Defensin Alpha

The AMP-antibiotic-microbiota triad in IBD: a mechanistic framework for dysregulated antimicrobial defense.

Yuyuan Hu, Yan Li, Qiang Zhang +7 more

Inflammatory bowel disease (IBD) represents a chronic relapsing disorder driven by a loss of homeostatic balance between the host immune system and the intestinal microbiota. Endogenous antimicrobial peptides (AMPs), produced primarily by epithelial and immune cells, function in concert with commensal microorganisms to preserve mucosal integrity and barrier function. Disruption of this antimicrobial equilibrium-through genetic susceptibility such as NOD2 mutations or environmental perturbations including antibiotic overuse-can impair antimicrobial defense, distort microbial composition, and initiate chronic inflammation. Recent investigations have revealed distinct alterations in AMP expression across IBD subtypes. In Crohn's disease, Paneth cell-derived α-defensins (HD5 and HD6) are markedly diminished in the ileal mucosa, whereas colonic, segmental IBD exhibits inadequate induction of β-defensins and LL-37. Conversely, in actively inflamed regions, certain AMPs such as human β-defensin-2 (HBD2) and lysozyme are strongly upregulated, reflecting a compensatory response to inflammatory cell infiltration and microbial invasion. Beyond host-derived peptides, broad-spectrum antibiotic exposure profoundly reshapes commensal communities, attenuates basal pattern-recognition receptor signaling, and secondarily perturbs AMP regulation-creating a feedback loop that amplifies dysbiosis. Here, we conceptualize these interactions as an integrated AMP-antibiotic-microbiota triad, in which endogenous antimicrobial regulation, exogenous antimicrobial pressure, and microbial ecological resilience dynamically co-determine mucosal stability. By positioning AMPs within this tripartite regulatory framework, this review delineates how antimicrobial imbalance arises across IBD subtypes, compares emerging therapeutic strategies-including AMP enhancement, microbiota-sparing antibiotic regimens, fecal microbiota transplantation, and metabolite-guided interventions-and highlights implications for precision recalibration of antimicrobial homeostasis in IBD.

VGLL4 modulates Paneth cells and sustains intestinal homeostasis.

Haoen Zhang, Zuoyun Wang, Xiaodong Wang +22 more

Paneth cells are defensive cells in the intestinal tract, which secrete niche factors and antimicrobial peptides (AMPs) to maintain the small intestinal stem cell niche and immune homeostasis. Here, we show that Vestigial-like family member 4 (VGLL4) plays a pivotal role in maintaining small intestinal homeostasis and in regulating Paneth cells. VGLL4 expression is downregulated in response to irradiation and DSS-induced colitis. Consistently, public datasets of human colitis show reduced VGLL4 expression. Loss of VGLL4 in the intestinal epithelium decreases Paneth cell numbers and AMPs production, and triggers gut microbiota dysbiosis, impairing intestinal regenerative capacity. Mechanistically, VGLL4 forms a complex with TEAD4 and ATOH1, stimulating GFI1 expression and promoting Paneth cell differentiation. Furthermore, VGLL4 forms a complex with TEAD4 and TCF4 to induce defensin expression, thereby maintaining microbiota composition. Collectively, our findings uncover novel roles for VGLL4 in intestinal homeostasis.

Antimicrobial Peptides (AMPs) Are Not Increased in Asymptomatic Bacteriuria in Healthy Older Adult Patients.

Katherine M Hunold, Andrew Schwaderer, Julie A Stephens +8 more

Antimicrobial peptides have demonstrated promise as biomarkers for urinary tract infection (UTI) in older adults (age ≥ 65 years). However, it is unknown if urinary AMP levels also increase in asymptomatic bacteriuria. Our objective was to determine if AMP levels vary between older adult patients with and without asymptomatic bacteriuria.

Microbiota modulation by a human Paneth cell α-defensin fragment protects against DSS-induced colitis.

Erica Bonazzi, Fuhua Hao, Andrew D Patterson +3 more

The gastrointestinal tract hosts a diverse microbial community, named the intestinal microbiota, which plays a vital role in gut health. Host defense peptides (HDPs), such as defensins, help regulate interactions between the host and its microbiota. Dysfunction in HDPs expression has been associated with a greater risk of inflammatory bowel diseases (IBDs), while some HDPs may positively shape the gut environment. Human alpha defensin 5 (HD5) can be cleaved by intestinal enzymes into bioactive fragments such as HD51-9. In this study, we screened various HDPs (LL-37, hBD2, HD5, and HD51-9) for their modulatory effects on healthy human microbiotas in vitro and found distinct, donor-specific effects, with HD51-9 showing the strongest functional impact. Testing HD51-9 in a mouse colitis model revealed potent and microbiota-dependent protection against DSS-induced inflammation, partly through the beneficial of mucus-microbiota interactions. These findings highlight the potential of HDPs, especially HD51-9, in modulating gut microbiota and treating intestinal inflammation.

Scalable recombinant production of bioactive human neutrophil peptide-1 in Komagataella phaffii.

Mohd Sadeeq, Chaozhi Wang, Ke Yu +5 more

The escalating crisis of antimicrobial resistance demands novel therapeutics that overcome the limitations of conventional antibiotics. Human α-defensins, such as Human Neutrophil Peptide-1 (HNP-1), represent compelling candidates due to their potent, broad-spectrum antimicrobial activity and membrane-disrupting mechanism. However, clinical translation has been hindered by the absence of scalable production systems capable of delivering high yields of functional peptide. To address this challenge, we developed an efficient expression platform in the yeast Komagataella phaffii. In this system, a codon-optimized HNP-1 sequence was fused to a His6-SUMO tag downstream of the α-factor secretion signal to enhance solubility, folding, and recovery. Initial shake-flask optimization identified pH 6.0 with 96 h of induction as optimal fermentation conditions, yielding 19.75 ± 1.1 mg/L of recombinant protein. Subsequent scale-up to a controlled 5 L bioreactor significantly enhanced production, achieving 122 mg/L of the fusion protein. Following purification and precise cleavage, this process delivered 15.25 mg/L of pure, mature HNP-1 representing, to our knowledge, the highest yield of recombinant HNP-1 reported. The final product demonstrated potent antibacterial activity against Staphylococcus aureus and Escherichia coli while exhibiting excellent hemocompatibility, confirming preservation of native structure and function. This work establishes a scalable production platform for HNP-1 and provides an adaptable framework for expressing other structurally complex antimicrobial peptides with therapeutic potential.

Cerebrospinal Fluid Human Neutrophil Peptides 1-3: A Potential Prognostic Marker in Intracerebral Hemorrhage.

Zhi He, Jun Xie, Fu-Ling Yan +4 more

Background: This study aims to investigate whether elevated human neutrophil peptides 1-3 (HNP 1-3) levels in cerebrospinal fluid (CSF) are associated with disease severity and clinical outcomes in patients with intracerebral hemorrhage (ICH). Materials and Methods: HNP 1-3 levels were measured in CSF samples collected within 3 days after hemorrhage onset in ICH patients and control subjects. Results: HNP 1-3 levels were significantly higher in ICH patients with moderate-severe coma and hematoma volume >30 mL group. Univariate and multivariate logistic regression analyses demonstrated that CSF HNP 1-3 levels were associated with unfavorable outcomes. Receiver operating characteristic analysis revealed that CSF HNP 1-3 concentrations >466.95 ng/mL could distinguish ICH patients at risk for an unfavorable prognosis. Conclusions: HNP 1-3 exhibit satisfactory diagnostic efficiency for predicting the prognosis of ICH patients.

Multiplexed MALDI fingerprints for rapid detection of spontaneous bacterial peritonitis.

Qiong Wu, Bo Wei, Han Zhang +9 more

The efficient diagnosis of spontaneous bacterial peritonitis (SBP) has been hindered by the low sensitivity of current clinical indicators. A rapid and accurate detection method for SBP is essential to enable early diagnosis and prompt intervention.

Key Factors Influencing Caries Development in Preschoolers: A Focus on Socio-Demographic, Maternal Health, and Salivary Biomarkers in 3-Year-Olds.

Branislava Stojkovic, Marija Igic, Tatjana Jevtovic Stoimenov +7 more

BACKGROUND The study examined and compared the significance of sociodemographic, oral health behavior, and maternal factors, as well as salivary pH and salivary levels of human neutrophil peptide 1 (HNP-1), human b defensin 2 (hBD-2), and human cathelicidin (LL-37) as early caries predictors in 3-year-olds. MATERIAL AND METHODS A 1-year observational prospective study was conducted. The study included 165 caries-free children aged 36-48 months and their mothers. At baseline data were collected through a questionnaire for mothers and clinical examination of the children. For certain children (N=35), unstimulated saliva samples were collected to determine salivary pH using a digital portable pH meter and salivary levels of HNP-1, hBD-2, and LL-37 peptides using ELISA. After 12 months, caries incidence was determined. The caries-predictive significance of factors was estimated by logistic regression analysis. RESULTS After 1 year, caries was diagnosed in 29.1% of the children. Univariate logistic regression analysis revealed that the potentially most significant caries predictors in 3-year-olds were debris index >1 (OR 6.324, P<0.001), breastfeeding duration (OR 1.017, P=0.001), lack of a personal dentist (OR 2.454, P=0.012), and poor dental health of the mother (OR 10.521, P<0.001). The multivariate model confirmed that these variables are the potentially most significant caries predictors in 3-year-olds. CONCLUSIONS This 1-year study showed that the most significant early caries predictors in 3-year-olds are debris index, breastfeeding length, lack of a personal dentist, and poor dental health of mothers. The tested salivary parameters did not show caries-predictive significance.

Anti-virulence and anti-efflux pump activity of synthetic defensins and histatin in Candida auris.

Siham Shaban, Mrudula Patel, Aijaz Ahmad

Novel antifungal therapies are needed to combat multidrug-resistant Candida auris, a pathogen with significant virulence and efflux pump activity. Building on our previous work demonstrating the fungicidal activity of selected antimicrobial peptides (AMPs) towards C. auris, this study investigates their effects against virulence factors and efflux pump. We evaluated the impact of human β-Defensin-3 (hBD-3), Human neutrophil peptide-1 (HNP-1) and human salivary histatin 5 (His 5) on C. auris virulence and resistance, using two clinical isolates. Their effect on adhesion and proteinase activity was studied using adherence assay and BSA plates, respectively. Their impact on biofilm formation and mature biofilm was studied using the MTT reduction test and further visualized using Confocal laser scanning microscope. Intracellular accumulation and external efflux of rhodamine-6-G was studied to establish their action on efflux pump activity. In addition, their effect on the expression of related genes was studied using RT-qPCR. At sub-minimum inhibitory concentrations (sub-MICs) and minimum inhibitory concentrations (MICs), all three test AMPs significantly reduced both adherence and proteinase activity. All the test peptides substantially inhibited the metabolic activity and decreased the density of growing and mature biofilms. Moreover, the tested peptides effectively obstructed efflux pumps and downregulated the genes linked to virulence and efflux pumps, including CDR1, CDR2, MDR1, SAP3, SNQ2, PGA26, PGA7 and PGA52. Finally, the safety of these peptides was verified by a haemolytic test. The finding of this study demonstrated the anti-virulence and anti-efflux pump properties of the tested AMPs. Therefore, these peptides can be a potentially effective alternative for managing infections caused by C. auris.

Anti-β2GPI/β2GPI complex promotes thrombosis by activating the P2Y2/MAPKs pathway to increase human neutrophil peptides.

Xin Guan, Wen Liu, Tianfeng Gao +5 more

Anti-β2 glycoprotein I (Anti-β2GPI) antibodies are a heterogeneous group of antiphospholipid antibodies targeting β2 glycoprotein I (β2GPI). High titer of anti-β2GPI antibodies is a risk factor for thrombosis in antiphospholipid syndrome (APS). Although it has been shown that anti-β2GPI antibodies can induce neutrophil activation involved in thrombosis, the underlying mechanism remains unclear. In this study, we analyzed the clinical data of thrombotic patients who were positive or negative for anti-β2GPI antibodies, as well as healthy individuals. The results showed that the percentage and absolute count of neutrophils, serum levels of human neutrophil peptides (HNPs), and HNP mRNA levels were significantly higher in the anti-β2GPI-positive patient group compared to the healthy control group. Notably, when compared to the anti-β2GPI-negative patient group with similar neutrophil percentages and counts, the serum HNPs levels were also significantly elevated in the anti-β2GPI-positive patient group. In vitro, we further showed that anti-β2GPI and β2GPⅠ complex (anti-β2GPI/ β2GPⅠ complex) induced a concentration - and time-dependent increase in HNPs, which was mediated through P2Y2 receptors on the surface of neutrophils. Meanwhile, we found that intracellular signaling pathways P38MAPK (P38 mitogen-activated protein kinase) and ERK (extracellular signal-regulated kinase) were also involved in the generation of HNPs. We also found that high levels of human neutrophil peptide-1 (HNP-1) could induce the production of procoagulant factors von Willebrand factor (vWF) and P-selectin in endothelial cells through the nuclear factor-κB (NF-κB) signaling pathway, which increased the risk of thrombosis.

Detection of chimeric alpha-defensin transcripts and peptides in mouse Paneth cells.

Steven Timmermans, Charlotte Wallaeys, Somara De Beul +2 more

In mammals, Paneth cells, located in the crypts of the small intestine, produceantimicrobial peptides that serve to keep the intestinal microbiome under control. a-Defensins are the primary antimicrobial peptides produced by these cells.

AhR Activation Transcriptionally Induces Anti-Microbial Peptide Alpha-Defensin 1 Leading to Reversal of Gut Microbiota Dysbiosis and Colitis.

Manikandan Palrasu, Khadija Kakar, Amarnath Marudamuthu +9 more

Alpha-defensin 1 is a small antimicrobial peptide that acts as the first line of defense against pathogens. It is induced following microbial cues and inflammatory signals in neutrophils and Paneth cells in the small intestine, which suggests that it plays a role in microbial homeostasis in the gut. The gut microbial products also serve as ligands for the aryl hydrocarbon receptor (AhR), an environmental sensor. In the current study, we investigated if there is any crosstalk between AhR and alpha-defensin 1. Interestingly, we found a positive correlation between AhR and alpha-defensin 1 protein levels in ileal tissues from active Crohn's' (CD) patients and epithelial cells (IECs) from multiple models of murine colitis. In vitro downregulation of AhR led to inhibition of α-defensin 1, while activation of AhR induced α-defensin 1 in IECs. AhR directly targeted the dioxin response element 3 (DRE3) region on the α-defensin 1 promoter in IECs. AhR-mediated induction of α-defensin 1 in colitis mice reversed the gut microbial dysbiosis and alleviated colitis. Our data identify a novel signaling pathway in which AhR acts as a transcription factor for α-defensin 1, leading to regulation of homeostasis between gut microbiota, intestinal mucosa, and mucosal immunity.

Tcf4 regulates secretory cell fate decisions in the small intestine and colon tumors: insights from transcriptomic, histological, and microbiome analyses.

Lucie Janeckova, Monika Stastna, Dusan Hrckulak +16 more

The canonical Wnt signaling pathway controls the continuous renewal of the intestinal epithelium and the specification of epithelial cell lineages. Tcf4, a nuclear mediator of Wnt signaling, is essential for the differentiation and maintenance of Paneth cells in the small intestine. Its deficiency is associated with reduced expression of key α-defensins, highlighting its role in host-microbe interactions. However, the exact function of Tcf4 in specifying the secretory lineage and its contribution to antimicrobial peptide production remain incompletely understood. Remarkably, α-defensin expression has also been detected in human colon adenomas, where aberrant Wnt signaling is a hallmark. This raises important questions: What is the role of these Paneth-like cells in tumor biology, and how does Tcf4 influence their identity and function?

Synovial fluid alpha-defensins in Lyme arthritis-a useful marker.

Pavel Melicherčík, Matěj Mazura, Martin Hodík +7 more

Lyme arthritis, one of the possible late manifestations of Lyme borreliosis, predominantly affects the supporting joints and in adults most often occurs in the form of monoarthritis of the knee. Early diagnosis is based on clinical findings and serology. PCR detection of Borrelia in synovial fluid has become an integral part of the laboratory testing algorithm. The clinical presentation and inflammatory markers in Lyme arthritis can resemble septic arthritis. Determining the levels of alpha-defensins (human neutrophil peptide (HNP 1-3)) in synovial fluid by liquid chromatography is a highly sensitive method revealing the presence of inflammatory process. Between 2020 and 2022, we examined eleven patients with Lyme arthritis of the knee. We measured levels of HNP 1-3 from synovial fluid by HPLC in patients, and we compared it with the corresponding C-reactive protein (CRP) levels in paired serum samples. In patients diagnosed with Lyme arthritis, HNP 1-3 levels in synovial fluid ranged from 2.5 to 261 mg/L, with a median of 46.5 mg/L. Average serum CRP was 43 mg/L. The results show that elevated HNP 1-3 can be consistent with not only septic arthritis or systemic disease, but also with Lyme arthritis, especially in patients with negative culture and 16S PCR from synovial fluid. Final diagnosis must be verified by examination for anti-Borrelia antibodies from serum and synovial fluid. The aim of this work is to introduce an HPLC method for the determination of alpha-defensins as one of the possible diagnostic markers.

Supplementation of Vitamin D3 and Fructooligosaccharides Downregulates Intestinal Defensins and Reduces the Species Abundance of Romboutsia ilealis in C57BL/6J Mice.

Tyler Hanson, Ethan Constantine, Zack Nobles +4 more

The activation of the vitamin D receptor (VDR) in the ileum has been shown to regulate Paneth cell-specific defensins, a large family of antimicrobial peptides; hence, this may serve as a potential mechanism to maintain intestinal homeostasis. Previously, we have demonstrated that a combination of vitamin D3 (VD) and fructooligosaccharides (FOSs) upregulates colonic Vdr in mice. Here, we aim to examine the effect of VD, alone or in combination with FOSs, on intestinal barrier integrity and the secretion of antimicrobial peptides, as well as the gut microbial community. Male and female C57BL/6J mice at 6 weeks old were randomized into three groups to receive the following dietary regimens (n = 10/sex/group) for 8 weeks: (1) standard AIN-93G control diet (CTR), (2) CTR + 5000 IU vitamin D3 (VD), and (3) VD + 5% fructooligosaccharides (VF). VD and VF differentially regulated the mRNA expressions of tight junction proteins in the colon and ileum. VF suppressed the upregulation of colonic ZO-1 and occludin, which was induced by VD supplementation alone. In the ileum, occludin but not ZO-1 was upregulated 20-fold in the VF-treated mice. While VD did not alter the mRNA expressions of Vdr and defensins in the ileum, these targets were downregulated by VF. Microbial analysis further reveals a shift of microbial beta diversity and a reduction in Romboutsia ilealis, a pathobiont, in VF-treated mice. Though the implications of these phenotypical and microbial changes remain to be determined, the administration of FOSs in the presence of VD may serve as an effective dietary intervention for maintaining intestinal homeostasis.

A paper-based aptamer-sandwich assay for detection of HNP 1 as a biomarker for periprosthetic joint infections on an integrated microfluidic platform.

Rishabh Gandotra, Feng-Chih Kuo, Mel S Lee +1 more

Total joint arthroplasty (TJA) has significantly improved the quality of life for millions suffering from end-stage arthritis. However, periprosthetic joint infections (PJI) remain a serious complication, necessitating extensive interventions and prolonged antimicrobial treatments. The aging population is expected to lead to a rise in TJA cases, subsequently increasing the incidence of PJI, particularly in the elderly who face higher mortality rates. Current diagnostic methods for suspected PJI, such as radiographs and biochemical markers like CRP and ESR, exhibit limited sensitivity. Therefore, there is a critical need for a specific synovial fluid biomarker assay to enhance PJI diagnosis using specific SF-based assay.

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

Siham Shaban, Mrudula Patel, Aijaz Ahmad

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 β-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.

Establishment of Baseline Urinary Antimicrobial Peptide Levels by Age: A Prospective Observational Study.

Jeffrey M Caterino, Julie A Stephens, Randell Wexler +7 more

Antimicrobial peptides (AMPs) are key effectors of urinary tract innate immunity. Identifying differences in urinary AMP levels between younger and older adults is important in understanding older adults' susceptibility and response to urinary tract infections (UTI) and AMP use as diagnostic biomarkers. We hypothesized that uninfected older adults have higher urinary human neutrophil peptides 1-3 (HNP 1-3), human alpha-defensin-5 (HD-5), and human beta-defensin-2 (hBD-2), but lower urinary cathelicidin (LL-37) than younger adults.

Advancing the Diagnosis of Periprosthetic Joint Infections: Integrated Microfluidic Platform for Alpha-Defensins-Specific Aptamer Selection and Its Analytical Applications.

Rishabh Gandotra, Hung-Bin Wu, Feng-Chih Kuo +2 more

Periprosthetic joint infections (PJIs) pose a significant challenge in orthopedic surgery, particularly total joint arthroplasty (TJA), due to the potential for implant failure and increased patient morbidity. Early and accurate detection of PJIs is crucial for timely intervention and better patient prognosis. Herein, we successfully screened a high-affinity aptamer targeting alpha-defensin complex human neutrophil protein 1-3 (HNP 1-3; potential PJI biomarkers in synovial fluid [SF]) for the first time using systematic evolution of ligands by exponential enrichment (SELEX) on an integrated microfluidic platform. The compact microfluidic device enabled efficient screening, with each round completed within <2 h, comprising five rounds of positive selection, two rounds of negative selection, and one round of competitive selection. A novel one-aptamer-one-antibody assay was further developed from the optimal aptamer screened, and it could accurately quantify HNP 1-3 in SF within 3 h with only ∼50 μL of SF. The assay demonstrated strong binding affinity and specificity for the target protein in SF. Thirteen PJI SF samples were accurately diagnosed and the assay was accurate over a wide dynamic range (0.32-100 mg/L). This study has showcased a rapid and accurate diagnostic tool for PJI detection, which should see widespread use in the clinic, holding promise for potential analytical applications in orthopedic surgery and improving patient care.

In vitro activity of human defensins HNP-1 and hBD-3 against multidrug-resistant ESKAPE Gram-negatives of clinical origin and selected peptidoglycan recycling-defective mutants.

María Escobar-Salom, Isabel María Barceló, Estrella Rojo-Molinero +4 more

The use of immune compounds as antimicrobial adjuvants is a classic idea recovering timeliness in the current antibiotic resistance scenario. However, the activity of certain antimicrobial peptides against ESKAPE Gram-negatives has not been sufficiently investigated. The objective of this study was to determine the activities of human defensins HNP-1 and hBD-3 alone or combined with permeabilizing/peptidoglycan-targeting agents against clinical ESKAPE Gram-negatives [Acinetobacter baumannii (AB), Enterobacter cloacae (EC), Klebsiella pneumoniae (KP), and acute/chronic Pseudomonas aeruginosa (PA)]. Lethal concentrations (LCs) of HNP-1 and hBD-3 were determined in four collections of multidrug resistant EC, AB, KP, and PA clinical strains (10-36 isolates depending on the collection). These defensins act through membrane permeabilization plus peptidoglycan building blockade, enabling that alterations in peptidoglycan recycling may increase their activity, which is why different recycling-defective mutants were also included. Combinations with physiological lysozyme and subinhibitory colistin for bactericidal activities determination, and with meropenem for minimum inhibitory concentrations (MICs), were also assessed. HNP-1 showed undetectable activity (LC > 32 mg/L for all strains). hBD-3 showed appreciable activities: LC ranges 2-16, 8-8, 8->32, and 8->32 mg/L for AB, EC, KP, and PA, being PA strains from cystic fibrosis significantly more resistant than acute origin ones. None of the peptidoglycan recycling-defective mutants showed greater susceptibility to HNP-1/hBD-3. Combination with colistin or lysozyme did not change their bactericidal power, and virtually neither did meropenem + hBD-3 compared to meropenem MICs. This is the first study comparatively analyzing the HNP-1/hBD-3 activities against the ESKAPE Gram-negatives, and demonstrates interesting bactericidal capacities of hBD-3 mostly against AB and EC.

Effects of exposure to the neonicotinoid pesticide clothianidin on α-defensin secretion and gut microbiota in mice.

Sakura Yonoichi, Yukako Hara, Yuya Ishida +14 more

The mechanism by which the neonicotinoid pesticide clothianidin (CLO) disrupts the intestinal microbiota of experimental animals is unknown. We focused on α-defensins, which are regulators of the intestinal microbiota. Subchronic exposure to CLO induced dysbiosis and reduced short-chain fatty acid-producing bacteria in the intestinal microbiota of mice. Levels of cryptdin-1 (Crp1, a major α-defensin in mice) in feces and cecal contents were lower in the CLO-exposed groups than in control. In Crp1 immunostaining, Paneth cells in the jejunum and ileum of the no-observed-adverse-effect-level CLO-exposed group showed a stronger positive signal than control, likely due to the suppression of Crp1 release. Our results showed that CLO exposure suppresses α-defensin secretion from Paneth cells as part of the mechanism underlying CLO-induced dysbiosis.

Enteric α-Defensin Contributes to Recovery of Radiation-Induced Intestinal Injury by Modulating Gut Microbiota and Fecal Metabolites.

Jie Wu, Xi Ran, Tao Wang +5 more

The effect of ionizing radiation on the gastrointestinal tract is a common complication of abdominal and pelvic radiotherapy. However, the pathological features of radiation enteropathy and its effective medical intervention regimen is still a global challenge. Here, we explored the role and mechanism of enteric alpha-defensins (EαDs) in protecting against radiation enteropathy. To address this, we utilized EαDs-deficiency mice, in which the matrix metallopeptidase 7 to activate Paneth cell α-defensins was knockout (KO) mice, and the complementary wild-type (WT) control mice for this study. Remarkably, the KO mice were more susceptible to 5.0 Gy total-body irradiation, resulting in worse clinic scores and lower survival rate, compared with the wild-type mice. Histological examination indicated that the KO mice were subjected to slow recovery of intestinal villus and mucosa function, characterized by the reduced expression of TFF3, Glut1 and Muc2. In addition, compared with the wild-type controls, the KO mice experienced serious inflammation response in intestinal tissue, indicated by the remarkably increased expression level of IL-1β, IL-6 and IL-12. Using high-throughput sequencing analysis, we found that the intestinal bacterial community of the KO mice was more prone to dysbiosis than that of the WT mice, with significantly increased abundance of opportunistic pathogenic bacteria, such as Streptococcus sp. and Escherichia-Shigella sp., whereas remarkably decreased probiotics harboring Lactobacillus sp., Desulfovibrio sp. etc. Fecal metabolomics analysis indicated that the relative abundance of 31 metabolites arose significantly different between WT and KO mice on day 10 after radiation exposure. A subset of differential metabolites to regulate host metabolism and immunity, such as acetic acid, acetate, butanoic acid, was negatively correlated with the alteration of gut microbiota in the irradiated KO mice. This study provides new insight into EαDs contribution to the recovery of radiation-induced intestinal damage, and suggests a potential novel target to prevent the adverse effects of radiotherapy.

Regulation of dermal fibroblasts by human neutrophil peptides.

Nattarika Niwetbowornchai, Thanawat Chaisirirat, Sira Sriswasdi +6 more

Human neutrophil peptides (HNPs) can induce cell proliferation and activation so their growth promoting activities may have potential clinical benefit. This study investigated the effects of HNPs on human dermal fibroblasts. Differential gene expression in HNP-treated cells and genes involved in regulating intracellular pathways were explored. Dermal fibroblasts were isolated from healthy neonatal foreskin and treated with HNPs in 2D and 3D cell culture systems. The expression of cell proliferation (Ki-67) gene and cell activation (COL1A1) gene plus their proteins was measured. Differential gene expression was determined using RNA-seq, and upregulated and downregulated genes were mapped onto intracellular pathways by KEGG analysis and Gene Ontology databases. HNPs significantly increased cell proliferation without cytotoxicity whilst HNP1 enhanced expression of COL1A1 and type I collagen production in 2D cells and 3D spheroids. RNA-sequencing analysis showed gene clustering with clear separation between HNP1-treated and control groups. A heatmap of top 50 differentially expressed genes was consistent among HNP1-treated samples. Most upregulated genes were associated with cell proliferation and activation as mapped into intracellular pathways whilst most downregulated genes belonged to steroid/arachidonic acid metabolism and inflammatory signaling pathways. HNP1 increased cell proliferation and activation but reduced lipid metabolism and inflammation.

Diagnostic Value of Synovial Fluid Biomarkers for Periprosthetic Joint Infection: A Prospective, Double-blind Trial.

Ying Xu, Xueting Ma, Haoran Guo +4 more

BACKGROUND This prospective, double-blind study investigated the clinical diagnostic value of synovial fluid S100 calcium-binding protein A8 (S100A8) and S100 calcium-binding protein A9 (S100A9) in periprosthetic joint infection (PJI) and investigated the subtypes of a-defensin that have diagnostic value for PJI. MATERIAL AND METHODS Synovial fluid samples were collected from 82 patients with suspected PJI after total joint arthroplasty. Patients were divided into a PJI group (n=39) and non-PJI group (n=43). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to determine S100A8, S100A9, alpha-defensin, and internal reference standards in synovial fluid. The receiver operating characteristic (ROC) curve was used to analyze the diagnostic efficiency of S100A8, S100A9, and alpha-defensin for PJI, as well as the diagnostic value in combination with common biomarkers of infection. RESULTS S100A8, 3 variants of S100A9, and 3 alpha-defensins (human neutrophil peptides [HNP]1-3) in synovial fluid were significantly higher in the PJI group than in the non-PJI group (P<0.001). The sensitivity, specificity, and the area under ROC curve (AUC) for diagnosing PJI were 97.4%, 86.0%, and 0.964 (95% CI: 0.929-0.998), respectively, for synovial fluid S100A8; 87.2%, 88.4% and 0.902 (95% CI: 0.823-0.980), respectively, for S100A9; and 89.7%, 83.7%, and 0.933 (95% CI: 0.884-0.982), respectively, for HNP1-3. The diagnostic efficiency was improved when combined with synovial fluid white blood cell count and percentage of polymorphonuclear neutrophils. CONCLUSIONS Synovial fluid S100A8, S100A9, and HNP1-3 have satisfactory diagnostic efficiency for the diagnosis of PJI, which will help clinicians to accurately diagnose PJI.

Paneth cell dysfunction in radiation injury and radio-mitigation by human α-defensin 5.

Pradeep K Shukla, Roshan G Rao, Avtar S Meena +10 more

The mechanism underlying radiation-induced gut microbiota dysbiosis is undefined. This study examined the effect of radiation on the intestinal Paneth cell α-defensin expression and its impact on microbiota composition and mucosal tissue injury and evaluated the radio-mitigative effect of human α-defensin 5 (HD5).

Evaluation of Rouxiella badensis Subsp Acadiensis (Canan SV-53) as a Potential Probiotic Bacterium.

Ivanna Novotny-Nuñez, Gabriela Perdigón, Chantal Matar +4 more

The advent of omic platforms revealed the significant benefits of probiotics in the prevention of many infectious diseases. This led to a growing interest in novel strains of probiotics endowed with health characteristics related to microbiome and immune modulation. Therefore, autochthonous bacteria in plant ecosystems might offer a good source for novel next-generation probiotics. The main objective of this study was to analyze the effect of Rouxiella badensis acadiensis Canan (R. acadiensis) a bacterium isolated from the blueberry biota, on the mammalian intestinal ecosystem and its potential as a probiotic microorganism. R. acadiensis, reinforced the intestinal epithelial barrier avoiding bacterial translocation from the gut to deep tissues, even after feeding BALB/c mice for a prolonged period of time. Moreover, diet supplementation with R. acadiensis led to increases in the number of Paneth cells, well as an increase in the antimicrobial peptide α defensin. The anti-bacterial effect of R. acadiensis against Staphylococcus aureus and Salmonella enterica serovar Typhimurium was also reported. Importantly, R. acadiensis-fed animals showed better survival in an in vivo Salmonella enterica serovar Typhimurium challenge compared with those that received a conventional diet. These results demonstrated that R. acadiensis possesses characteristics of a probiotic strain by contributing to the reinforcement and maintenance of intestinal homeostasis.

An aptamer-based sandwich assay for detection of alpha-defensin human neutrophil protein 1 on a microfluidic platform.

Rishabh Gandotra, To-Wen Chen, Feng-Chih Kuo +2 more

The diagnosis of periprosthetic joint infection (PJI) remains a labor-intensive and challenging issue, with life-threatening complications associated with misdiagnoses. Superior diagnostic approaches are therefore urgently needed, and synovial biomarkers are gaining substantial attention in this capacity. A new aptamer-based sandwich assay was developed where the aptamer probes specific to one such biomarker, alpha-defensin human neutrophil protein 1 (HNP 1), was integrated herein into a new microfluidic platform. The magnetic beads coated with the primary aptamer probe were able to bind the target protein with high affinity and high specificity in synovial fluid and a fluorescent-labelled secondary aptamer were further used to quantify HNP 1 in a sandwich approach. Up to four clinical samples with low volume (∼50 μL each) in a much faster assay including detection within <60 min with 100% accuracy (with totally 13 clinical samples without the need of sample pretreatment) through the use of the aptamer-based sandwich assay were automatically detected on a single chip. The wide dynamic range of this compact device, 0.5-100 mg/L, highlights its utility for future PJI diagnostics in the clinic.

Shorter sleep time relates to lower human defensin 5 secretion and compositional disturbance of the intestinal microbiota accompanied by decreased short-chain fatty acid production.

Yu Shimizu, Ryodai Yamamura, Yuki Yokoi +8 more

Sleep is essential for our health. Short sleep is known to increase disease risks via imbalance of intestinal microbiota, dysbiosis. However, mechanisms by which short sleep induces dysbiosis remain unknown. Small intestinal Paneth cell regulates the intestinal microbiota by secreting antimicrobial peptides including α-defensin, human defensin 5 (HD5). Disruption of circadian rhythm mediating sleep-wake cycle induces Paneth cell failure. We aim to clarify effects of short sleep on HD5 secretion and the intestinal microbiota. Fecal samples and self-reported sleep time were obtained from 35 healthy middle-aged Japanese (41 to 60-year-old). Shorter sleep time was associated with lower fecal HD5 concentration (r = 0.354, p = 0.037), lower centered log ratio (CLR)-transformed abundance of short-chain fatty acid (SCFA) producers in the intestinal microbiota such as [Ruminococcus] gnavus group (r = 0.504, p = 0.002) and Butyricicoccus (r = 0.484, p = 0.003), and lower fecal SCFA concentration. Furthermore, fecal HD5 positively correlated with the abundance of these genera and SCFA concentration. These findings suggest that short sleep relates to disturbance of the intestinal microbiota via decreased HD5 secretion.

Decreased Paneth cell α-defensins promote fibrosis in a choline-deficient L-amino acid-defined high-fat diet-induced mouse model of nonalcoholic steatohepatitis via disrupting intestinal microbiota.

Shunta Nakamura, Kiminori Nakamura, Yuki Yokoi +10 more

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by fibrosis that develops from fatty liver. Disruption of intestinal microbiota homeostasis, dysbiosis, is associated with fibrosis development in NASH. An antimicrobial peptide α-defensin secreted by Paneth cells in the small intestine is known to regulate composition of the intestinal microbiota. However, involvement of α-defensin in NASH remains unknown. Here, we show that in diet-induced NASH model mice, decrease of fecal α-defensin along with dysbiosis occurs before NASH onset. When α-defensin levels in the intestinal lumen are restored by intravenous administration of R-Spondin1 to induce Paneth cell regeneration or by oral administration of α-defensins, liver fibrosis is ameliorated with dissolving dysbiosis. Furthermore, R-Spondin1 and α-defensin improved liver pathologies together with different features in the intestinal microbiota. These results indicate that decreased α-defensin secretion induces liver fibrosis through dysbiosis, further suggesting Paneth cell α-defensin as a potential therapeutic target for NASH.

Host Cationic Antimicrobial Molecules Inhibit S. aureus Exotoxin Production.

Patrick M Schlievert, Samuel H Kilgore, Lisa A Beck +3 more

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 β-defensin human β-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 β-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.