The Antimicrobial Activity of Human Defensins at Physiological Non-Permeabilizing Concentrations Is Caused by the Inhibition of the Plasma Membrane H+-ATPases.

Human defensins are cysteine-rich peptides (Cys-rich peptides) of the innate immune system. Defensins contain an ancestral structural motif (i.e., γ-core motif) associated with the antimicrobial activity of natural Cys-rich peptides. In this study, low concentrations of human α- and ß-defensins showed microbicidal activity that was not associated with cell membrane permeabilization. The cell death pathway was similar to that previously described for human lactoferrin, also an immunoprotein containing a γ-core motif. The common features were (1) cell death not related to plasma membrane (PM) disruption, (2) the inhibition of microbicidal activity via extracellular potassium, (3) the influence of cellular respiration on microbicidal activity, and (4) the influence of intracellular pH on bactericidal activity. In addition, in yeast, we also observed (1) partial K+-efflux mediated via Tok1p K+-channels, (2) the essential role of mitochondrial ATP synthase in cell death, (3) the increment of intracellular ATP, (4) plasma membrane depolarization, and (5) the inhibition of external acidification mediated via PM Pma1p H+-ATPase. Similar features were also observed with BM2, an antifungal peptide that inhibits Pma1p H+-ATPase, showing that the above coincident characteristics were a consequence of PM H+-ATPase inhibition. These findings suggest, for the first time, that human defensins inhibit PM H+-ATPases at physiological concentrations, and that the subsequent cytosolic acidification is responsible for the in vitro microbicidal activity. This mechanism of action is shared with human lactoferrin and probably other antimicrobial peptides containing γ-core motifs.

Human ß-defensins: The multi-functional natural peptide.

The increasing threat of antibiotic resistance among pathogenic microorganisms and the urgent demand for new antibiotics require immediate attention. Antimicrobial peptides exhibit effectiveness against microorganisms, fungi, viruses, and protozoa. The discovery of human ß-defensins represents a major milestone in biomedical research, opening new avenues for scientific investigation into the innate immune system and its resistance mechanisms against pathogenic microorganisms. Multiple defensins present a promising alternative in the context of antibiotic abuse. However, obstacles to the practical application of defensins as anti-infective therapies persist due to the unique properties of human ß-defensins themselves and serious pharmacological and technical challenges. To overcome these challenges, diverse delivery vehicles have been developed and progressively improved for the conjugation or encapsulation of human ß-defensins. This review briefly introduces the biology of human ß-defensins, focusing on their multistage structure and diverse functions. It also discusses several heterologous systems for producing human ß-defensins, various delivery systems created for these peptides, and patent applications related to their utilization, concluding with a summary of current challenges and potential solutions.

A Novel Beta-Defensin Isoform from Malabar Trevally, Carangoides malabaricus (Bloch & Schneider, 1801), an Arsenal Against Fish Bacterial Pathogens: Molecular Characterization, Recombinant Production, and Mechanism of Action.

Antimicrobial peptides (AMPs), including beta-defensin from fish, are a crucial class of peptide medicines. The focus of the current study is the molecular and functional attributes of CmDef, a 63-amino acid beta-defensin AMP from Malabar trevally, Carangoides malabaricus. This peptide demonstrated typical characteristics of AMPs, including hydrophobicity, amphipathic nature, and +2.8 net charge. The CmDef was recombinantly expressed and the recombinant peptide, rCmDef displayed a strong antimicrobial activity against bacterial fish pathogens with an MIC of 8 µM for V. proteolyticus and 32 µM for A. hydrophila. The E. tarda and V. harveyi showed an inhibition of 94% and 54%, respectively, at 32 µM concentration. No activity was observed against V. fluvialis and V. alginolyticus. The rCmDef has a multimode of action that exerts an antibacterial effect by membrane depolarization followed by membrane permeabilization and ROS production. rCmDef also exhibited anti-cancer activities in silico without causing hemolysis. The peptide demonstrated stability under various conditions, including different pH levels, temperatures, salts, and metal ions (KCl and CaCl2), and remained stable in the presence of proteases such as trypsin and proteinase K at concentrations up to 0.2 µg/100 µl. The strong antibacterial efficacy and non-cytotoxic nature suggest that rCmDef is a single-edged sword that can contribute significantly to aquaculture disease management.

Antifungal activity of human antimicrobial peptides targeting apoptosis in Candida auris.

Introduction. Innovative antifungal therapies are of crucial importance to combat the potentially life-threatening infections linked to the multidrug-resistant fungal pathogen Candida auris. Induction of regulated cell death, apoptosis, could provide an outline for future therapeutics. Human antimicrobial peptides (AMPs), well-known antifungal compounds, have shown the ability to induce apoptosis in pathogenic fungi.Hypothesis/Gap Statement . Although it is known that AMPs possess antifungal activity against C. auris, their ability to induce apoptosis requires further investigations.Aim. This study evaluated the effects of AMPs on the induction of apoptosis in C. auris.Methods. Human neutrophil peptide-1 (HNP-1), human ß-Defensins-3 (hBD-3) and human salivary histatin 5 (His 5) were assessed against two clinical C. auris isolates. Apoptosis hallmarks were examined using FITC-Annexin V/PI double labelling assay and terminal deoxynucleotidyl transferase deoxynucleotidyl transferase nick-end labelling (TUNEL) to detect phosphatidylserine externalization and DNA fragmentation, respectively. Then, several intracellular triggers were studied using JC-10 staining, spectrophotometric assay and 2',7'-dichlorofluorescin diacetate staining to measure the mitochondrial membrane potential, cytochrome-c release and reactive oxygen species (ROS) production, respectively.Results and conclusion. FITC-Annexin V/PI staining and TUNEL analysis revealed that exposure of C. auris cells to HNP-1 and hBD-3 triggered both early and late apoptosis, while His 5 caused significant necrosis. Furthermore, HNP-1 and hBD-3 induced significant mitochondrial membrane depolarization, which resulted in substantial cytochrome c release. In contrast to His 5, which showed minimal mitochondrial depolarization and no cytochrome c release. At last, all peptides significantly increased ROS production, which is related to both types of cell death. Therefore, these peptides represent promising and effective antifungal agents for treating invasive infections caused by multidrug-resistant C. auris.

Human ß-defensins and their synthetic analogs: Natural defenders and prospective new drugs of oral health.

As a family of cationic host defense peptides, human ß-defensins (HBDs) are ubiquitous in the oral cavity and are mainly synthesized primarily by epithelial cells, serving as the primary barrier and aiming to prevent microbial invasion, inflammation, and disease while maintaining physiological homeostasis. In recent decades, there has been great interest in their biological functions, structure-activity relationships, mechanisms of action, and therapeutic potential in oral diseases. Meanwhile, researchers are dedicated to improving the properties of HBDs for clinical application. In this review, we first describe the classification, structural characteristics, functions, and mechanisms of HBDs. Next, we cover the role of HBDs and their synthetic analogs in oral diseases, including dental caries and pulp infections, periodontitis, peri-implantitis, fungal/viral infections and oral mucosal diseases, and oral squamous cell carcinoma. Finally, we discuss the limitations and challenges of clinical translation of HBDs and their synthetic analogs, including, but not limited to, stability, bioavailability, antimicrobial activity, resistance, and toxicity. Above all, this review summarizes the biological functions, mechanisms of action, and therapeutic potential of both natural HBDs and their synthetic analogs in oral diseases, as well as the challenges associated with clinical translation, thus providing substantial insights into the laboratory development and clinical application of HBDs in oral diseases.

ERK1/2-CEBPB Axis-Regulated hBD1 Enhances Anti-Tuberculosis Capacity in Alveolar Type II Epithelial Cells.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), remains a global health crisis with substantial morbidity and mortality rates. Type II alveolar epithelial cells (AEC-II) play a critical role in the pulmonary immune response against Mtb infection by secreting effector molecules such as antimicrobial peptides (AMPs). Here, human ß-defensin 1 (hBD1), an important AMP produced by AEC-II, has been demonstrated to exert potent anti-tuberculosis activity. HBD1 overexpression effectively inhibited Mtb proliferation in AEC-II, while mice lacking hBD1 exhibited susceptibility to Mtb and increased lung tissue inflammation. Mechanistically, in A549 cells infected with Mtb, STAT1 negatively regulated hBD1 transcription, while CEBPB was the primary transcription factor upregulating hBD1 expression. Furthermore, we revealed that the ERK1/2 signaling pathway activated by Mtb infection led to CEBPB phosphorylation and nuclear translocation, which subsequently promoted hBD1 expression. Our findings suggest that the ERK1/2-CEBPB-hBD1 regulatory axis can be a potential therapeutic target for anti-tuberculosis therapy aimed at enhancing the immune response of AEC-II cells.

Effects of Schumann resonance on the proliferation and migration of normal human epidermal keratinocytes and the expression of DEFB1 and SIRT1.

BACKGROUND: When the skin is damaged and its barrier function is disrupted, the proliferation and migration of epidermal keratinocytes are vital for repairing the damaged area. The Schumann resonance at 7.8 Hz has been reported to protect rat cardiomyocytes against oxidative stress and inhibit the proliferation of B16 mouse melanoma cells. However, its effect on the skin is unknown. AIMS: In this study, we applied 7.8-Hz electromagnetic waves to normal human epidermal keratinocytes (NHEKs) and investigated its effects on cell proliferation and migration, ß-defensin (DEFB1) and sirtuin 1 (SIRT1) expression. METHODS: We performed cell proliferation assay, cell migrationassay and gene expression analysis of DEFB1 and SIRT1. RESULTS: We found that the application of 7.8-Hz electromagnetic waves caused a 2.8-fold increase in NHEK proliferation, enhanced cell migration, and increased the expression of DEFB1 and SIRT1 by 2.4-fold and 4.9-fold, respectively. CONCLUSIONS: These results suggest that the application of 7.8-Hz electromagnetic waves may contribute to improving the skin barrier function and skin ulcer.

Antimicrobial activity of yak beta-defensin 116 against Staphylococcus aureus and its role in gut homeostasis.

Staphylococcus aureus (S. aureus) is one of the most common food-borne poisoning microbial agent. However, the antimicrobial activity of ß-defensin 116 in yak and its application in S. aureus-induced diarrheal disease have not been reported. In this study, 303 bp cDNA sequence of yak DEFB116 gene was obtained. In addition, the prokaryotic expression vector of DEFB116 protein with a molecular weight of 16 kDa was successfully constructed and expressed. The yak DEFB116 gene can encode 19 amino acids, the percentage of hydrophobic amino acids is 36 % and the total positive charge is 6, which has potential antibacterial potential. Sufficient DEFB116 protein concentration and time can destroy the integrity of the bacterial cell membrane, resulting in leakage of intracellular solutes and thus killing S. aureus. The intestinal histopathological features and the number of inflammatory cells were improved in the diarrhea mouse model under the action of DEFB116 protein. The decrease of goblet cells was reversed, the expression of mucoprotein was increased. DEFB116 protein increased the abundance of Lactobacillus johnsonii, Lactobacillus reuteri and Desulfovibrio, and inhibited the reproduction of pathogenic bacteria. These findings provide new insights into the potential future applications of yak ß-defencins in the food industry and medical fields.

Extract of Sophorae flavescentis radix-Cnidii fructus couplet medicines treats vulvovaginal candidiasis by affecting the vaginal mucosal barrier.

Aim: This study investigated the inhibition of extract of Sophorae flavescentis radix-Cnidii fructus couplet medicines (ESCC) on Candida albicans (C. albicans) in vitro and the effect of ESCC on the vaginal mucosal barrier in vivo. Materials & methods: Susceptibility testing was performed with C. albicans SC5314. A vulvovaginal candidiasis mouse model was successfully established. The plate method, Gram staining, hematoxylin and eosin staining and ELISA were used to detect relevant inflammatory indexes: IFN-γ, IL-1 and TNF-α. Quantitative real-time PCR and western blot were used to detect mucosal immune-related factors: MUC1, MUC4, DEFB1 and DEFB2. Results: ESCC was able to inhibit the proliferative activity of C. albicans, and it affected inflammation-related factors and indicators of vaginal mucosal immunity. Conclusion: ESCC showed potential value in the treatment of vulvovaginal candidiasis.

Expression of Human ß-defensin 2 (hBD-2) in Pichia Pastoris and Investigation of Its Binding Efficiency with ACE-2.

COVID-19 is a disease that have affected the entire world, and it continues to spread with new variants. A patient's innate immune system plays a critical role in the mild and severe transition of COVID-19. Antimicrobial peptides (AMPs), which are important components of the innate immune system, are potential molecules to fight pathogenic bacteria, fungi, and viruses. Human ß-defensin 2 (hBD-2), a 41-amino-acid antimicrobial peptide, is one of the defensins inducibly expressed in the skin, lungs, and trachea in humans. In this study, it was aimed to investigate the interaction of hBD-2 produced recombinantly in Pichia pastoris with the human angiotensin-converting enzyme 2 (ACE-2) under in vitro conditions. First, hBD-2 was cloned in P. pastoris X-33 via the pPICZαA vector, a yeast expression platform, and its expression was confirmed by SDS-PAGE, western blotting, and qRT-PCR. Then, the interaction between recombinant hBD-2 and ACE-2 proteins was revealed by a pull-down assay. In light of these preliminary experiments, we suggest that the recombinantly produced hBD-2 may be protective against SARS-CoV-2 and be used as a supplement in treatment. However, current findings need to be supported by cell culture studies, toxicity analyses, and in vivo experiments.

Expression, purification and investigation of antibacterial activity of a novel hybrid peptide LL37/hBD-129 by applied comprehensive computational and experimental approaches.

Antibiotic-resistant pathogens have become a great universal health concern. Antimicrobial peptides (AMPs) are small amphipathic and cationic polypeptides with high therapeutic potential against various microorganisms containing drug-resistant strains. Two major groups of these peptides, which have antibacterial activity against Gram-positive and Gram-negative bacteria, antiviral activity, and even antifungal activity, are defensins and cathelicidins. Hybridization of various AMPs is an appropriate approach to achieving new fusion AMPs with high antibacterial activity but low cellular toxicity. In the current research, the amino-acid sequence of human cathelicidin LL-37 (2-31) and Human beta-defensin (hBD)-129 were combined, and the fusion protein was evaluated by bioinformatics tool. The designed AMP gene sequence was commercially synthesized and cloned in the pET-28a expression vector. The LL-37/hBD-129 fusion protein was expressed in E.coli BL21-gold (DE3). The expression of the recombinant protein was evaluated using the SDS-PAGE method. The LL37/hBD-129 was successfully expressed as a recombinant hybrid AMP in E.coli BL21-gold (DE3) strain. Purification of the expressed AMP was performed by Ni-NTA column affinity chromatography, and the purified AMP was validated using the Western blot technic. Finally, the antimicrobial activity of the fusion AMP against Staphylococcus aureus and Escherichia coli bacteria was assessed. Based on the in silico analysis and experimental evaluations, the fusion AMP showed a significant antimicrobial effect on E. coli and Staphylococcus aureus bacteria.

A mechanistic evaluation of human beta defensin 2 mediated protection of human skin barrier in vitro.

The human skin barrier, a biological imperative, is impaired in inflammatory skin diseases such as atopic dermatitis (AD). Staphylococcus aureus is associated with AD lesions and contributes to pathological inflammation and further barrier impairment. S. aureus secretes extracellular proteases, such as V8 (or 'SspA'), which cleave extracellular proteins to reduce skin barrier. Previous studies demonstrated that the host defence peptide human beta-defensin 2 (HBD2) prevented V8-mediated damage. Here, the mechanism of HBD2-mediated barrier protection in vitro is examined. Application of exogenous HBD2 provided protection against V8, irrespective of timeline of application or native peptide folding, raising the prospect of simple peptide analogues as therapeutics. HBD2 treatment, in context of V8-mediated damage, modulated the proteomic/secretomic profiles of HaCaT cells, altering levels of specific extracellular matrix proteins, potentially recovering V8 damage. However, HBD2 alone did not substantially modulate cellular proteomic/secretomics profiles in the absence of damage, suggesting possible therapeutic targeting of lesion damage sites only. HBD2 did not show any direct protease inhibition or induce expression of known antiproteases, did not alter keratinocyte migration or proliferation, or form protective nanonet structures. These data validate the barrier-protective properties of HBD2 in vitro and establish key protein datasets for further targeted mechanistic analyses.

ß-defensin 2 synthesized by a cell-free protein synthesis system and encapsulated in liposomes inhibits adhesion of Porphyromonas gingivalis to oral epithelial cells.

ß-defensin 2 (BD-2), an antimicrobial peptide (AMP), is expressed by oral epithelial cells and plays an important role in innate immunity of the oral cavity. Cell-free protein synthesis (CFPS) systems have been studied for the synthesis of various proteins, however, the synthesis of BD-2 by a CFPS system has not been extensively explored. Liposomes have been developed as tools for drug delivery. A delivery of liposome-encapsulated AMP to oral epithelium may be useful to prevent oral infectious diseases. In the present study, we investigated the antimicrobial activity of the BD-2 protein, artificially synthesized using a CFPS system and encapsulated in liposomes. BD-2 protein was artificially synthesized using template DNA and a reconstituted CFPS system and was identified by western blotting. Bilayer liposomes were prepared using 1,2-dioleoyl-sn-glycero-3-phospho-choline and 3-sn-phosphatidylcholine from egg yolk. The artificially synthesized BD-2 was encapsulated in liposomes, collected by ultrafiltration, and detected by western blotting. Human oral epithelial cells were cultured with the liposome-encapsulated BD-2 and the concentration of BD-2 in the cell lysate of the culture with the synthesized BD-2 was higher than that of the control cultures. The antimicrobial activity of the synthesized BD-2 was investigated by an adhesion assay of Porphyromonas gingivalis to oral epithelial cells. The artificially synthesized BD-2 and its liposome significantly inhibited adhesion of P. gingivalis to oral epithelial cells. These results suggest that artificially synthesized BD-2 and liposome-encapsulated BD-2 show antimicrobial activity and can potentially play a role in oral healthcare for periodontal diseases.

Host Cationic Antimicrobial Molecules Inhibit S. aureus Exotoxin Production.

Innate immune molecules, including antimicrobial peptides (for example, defensins) and lysozyme, function to delay or prevent bacterial infections. These molecules are commonly found on mucosal and skin surfaces. Staphylococcus aureus is a common pathogen and causes millions of infections annually. It is well known that innate immune molecules, such as defensins and lysozyme, either poorly inhibit or do not inhibit the growth of S. aureus. Our current studies show that the α-defensin human neutrophil α-defensin-1 (HNP-1) and lysozyme inhibit exotoxin production, both hemolysins and superantigens, which are required for S. aureus infection. HNP-1 inhibited exotoxin production at concentrations as low as 0.001 µg/mL. Lysozyme inhibited exotoxin production at 0.05 to 0.5 µg/mL. Both HNP-1 and lysozyme functioned through at least one two-component system (SrrA/B). The ß-defensin human ß-defensin 1 (HBD-1) inhibited hemolysin but not superantigen production. The cation chelator S100A8/A9 (calprotectin), compared to EDTA, was tested for the ability to inhibit exotoxin production. EDTA at high concentrations inhibited exotoxin production; these were the same concentrations that interfered with staphylococcal growth. S100A8/A9 at the highest concentration tested (10 µg/mL) had no effect on S. aureus growth but enhanced exotoxin production. Lower concentrations had no effect on growth or exotoxin production. Lysostaphin is regularly used to lyse S. aureus. The lytic concentrations of lysostaphin were the only concentrations that also inhibited growth and exotoxin production. Our studies demonstrate that a major activity of innate defensin peptides and lysozyme is inhibition of staphylococcal exotoxin production but not inhibition of growth. IMPORTANCE Staphylococcus aureus causes large numbers of both relatively benign and serious human infections, which are mediated in large part by the organisms' secreted exotoxins. Since 1921, it has been known that lysozyme and, as shown later in the 1900s, other innate immune peptides, including human neutrophil α-defensin-1 (HNP-1) and human ß-defensin 1 (HBD-1), are either not antistaphylococcal or are only weakly inhibitory to growth. Our study confirms those findings but, importantly, shows that at subgrowth inhibitory concentrations, these positively charged innate immune peptides inhibit exotoxin production, including both hemolysins and the superantigen toxic shock syndrome toxin-1. The data show that the principal activity of innate immune peptides in the host is likely to be inhibition of exotoxin production required for staphylococcal mucosal or skin colonization rather than growth inhibition.

Chicken ß-defensin-1 peptide as a candidate anticoccidial agent in broiler chickens.

The current study aimed to investigate the potentiality of using avian ß-defensin-1 peptide as a candidate agent against coccidiosis infection in broiler chicken.We employed an in-silico analysis to study the primary structure of ß-defensin-1 peptide as well as its 3-D and molecular dynamic structures. This will also enable obtaining adequate information about the mode of action of these peptides and the intra-cellular transduction pathways. The results revealed no significant difference among groups of broiler chicken in terms of body weight before the Eimeria challenge.The results of our study indicated a significant reduction in oocyst count in birds administered ß-defensin-1 peptide treatment, vis-a-vis healthy birds. The treated group showed a 2-3 times reduction in oocyst count, compared to the positive control group. The Eimeria oocysts count evaluated for birds administered with ß-defensin-1 after the Eimeria challenge showed a significant difference. The study indicated significant reduction and down-regulation in the level of expression of ß-defensin 1 and 4 in the control and treatment groups.This electrostatic profile and hydrophobicity regulate the functioning of this peptide. The results may help in the development of novel approaches that could be used as alternatives or adjunct to the existing means of coccidiosis control in broilers.

Effect of platelet-rich fibrin (PRF) membranes on the healing of infected skin wounds.

Tissue engineering focuses on wound healing and tissue regeneration. Platelet-rich fibrin (PRF) is a fibrin matrix containing cytokines, growth factors and cells that are gradually released into the wound over time. This study aimed to evaluate the effect of PRF membranes on wound repair and microbial control in infected wounds. Skin wounds were performed on the dorsum of rats using a 6 mm diameter metal punch. The defects were randomly assigned into four groups (n = 12/each) accordingly to the treatment: G1, noninfected wound filled only with clot; G2, noninfected wound with PRF; G3, infected wound (S. aureus) without PRF; G4, infected wound (S. aureus) with PRF. After 7 and 14 days, macroscopic and histological analyses of the wounds were performed. Furthermore, the quantification of ß-defensin in PRF was measured by ELISA. At 14 days, the groups with PRF (G2 and G4) had wound sizes significantly smaller than the original defects (6 mm) (p < 0.05) and significantly smaller than those not treated with PRF, in both the infected and noninfected groups (p < 0.05). Furthermore, the groups with infected wounds (G3 and G4) demonstrated a significantly lower inflammation score in the PRF group than in the noninfected groups (p < 0.05). In vitro analysis of ß-defensin was performed in all PRF membrane groups, and the median value was 1.444 pg/mL. PRF in the wounds of both control and infected rats played an important role in the modulation of tissue healing, most notably in infected sites.

Defensins: defenders of human reproductive health.

BACKGROUND: Reproductive tract infection is an important factor leading to male and female infertility. Among female infertility factors, microbial and viral infections are the main factors affecting female reproductive health and causing tubal infertility, ectopic tubal pregnancy and premature delivery. Among male infertility factors, 13-15% of male infertility is related to infection. Defensins are cationic antibacterial and antiviral peptides, classified into α-defensins, ß-defensins and θ-defensins. Humans only have α-defensins and ß-defensins. Apart from their direct antimicrobial functions, defensins have an immunomodulatory function and are involved in many physiological processes. Studies have shown that defensins are widely distributed in the female reproductive tract (FRT) and male reproductive tract (MRT), playing a dual role of host defence and fertility protection. However, to our knowledge, the distribution, regulation and function of defensins in the reproductive tract and their relation to reproduction have not been reviewed. OBJECTIVE AND RATIONALE: This review summarizes the expression, distribution and regulation of defensins in the reproductive tracts to reveal the updated research on the dual role of defensins in host defence and the protection of fertility. SEARCH METHODS: A systematic search was conducted in PubMed using the related keywords through April 2022. Related data from original researches and reviews were integrated to comprehensively review the current findings and understanding of defensins in the human reproductive system. Meanwhile, female and male transcriptome data in the GEO database were screened to analyze defensins in the human reproductive tracts. OUTCOMES: Two transcriptome databases from the GEO database (GSE7307 and GSE150852) combined with existing researches reveal the expression levels and role of the defensins in the reproductive tracts. In the FRT, a high expression level of α-defensin is found, and the expression levels of defensins in the vulva and vagina are higher than those in other organs. The expression of defensins in the endometrium varies with menstrual cycle stages and with microbial invasion. Defensins also participate in the local immune response to regulate the risk of spontaneous preterm birth. In the MRT, a high expression level of ß-defensins is also found. It is mainly highly expressed in the epididymal caput and corpus, indicating that defensins play an important role in sperm maturation. The expression of defensins in the MRT varies with androgen levels, age and the status of microbial invasion. They protect the male reproductive system from bacterial infections by neutralizing lipopolysaccharide and downregulating pro-inflammatory cytokines. In addition, animal and clinical studies have shown that defensins play an important role in sperm maturation, motility and fertilization. WIDER IMPLICATIONS: As a broad-spectrum antimicrobial peptide without drug resistance, defensin has great potential for developing new natural antimicrobial treatments for reproductive tract infections. However, increasing evidence has shown that defensins can not only inhibit microbial invasion but can also promote the invasion and adhesion of some microorganisms in certain biological environments, such as human immunodeficiency virus. Therefore, the safety of defensins as reproductive tract anti-infective drugs needs more in-depth research. In addition, the modulatory role of defensins in fertility requires more in-depth research since the current conclusions are based on small-size samples. At present, scientists have made many attempts at the clinical transformation of defensins. However, defensins have problems such as poor stability, low bioavailability and difficulties in their synthesis. Therefore, the production of safe, effective and low-cost drugs remains a challenge.

Human ß-defensin 2 ameliorates acute GVHD by limiting ileal neutrophil infiltration and restraining T cell receptor signaling.

Acute graft-versus-host disease (aGVHD), which is driven by allogeneic T cells, has a high mortality rate and limited treatment options. Human ß-defensin 2 (hBD-2) is an endogenous epithelial cell-derived host-defense peptide. In addition to its antimicrobial effects, hBD-2 has immunomodulatory functions thought to be mediated by CCR2 and CCR6 in myeloid cells. In this study, we analyzed the effect of recombinant hBD-2 on aGVHD development. We found that intestinal ß-defensin expression was inadequately induced in response to inflammation in two independent cohorts of patients with aGVHD and in a murine aGVHD model. Treatment of mice with hBD-2 reduced GVHD severity and mortality and modulated the intestinal microbiota composition, resulting in reduced neutrophil infiltration in the ileum. Furthermore, hBD-2 treatment decreased proliferation and proinflammatory cytokine production by allogeneic T cells in vivo while preserving the beneficial graft-versus-leukemia effect. Using transcriptome and kinome profiling, we found that hBD-2 directly dampened primary murine and human allogeneic T cell proliferation, activation, and metabolism in a CCR2- and CCR6-independent manner by reducing proximal T cell receptor signaling. Furthermore, hBD-2 treatment diminished alloreactive T cell infiltration and the expression of genes involved in T cell receptor signaling in the ilea of mice with aGVHD. Together, we found that both human and murine aGVHD were characterized by a lack of intestinal ß-defensin induction and that recombinant hBD-2 represents a potential therapeutic strategy to counterbalance endogenous hBD-2 deficiency.

Immunomodulatory effects of ß-defensin 2 on macrophages induced immuno-upregulation and their antitumor function in breast cancer.

BACKGROUND: Macrophages are mononuclear CD34+ antigen-presenting cells of defense mechanism and play dual roles in tumor burden. The immunomodulatory and their antitumor function of ß-defensin 2 is still unclear, despite the accumulating evidence of the response in infection. So, the aim of present study is to elucidate the role of ß-defensin 2 on the level of ROS, cytokines, chemokine expression in macrophages and antitumor function in breast cancer. METHOD: Swiss albino mice were used to harvest PEC macrophages and C127i breast cancer cells line for tumor model was used in this study. Macrophages were harvested and characterized by flow-cytometry using F4/80 and CD11c antibodies. MTT was performed to estimate cytotoxicity and dose optimization of ß-defensin 2. Oxidative stress was analyzed by H2O2 and NO estimation followed by iNOS quantified by q-PCR. Cytokines and chemokines estimation was done using q-PCR. Co-culture experiment was performed to study anti-tumor function using PI for cell cycle, Annexin -V and CFSE analysis for cell proliferation. RESULTS: PEC harvested macrophages were characterized by flow-cytometry using F4/80 and CD11c antibodies with the purity of 8% pure population of macrophages. It was found that 99% of cells viable at the maximum dose of 100 ng/ml of ß-defensin 2 in MTT. Levels of NO and H2O2 were found to be decreased in ß-defensin 2 as compared to control. Expression of cytokines of IFN-γ, IL-1α, TNF-α, TGF-ßwas found to be increased while IL-3 was decreased in ß-defensin 2 group as compared to control. Levels of chemokines CXCL-1, CXCL-5 and CCL5 increased in treated macrophages while CCL24 and CXCL-15 expression decreased. Adhesion receptor (CD32) and fusion receptor (CD204) were decreased in the ß-defensin 2 group as compared to control. Anti-tumor experiment was performed using co-culture experiment apoptosis (Annexin-V) was induced, cell cycle arrest in phage and cell proliferation of C127i cells was decreased. CONCLUSION: This is the first report of ß-defensin 2 modulates macrophage immunomodulatory and their antitumor function in breast cancer. ß-defensin 2 as a new therapeutic target for immunotherapy as an adjuvant in vaccines.

Antimicrobial peptide human ß-defensin-2 improves in vitro cellular viability and reduces pro-inflammatory effects induced by enteroinvasive Escherichia coli in Caco-2 cells by inhibiting invasion and virulence factors' expression.

Escherichia coli is one of the commensal species most represented in the intestinal microbiota. However, there are some strains that can acquire new virulence factors that enable them to adapt to new intestinal niches. These include enteroinvasive E. coli (EIEC) that is responsible for the bacillary dysentery that causes severe diarrheal symptoms in both children and adults. Due to the increasing onset of antibiotic resistance phenomena, scientific research is focused on the study of other therapeutic approaches for the treatment of bacterial infections. A promising alternative could be represented by antimicrobial peptides (AMPs), that have received widespread attention due to their broad antimicrobial spectrum and low incidence of bacterial resistance. AMPs modulate the immune defenses of the host and regulate the composition of microbiota and the renewal of the intestinal epithelium. With the aim to investigate an alternative therapeutic approach, especially in the case of antibiotic resistance, in this work we created a line of intestinal epithelial cells able to express high concentrations of AMP human ß-defensin-2 (HBD-2) in order to test its ability to interfere with the pathogenicity mechanisms of EIEC. The results showed that HBD-2 is able to significantly reduce the expression of the proinflammatory cytokines by intestinal epithelial cells, the invasiveness ability of EIEC and the expression of invasion-associated genes.