Search Results (47)

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 β-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. Full article

β-Casomorphin-7 (BCM), a breakdown product of milk β-casein, exhibits opioid activity. Opioids are known to affect the immune system, but the effects of BCM on ulcerative colitis (UC) are not clear. We examined the effects of BCM on mucosal immunity using a mouse dextran sulfate sodium-induced colitis model and an in vitro CD8+ T cell activation model. Human UC patients were examined to reveal the relationship between CD10 and mucosal immunity. Combined treatment of the colitis model with thiorphan (TOP) inhibited BCM degradation by suppressing CD10 in the intestinal mucosa, activating mouse mucosal CD8, and suppressing CD4 and Treg. In the CD8+ T cell in vitro activation assay using mouse splenocytes, BCM inhibited the oxidative phosphorylation (OXPHOS) of CD8+ T cells and induced the glycolytic pathway, promoting their activation. Conversely, in a culture system, BCM suppressed OXPHOS and decreased defensin α production in IEC6 mouse intestinal epithelial cells. In the mouse model, BCM reduced defensin α and butyrate levels in the colonic mucosa. During the active phase of human ulcerative colitis, the downward regulation of ileal CD10 expression by CpG methylation of the gene promoter was observed, resulting in increased CD8 activation and decreased defensin α and butyrate levels. BCM is a potential aggravating factor for UC and should be considered in the design of dietary therapy. In addition, decreased CD10 expression may serve as an indicator of UC activity and recurrence, but further clinical studies are needed. Full article

Antimicrobial peptides (AMPs), as well as host defense peptides (HDPs), constitute the first line of defense as part of the innate immune system. Humans are known to express antimicrobial precursor proteins, which are further processed to generate AMPs, including several types of α/β defensins, histatins, and cathelicidin-derived AMPs like LL37. The broad-spectrum activity of AMPs is crucial to defend against infections caused by pathogenic bacteria, viruses, fungi, and parasites. The emergence of multi-drug resistant pathogenic bacteria is of global concern for public health. The prospects of targeting antibiotic-resistant strains of bacteria with AMPs are of high significance for developing new generations of antimicrobial agents. The 37-residue long LL37, the only cathelicidin family of AMP in humans, has been the major focus for the past few decades of research. The host defense activity of LL37 is likely underscored by its expression throughout the body, spanning from the epithelial cells of various organs—testis, skin, respiratory tract, and gastrointestinal tract—to immune cells. Remarkably, apart from canonical direct killing of pathogenic organisms, LL37 exerts several other host defense activities, including inflammatory response modulation, chemo-attraction, and wound healing and closure at the infected sites. In addition, LL37 and its derived peptides are bestowed with anti-cancer and anti-amyloidogenic properties. In this review article, we aim to develop integrative, mechanistic insight into LL37 and its derived peptides, based on the known biophysical, structural, and functional studies in recent years. We believe that this review will pave the way for future research on the structures, biochemical and biophysical properties, and design of novel LL37-based molecules. Full article

Obesity and metabolic comorbidities are associated with gut permeability. While high-fructose and Western-style diet (WSD) disrupt intestinal barrier function, oral administration of human α-defensin 5 (HD5) and β-defensin 2 (hBD2) is believed to improve intestinal integrity and metabolic disorders. Eighty-four male C57BL/6J mice were fed a WSD or a control diet (CD) ± fructose (F) for 18 weeks. In week 13, mice were randomly divided into three intervention groups, receiving defensin fragment HD5_1–9, full-length hBD2, or bovine serum albumin (BSA)-control for six weeks. Subsequently, parameters of hepatic steatosis, glucose metabolism, and gut barrier function were assessed. WSDF increased body weight and hepatic steatosis (p < 0.01) compared to CD-fed mice, whereas peptide intervention decreased liver fat (p < 0.05) and number of hepatic lipid droplets (p < 0.01) compared to BSA-control. In addition, both peptides attenuated glucose intolerance by reducing blood glucose curves in WSDF-fed mice. Evaluation of gut barrier function revealed that HD5_1–9 and hBD2 improve intestinal integrity by upregulating tight junction and mucin expression. Moreover, peptide treatment restored ileal host defense peptides (HDP) expression, likely by modulating the Wnt, Myd88, p38, and Jak/STAT pathways. These findings strongly suggest that α- and β-defensin treatment improve hepatic steatosis, glucose metabolism, and gut barrier function. Full article

Whooping cough is a severe childhood disease, caused by the bacterium Bordetella pertussis, which releases pertussis toxin (PT) as a major virulence factor. Previously, we identified the human antimicrobial peptides α-defensin-1 and -5 as inhibitors of PT and demonstrated their capacity to inhibit the activity of the PT enzyme subunit PTS1. Here, the underlying mechanism of toxin inhibition was investigated in more detail, which is essential for developing the therapeutic potential of these peptides. Flow cytometry and immunocytochemistry revealed that α-defensin-5 strongly reduced PT binding to, and uptake into cells, whereas α-defensin-1 caused only a mild reduction. Conversely, α-defensin-1, but not α-defensin-5 was taken up into different cell lines and interacted with PTS1 inside cells, based on proximity ligation assay. In-silico modeling revealed specific interaction interfaces for α-defensin-1 with PTS1 and vice versa, unlike α-defensin-5. Dot blot experiments showed that α-defensin-1 binds to PTS1 and even stronger to its substrate protein Gαi in vitro. NADase activity of PTS1 in vitro was not inhibited by α-defensin-1 in the absence of Gαi. Taken together, these results suggest that α-defensin-1 inhibits PT mainly by inhibiting enzyme activity of PTS1, whereas α-defensin-5 mainly inhibits cellular uptake of PT. These findings will pave the way for optimization of α-defensins as novel therapeutics against whooping cough. Full article

Both defensin and inflammation are part of the human innate immune system that responds rapidly to pathogens. The combination of defensins with pro- or anti-inflammatory effects can be a potential research direction for the treatment of infection by pathogens. This study aimed to identify whether MSF (Miracle Synergy material made using Filipendula glaberrima), a probiotic lysate of Filipendula glaberrima extracts fermented with Lactiplantibacillus plantarum K8, activates the expression of human β-defensin (HBD2 and HBD3) to protect the host against pathogens and inhibit inflammation caused by S. aureus, in vitro with Western blot analysis, qRT-PCR and in vivo studies with a mouse model were used to evaluate the effects of MSF. The MSF treatment induced HBD2 and HBD3 expression via the p38 and NF-κB pathways. Furthermore, MSF treatment significantly reduced the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-8), also through p38 and NF-κB in S. aureus-induced inflammatory condition. MSF treatment remarkably reduced erythema in mice ears caused by the injection of S. aureus, while K8 lysate treatment did not initiate a strong recovery. Taken together, MSF induced the expression of HBD2 and HDB3 and activated anti-inflammatory activity more than the probiotic lysates of L. plantarum K8. These findings show that MSF is a potential defensin inducer and anti-inflammatory agent. Full article

Proteinase 3 (PR3) is a neutrophil granulocyte enzyme and an autoantigen found in several forms of vasculitis. Due to the diagnostic and clinical importance of antibodies (Abs) to PR3, it is important to characterize the protein and the nature of its epitopes. Here, we have characterized PR3 monoclonal antibodies (MAbs) and disease-associated Abs and their dependency on the PR3 structure and modifications, especially interactions with α-defensins. Three MAbs (HYB 172-01, 172-04, 172-05), which bind to PR3 in its native and denatured forms and provide the disulphide bridges, were intact. α-1-antitrypsin (AT) binds to purified human neutrophil granulocyte PR3 and inhibits its proteolytic activity, towards a small synthetic peptide substrate and a large protein substrate (casein). AT also inhibited the binding of the three MAbs to PR3, indicating that they bind in a region affected by AT binding. However, the MAbs did not inhibit PR3 proteolytic activity with a small substrate, showing that they bound at the active site without restricting access to the substrate cleft. Patient-derived Abs showed essentially the same characteristics as the MAbs, with important implications for vasculitis diagnostics and pathophysiology. Current findings illustrate that PR3 epitopes depend on the three-dimensional structure of the PR3/defensin complex, and that the epitopes depend to a smaller or larger degree on PR3/defensin associations. Full article

Defensins have long been considered as merely antimicrobial peptides. Throughout the years, more immune-related functions have been discovered for both the α-defensin and β-defensin subfamily. This review provides insights into the role of defensins in tumor immunity. Since defensins are present and differentially expressed in certain cancer types, researchers started to unravel their role in the tumor microenvironment. The human neutrophil peptides have been demonstrated to be directly oncolytic by permealizing the cell membrane. Further, defensins can inflict DNA damage and induce apoptosis of tumor cells. In the tumor microenvironment, defensins can act as chemoattractants for subsets of immune cells, such as T cells, immature dendritic cells, monocytes and mast cells. Additionally, by activating the targeted leukocytes, defensins generate pro-inflammatory signals. Moreover, immuno-adjuvant effects have been reported in a variety of models. Therefore, the action of defensins reaches beyond their direct antimicrobial effect, i.e., the lysis of microbes invading the mucosal surfaces. By causing an increase in pro-inflammatory signaling events, cell lysis (generating antigens) and attraction and activation of antigen presenting cells, defensins could have a relevant role in activating the adaptive immune system and generating anti-tumor immunity, and could thus contribute to the success of immune therapy. Full article

Placentas from gestational diabetes mellitus (GDM) patients undergo significant metabolic and immunologic adaptations due to hyperglycemia, which results in an exacerbated synthesis of proinflammatory cytokines and an increased risk for infections. Insulin or metformin are clinically indicated for the treatment of GDM; however, there is limited information about the immunomodulatory activity of these drugs in the human placenta, especially in the context of maternal infections. Our objective was to study the role of insulin and metformin in the placental inflammatory response and innate defense against common etiopathological agents of pregnancy bacterial infections, such as E. coli and S. agalactiae, in a hyperglycemic environment. Term placental explants were cultivated with glucose (10 and 50 mM), insulin (50–500 nM) or metformin (125–500 µM) for 48 h, and then they were challenged with live bacteria (1 × 10⁵ CFU/mL). We evaluated the inflammatory cytokine secretion, beta defensins production, bacterial count and bacterial tissue invasiveness after 4–8 h of infection. Our results showed that a GDM-associated hyperglycemic environment induced an inflammatory response and a decreased beta defensins synthesis unable to restrain bacterial infection. Notably, both insulin and metformin exerted anti-inflammatory effects under hyperglycemic infectious and non-infectious scenarios. Moreover, both drugs fortified placental barrier defenses, resulting in reduced E. coli counts, as well as decreased S. agalactiae and E. coli invasiveness of placental villous trees. Remarkably, the double challenge of high glucose and infection provoked a pathogen-specific attenuated placental inflammatory response in the hyperglycemic condition, mainly denoted by reduced TNF-α and IL-6 secretion after S. agalactiae infection and by IL-1β after E. coli infection. Altogether, these results suggest that metabolically uncontrolled GDM mothers develop diverse immune placental alterations, which may help to explain their increased vulnerability to bacterial pathogens. Full article

Background and Objectives: Neutrophil infiltration is an established signature of Non-Alcoholic Fatty Liver Disease (NAFLD) and Steatohepatitis (NASH). The most abundant neutrophilic peptide, alpha-defensin, is considered a new evolving risk factor in the inflammatory milieu, intimately involved in lipid mobilization. Our objective is to assess for potential association between alpha-defensin immunostains and NAFLD severity. Materials and Methods: We retrospectively investigated the liver biopsies of NAFLD/NASH patients, obtained at Hillel Yaffe Medical center between the years 2012 and 2016. Patients’ characteristics were recorded, including relevant blood tests at the time of biopsy. Each biopsy was semi-quantitatively scored using NAFLD Activity Score (NAS) and NASH fibrosis stage. The biopsies were immunostained for alpha-defensin. The precipitation of alpha-defensin was correlated to NAS and fibrosis. Results: A total of 80 biopsies were evaluated: male ratio 53.2%, mean age 44.9 ± 13.2 years, 54 had fibrosis grades 0–2, and 26 were grade 3–4. Conventional metabolic risk factors were more frequent in the high-grade fibrosis group. Immunostaining for alpha-defensin disclosed higher intensity (a.u.) in grade 3–4 fibrosis relative to grades 0–2, 25% vs. 6.5%, p < 0.05, respectively. Moreover, alpha-defensin staining was nicely co-localized with fibrosis. Conclusions: In our group of NASH/NAFLD patients, higher metabolic risk profile was associated with higher fibrosis grade. Immunostaining for alpha-defensin showed patchy intense staining concordant with high fibrosis, nicely co-localized with histological fibrosis. Whether alpha-defensin is a profibrotic risk factor or merely risk marker for fibrosis must be clarified in future studies. Full article

Background: The goal of this study is the identification of potential marker molecules for characterizing different basal cell carcinoma entities, to help improve clinical decisions for surgical resection therapy. Methods: Three different entities, sclerodermiform, solid and superficial basal cell carcinomas, were subjected to immunohistochemical microscopy and histomorphometric analyses for human α- (DEFA1/3; DEFA4) and β-defensins (hBD1/2/3) and special S100 proteins (S100A4/7/8/9). Thirty specimens of the three entities were evaluated. Analyses were performed by comparing tissue and cellular localization and staining intensities of tumorous with non-tumorous areas. Staining intensities were semiquantitatively examined by using an RGB-based model. Results: Human defensins are present in all three entities of basal cell carcinomas. They all show cytoplasmic immunostaining in cells of the epithelium, stroma and tumor. Notably, human β-defensin3 is accumulated in the cell nuclei of sclerodermiform and superficial basal cell carcinomas. S100A4 and A7 are undetectable in tumor regions. However, S100A4 occurs in cancer-associated stroma cells with nuclear staining in superficial basal cell carcinomas. Conclusion: Two candidates, namely hBD3 and S100A4, might be used as potential clinical tools for evaluating successful surgical resection therapy to avoid aesthetic and functional facial deformation. Full article

Antimicrobial peptides (AMP) are essential components of innate immunity with a broad range of antimicrobial activities against bacteria, viruses, and fungi. The aim of this study was to investigate AMP expression in the upper gastrointestinal tract in normal and pathological metabolic states in humans. Furthermore, we examined the correlation between vitamin D levels and AMP expression in the same cohort. Serum concentrations of 25-hydroxyvitamin D3 were measured, and mRNA expression of β-defensins HBD-1, -2, -3, -4, α-defensins HD-5 and -6 and cathelicidin in the upper gastrointestinal tract epithelia were determined by quantitative RT-PCR in 31 individuals (10 with type 2 diabetes, 10 with insulin resistance, and 11 healthy controls). The majority of the cohort showed low vitamin D concentrations, which were negatively correlated with mRNA expression levels of HBD-3 in corpus mucosa. HBD-1 and HBD-3 mRNA were expressed in corpus mucosa, with the former significantly decreased in patients with diabetes. Hence, we conclude that type 2 diabetes is associated with reduced AMP expression in the upper gastrointestinal tract, which might contribute towards epithelial barrier dysfunction and increased bacterial translocation in these patients. Full article

The oral innate immune response may diminish with aging. In the present study, the aim was to examine human β-defensin (hBD) 1-3 and human neutrophil peptide (HNP)-1 levels in the saliva of an elderly population to establish the extent of periodontal disease and tooth loss. A total of 175 individuals aged ≥ 65 years were divided into five groups based on the number of teeth with a pocket depth ≥ 4 mm as follows: 17 pocket-free individuals (Control), 55 individuals having 1–6 pocket teeth (PerioA), 33 individuals having 7–13 pocket teeth (PerioB), 29 individuals having at least 14 pocket teeth (PerioC), and 41 edentulous individuals. Their salivary defensin levels were measured with ELISA kits. The salivary HNP-1 levels were significantly higher in the Perio groups (PerioB: p < 0.001 and PerioC: p < 0.001) in comparison to the Control. The associations between salivary HNP-1 levels and the number of pocket teeth remained significant after adjustments for age, gender, level of education, and number of teeth. The salivary HNP and hBD levels differed in terms of their correlation to the extent of periodontal disease and tooth loss in the elderly. Full article

Host defense against early-life infections such as chorioamnionitis, neonatal sepsis, or necrotizing enterocolitis (NEC) relies primarily on innate immunity, in which antimicrobial peptides (AMPs) play a major role. AMPs that are important for the fetus and neonate include α and β defensins, cathelicidin LL-37, antiproteases (elafin, SLPI), and hepcidin. They can be produced by the fetus or neonate, the placenta, chorioamniotic membranes, recruited neutrophils, and milk-protein ingestion or proteolysis. They possess antimicrobial, immunomodulating, inflammation-regulating, and tissue-repairing properties. AMPs are expressed as early as the 13th week and increase progressively through gestation. Limited studies are available on AMP expression and levels in the fetus and neonate. Nevertheless, existing evidence supports the role of AMPs in pathogenesis of chorioamnionitis, neonatal sepsis, and NEC, and their association with disease severity. This suggests a potential role of AMPs in diagnosis, prevention, prognosis, and treatment of sepsis and NEC. Herein, we present an overview of the antimicrobial and immunomodulating properties of human AMPs, their sources in the intrauterine environment, fetus, and neonate, and their changes during pre- and post-natal infections and NEC. We also discuss emerging data regarding the potential utility of AMPs in early-life infections, as diagnostic or predictive biomarkers and as therapeutic alternatives or adjuncts to antibiotic therapy considering the increase of antibiotic resistance in neonatal intensive care units. Full article

The gingival epithelium–capillary interface is a unique feature of periodontal soft tissue, preserving periodontal tissue homeostasis and preventing microorganism and toxic substances from entering the subepithelial tissue. However, the function of the interface is disturbed in periodontitis, and mechanisms of the breakdown of the interface are incompletely understood. To address these limitations, we developed a microfluidic epithelium–capillary barrier with a thin culture membrane (10 μm) that closely mimics the in vivo gingival epithelial barrier with an immune micro-environment. To test the validity of the fabricated gingival epithelial barrier model, epithelium–capillary interface-on-a-chip was cultured with human gingival epithelial cells (HGECs) and human vascular endothelial cells (HUVEC). Their key properties were tested using optical microscope, transepithelial/transendothelial electrical resistance (TEER), and permeability assays. The clear expression of VE-cadherin revealed the tight junctions in endothelial cells. Live/dead assays indicated a high cell viability, and the astrocytic morphology of HGE cells was confirmed by F-actin immunostaining. By the third day of cell culture, TEER levels typically exceeded in co-cultures. The resultant permeability coefficients showed a significant difference between 70 kDa and 40 kDa FITC-dextran. The expression of protein intercellular cell adhesion molecule (ICAM-1) and human beta defensin-2 (HBD2) decreased when exposed to TNF-α and LPS, but recovered with the NF-κB inhibitor treatment- Pyrrolidinedithiocarbamic acid (PDTC), indicating the stability of the fabricated chip. These results demonstrate that the developed epithelium-capillary interface system is a valid model for studying periodontal soft tissue function and drug delivery. Full article