Technische Hochschule University Hospital Aachen. In recent decades, several defensins able to rapidly kill mi- Conflict of interest statement: C.B. is a recipient of a Sanofi-funded PhD fellowship in the croorganisms have been identified. Although varying in their context of the French private–public partnership Convention Industrielle de Formation structure and length, these peptides show common features. par la Recherche (CIFRE) program. Defensins are mainly cationic and amphipathic, synthetized as Published under the PNAS license. propeptides and released in a mature form following hydrolysis 1E.S. and E.T. contributed equally to this work. by specific proteases. Defensins, like cytokines, are considered 2To whom correspondence may be addressed. Email: [email protected] or functional effectors of the innate immune system, also liaising [email protected]. with cells of the adaptive immune system, including monocytes, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. T cells, or immature dentritic cells, through their direct inter- 1073/pnas.1805298115/-/DCSupplemental. actions with receptors such as CCR2, CCR6, and FPLR1 (7, 8). www.pnas.org/cgi/doi/10.1073/pnas.1805298115 PNAS Latest Articles | 1of10 Downloaded by guest on September 30, 2021 the immune response, angiogenesis, tissue remodeling, and thus showing a synergistic effect of the two molecules on HBD3 ability to bind LPS in septic shock models (10). Moreover, dis- expression. We provided molecular insights supporting the syn- eases associated with defects in the expression of β-defensins ergistic effect of the molecule combination and confirmed our continue to be identified by clinical studies (11). However, the findings in primary human colonic cells using an ex vivo minigut limited knowledge accumulated about the circuits involved in the organoid model. Finally, we showed that andrographolide and regulation of AMP expression has hampered efforts to develop isoliquiritigenin synergism increased the antimicrobial activity of innovative therapies aimed at stimulating their expression. cells against pathogenic bacteria. This work opens the way to using The idea of using natural AMPs as antibiotics has been ex- molecular inducers of AMPs as an innovative therapeutic strategy plored in recent years and continues to be investigated. How- to treat infections and dysbiosis-driven diseases at a time of in- ever, current AMPs-based antiinfectious strategies show that creasing incidence of antibiotic resistance. local or systemic administration of modified or synthetic AMPs provides mixed results that are largely due to the intrinsic bio- Results chemical and complex bioavailability characteristics of these Andrographolide, Oridonin, and Isoliquiritigenin Induce Expression of peptides (12, 13). Several factors, including cost, toxicity, and the Human AMP β-Defensin-3. To identify molecules from the susceptibility to several parameters (salt and proteases con- pharmacopeia inducing the expression of genes encoding antimi- centrations, oxido-reduction status, spontaneous aggregation, crobial peptides (β-defensins HBD2 and -3 and cathelicidin LL-37), and difficulty in mimicking the optimal local condition of ac- we treated human colonic epithelial cells [Caco-2, subclone TC7 tion) have limited their use as therapeutic agents (12, 14). In (24)] with a library of 170 natural compounds isolated from plants addition is the need for AMPs to be expressed and secreted at used in traditional Chinese medicine, which were structurally di- high levels, along with other antimicrobial molecules and possibly verse, bioactive, and cell permeable. Treatments were performed cells, in relevant sites. The possibility of boosting transcription of on confluent cell monolayers using three different concentrations