Expanding the insect defensin landscape.

In this issue of Structure, Walker et al.1 determined the NMR structure of a recently discovered defensin, Pp19, from the venom of an assassin bug. This peptide adopts an α-defensin-like structure, which had not been observed in insects before. Unlike mammalian α-defensins, which are generally antimicrobial, Pp19 has insecticidal activity.

A Novel Dimeric Short Peptide Derived from α-Defensin-Related Rattusin with Improved Antimicrobial and DNA-Binding Activities.

Rattusin, an α-defensin-related antimicrobial peptide isolated from the small intestine of rats, has been previously characterized through NMR spectroscopy to elucidate its three-dimensional structure, revealing a C2 homodimeric scaffold stabilized by five disulfide bonds. This study aimed to identify the functional region of rattusin by designing and synthesizing various short analogs, subsequently leading to the development of novel peptide-based antibiotics. The analogs, designated as F1, F2, F3, and F4, were constructed based on the three-dimensional configuration of rattusin, among which F2 is the shortest peptide and exhibited superior antimicrobial efficacy compared to the wild-type peptide. The central cysteine residue of F2 prompted an investigation into its potential to form a dimer at neutral pH, which is critical for its antimicrobial function. This activity was abolished upon the substitution of the cysteine residue with serine, indicating the necessity of dimerization for antimicrobial action. Further, we synthesized ß-hairpin-like analogs, both parallel and antiparallel, based on the dimeric structure of F2, which maintained comparable antimicrobial potency. In contrast to rattusin, which acts by disrupting bacterial membranes, the F2 dimer binds directly to DNA, as evidenced by fluorescence assays and DNA retardation experiments. Importantly, F2 exhibited negligible cytotoxicity up to 515 µg/mL, assessed via hemolysis and MTT assays, underscoring its potential as a lead compound for novel peptide-based antibiotic development.

Alpha-defensin binding expands human adenovirus tropism.

Mammalian α-defensins are a family of abundant effector peptides of the mucosal innate immune system. Although primarily considered to be antimicrobial, α-defensins can increase rather than block infection by certain prominent bacterial and viral pathogens in cell culture and in vivo. We have shown previously that exposure of mouse and human adenoviruses (HAdVs) to α-defensins is able to overcome competitive inhibitors that block cell binding, leading us to hypothesize a defensin-mediated binding mechanism that is independent of known viral receptors. To test this hypothesis, we used genetic approaches to demonstrate that none of several primary receptors nor integrin co-receptors are needed for human α-defensin-mediated binding of HAdV to cells; however, infection remains integrin dependent. Thus, our studies have revealed a novel pathway for HAdV binding to cells that bypasses viral primary receptors. We speculate that this pathway functions in parallel with receptor-mediated entry and contributes to α-defensin-enhanced infection of susceptible cells. Remarkably, we also found that in the presence of α-defensins, HAdV tropism is expanded to non-susceptible cells, even when viruses are exposed to a mixture of both susceptible and non-susceptible cells. Therefore, we propose that in the presence of sufficient concentrations of α-defensins, such as in the lung or gut, integrin expression rather than primary receptor expression will dictate HAdV tropism in vivo. In summary, α-defensins may contribute to tissue tropism not only through the neutralization of susceptible viruses but also by allowing certain defensin-resistant viruses to bind to cells independently of previously described mechanisms.

Characterisation of defensins across the marsupial family tree.

Defensins are antimicrobial peptides involved in innate immunity, and gene number differs amongst eutherian mammals. Few studies have investigated defensins in marsupials, despite their potential involvement in immunological protection of altricial young. Here we use recently sequenced marsupial genomes and transcriptomes to annotate defensins in nine species across the marsupial family tree. We characterised 35 alpha and 286 beta defensins; gene number differed between species, although Dasyuromorphs had the largest repertoire. Defensins were encoded in three gene clusters within the genome, syntenic to eutherians, and were expressed in the pouch and mammary gland. Marsupial beta defensins were closely related to eutherians, however marsupial alpha defensins were more divergent. We identified marsupial orthologs of human DEFB3 and 6, and several marsupial-specific beta defensin lineages which may have novel functions. Marsupial predicted mature peptides were highly variable in length and sequence composition. We propose candidate peptides for future testing to elucidate the function of marsupial defensins.

Reduced expression of central innate defense molecules in pancreatic biopsies from subjects with Type 1 diabetes.

AIMS/HYPOTHESIS: Defensins play a crucial role in the innate immune system's first defense against microbial threats. However, little is known about the defensin system in the pancreas, especially in relation to Type 1 diabetes. We explore the expression of defensins in different disease stages of Type 1 diabetes and correlated obtained findings to the degree of inflammation, providing new insights into the disease and the innate immune system. MATERIAL AND METHODS: Pancreases from non-diabetic human organ donors of different age groups and donors with Type 1 diabetes with different disease duration were examined. Sections from head, body and tail of the pancreas were stained for eight different defensins and for immune cells; CD3+, CD45+, CD68+ and NES+ (granulocytes). RESULTS: In non-diabetic adult controls the level of expression for defensins Beta-1,Alpha-1, Cathelicidin and REG3A correlated with the level of inflammation. In contrast, individuals with Type  1 diabetes exhibit a reduction or absence of several central defensins regardless of the level of inflammation in their pancreas. The expression of Cathelicidin is present in neutrophils and macrophages but not in T-cells in subjects with Type 1 diabetes. CONCLUSIONS: Obtained findings suggest a pancreatic dysfunction in the innate immune system and the bridging to the adaptive system in Type 1 diabetes. Further studies on the role of the local innate immune system in Type 1 diabetes is needed.

The James A. Rand Young Investigator's Award: Keeping It Simple: Are all Musculoskeletal Infection Society Tests Useful to Diagnose Periprosthetic Joint Infection?

BACKGROUND: Current data evaluating the clinical value and cost-effectiveness of advanced diagnostic tests for periprosthetic joint infection (PJI) diagnosis, including alpha-defensin and synovial C-reactive protein (CRP), is conflicting. This study aimed to evaluate the adequacy of preoperative and intraoperative PJI workups without utilizing these tests. METHODS: This retrospective analysis identified all patients who underwent revision total knee or hip arthroplasty (rTKA and rTHA, respectively) for suspected PJI between 2018 and 2020 and had a minimum follow-up of 2 years. Perioperative data and lab results were collected, and cases were dichotomized based on whether they met the 2018 Musculoskeletal Infection Society (MSIS) criteria for PJI. In total, 204 rTKA and 158 rTHA cases suspected of PJI were reviewed. RESULTS: Nearly 100% of the cases were categorized as "infected" for meeting the 2018 MSIS criteria without utilization of alpha-defensin or synovial CRP (rTKA: n = 193, 94.6%; rTHA: n = 156, 98.7%). Most cases were classified as PJI preoperatively by meeting either the major MSIS or the combinational minor MSIS criteria of traditional lab tests (rTKA: n = 177, 86.8%; rTHA: n = 143, 90.5%). A subset of cases was classified as PJI by meeting combinational preoperative and intraoperative MSIS criteria (rTKA: 16, 7.8%; rTHA: 13, 8.2%). Only 3.6% of all cases were considered "inconclusive" using preoperative and intraoperative data. CONCLUSIONS: Given the high rate of cases satisfying PJI criteria during preoperative workup using our available tests, the synovial alpha-defensin and synovial CRP tests may not be necessary in the routine diagnostic workup of PJI. We suggest that the primary PJI workup process should be based on a stepwise algorithmic approach with the most economical testing necessary to determine a diagnosis first. The use of advanced, commercialized, and costly biomarkers should be utilized only when traditional testing is indeterminate.

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.

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

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.

DEFA1A3 DNA gene-dosage regulates the kidney innate immune response during upper urinary tract infection.

Antimicrobial peptides (AMPs) are host defense effectors with potent neutralizing and immunomodulatory functions against invasive pathogens. The AMPs α-Defensin 1-3/DEFA1A3 participate in innate immune responses and influence patient outcomes in various diseases. DNA copy-number variations in DEFA1A3 have been associated with severity and outcomes in infectious diseases including urinary tract infections (UTIs). Specifically, children with lower DNA copy numbers were more susceptible to UTIs. The mechanism of action by which α-Defensin 1-3/DEFA1A3 copy-number variations lead to UTI susceptibility remains to be explored. In this study, we use a previously characterized transgenic knock-in of the human DEFA1A3 gene mouse to dissect α-Defensin 1-3 gene dose-dependent antimicrobial and immunomodulatory roles during uropathogenic Escherichia coli (UPEC) UTI. We elucidate the relationship between kidney neutrophil- and collecting duct intercalated cell-derived α-Defensin 1-3/DEFA1A3 expression and UTI. We further describe cooperative effects between α-Defensin 1-3 and other AMPs that potentiate the neutralizing activity against UPEC. Cumulatively, we demonstrate that DEFA1A3 directly protects against UPEC meanwhile impacting pro-inflammatory innate immune responses in a gene dosage-dependent manner.

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.

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. IMPORTANCE: In the current scenario of critical need for new antimicrobials against multidrug-resistant bacteria, all options must be considered, including classic ideas such as the use of purified immune compounds. However, information regarding the activity of certain human defensins against ESKAPE Gram-negatives was incomplete. This is the first study comparatively assessing the in vitro activity of two membrane-permeabilizing/peptidoglycan construction-blocking defensins (HNP-1 and hBD-3) against relevant clinical collections of ESKAPE Gram-negatives, alone or in combination with permeabilizers, additional peptidoglycan-targeting attacks, or the blockade of its recycling. Our data suggest that hBD-3 has a notable bactericidal activity against multidrug-resistant Acinetobacter baumannii and Enterobacter cloacae strains that should be considered as potential adjuvant option. Our results suggest for the first time an increased resistance of Pseudomonas aeruginosa strains from chronic infection compared to acute origin ones, and provide new clues about the predominant mode of action of hBD-3 against Gram-negatives (permeabilization rather than peptidoglycan-targeting).

Antimicrobial peptides and other potential biomarkers of critical illness in SARS-CoV-2 patients with acute kidney injury. AMPAKI-CoV study.

Antimicrobial peptides (AMPs) constitute a complex network of 10-100 amino acid sequence molecules widely distributed in nature. While over 300 AMPs have been described in mammals, cathelicidins and defensins remain the most extensively studied. Some publications have explored the role of AMPs in COVID-19, but these findings are preliminary, and in vivo studies are still lacking. In this study, we report the plasma levels of five AMPs (LL-37, α-defensin 1, α-defensin 3, ß-defensin 1, and ß-defensin 3), using the ELISA technique (MyBioSource, San Diego, CA, United States, kits MBS2601339 (beta-defensin 1), MBS2602513 (beta-defensin 3), MBS703879 (alpha-defensin 1), MBS706289 (alpha-defensin 3), MBS7234921 (LL37)), and the measurement of six cytokines (tumor necrosis factor-α, interleukin-1ß, interleukin-6, interleukin-10, interferon-γ, and monocyte chemoattractant protein-1), through the magnetic bead immunoassay Milliplex® and the MAGPIX® System (MilliporeSigma, Darmstadt, Germany, kit HCYTOMAG-60 K (cytokines)), in 15 healthy volunteers, 36 COVID-19 patients without Acute Kidney Injury (AKI) and 17 COVID-19 patients with AKI. We found increased levels of α-defensin 1, α-defensin 3 and ß-defensin 3, in our COVID-19 population, when compared to healthy controls, along with higher levels of interleukin-6, interleukin-10, interferon-γ, and monocyte chemoattractant protein-1. These findings suggest that these AMPs and cytokines may play a crucial role in the systemic inflammatory response and tissue damage characterizing severe COVID-19. The levels of α-defensin 1 and α-defensin 3 were significantly higher in COVID-19 AKI group in comparison to the non-AKI group. Furthermore, IL-10 and the product IL-10 × IL-1B showed excellent performance in discriminating AKI, with AUCs of 0.86 and 0.88, respectively. Among patients with COVID-19, AMPs may play a key role in the inflammation process and disease progression. Additionally, α-defensin 1 and α-defensin 3 may mediate the AKI process in these patients, representing an opportunity for further research and potential therapeutic alternatives in the future.

Ameliorative Effects of Zingiber officinale Rosc on Antibiotic-Associated Diarrhea and Improvement in Intestinal Function.

The global increase in antibiotic consumption is related to increased adverse effects, such as antibiotic-associated diarrhea (AAD). This study investigated the chemical properties of Zingiber officinale Rosc (ZO) extract and its ameliorative effects using a lincomycin-induced AAD mouse model. Intestinal tissues were evaluated for the expression of lysozyme, claudin-1, and α-defensin-1, which are associated with intestinal homeostasis. The cecum was analyzed to assess the concentration of short-chain fatty acids (SCFAs). The chemical properties analysis of ZO extracts revealed the levels of total neutral sugars, acidic sugars, proteins, and polyphenols to be 86.4%, 8.8%, 4.0%, and 0.8%, respectively. Furthermore, the monosaccharide composition of ZO was determined to include glucose (97.3%) and galactose (2.7%). ZO extract administration ameliorated the impact of AAD and associated weight loss, and water intake also returned to normal. Moreover, treatment with ZO extract restored the expression levels of lysozyme, α-defensin-1, and claudin-1 to normal levels. The decreased SCFA levels due to induced AAD showed a return to normal levels. The results indicate that ZO extract improved AAD, strengthened the intestinal barrier, and normalized SCFA levels, showing that ZO extract possesses intestinal-function strengthening effects.

Regulation of Chloride Channels by Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor-Induced α-Defensin 5.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are used to treat non-small cell lung cancer with EGFR mutations. However, first-generation erlotinib and second-generation afatinib often cause diarrhea, which may develop because of the association between EGFR-TKIs and the chloride channel or abnormalities in the intestinal microbiota due to disruption of the intestinal immune system. As reports on the effects of EGFR-TKIs on intestinal immunity are lacking, we aimed to determine whether the intestinal immune system is involved in the molecular effects of EGFR-TKIs on chloride channels using Caco-2 cells. Initially, we evaluated the association of chloride channels with α-defensin 5 (DEFA5), a marker of intestinal immunity. Erlotinib and afatinib significantly increased the extracellularly secreted DEFA5 level and autophagy-related 16-like 1 and X-box binding protein 1 transcript levels, indicative of enhanced granule exocytosis. Conversely, intracellular DEFA5 and Toll-like receptor 4 protein expression and tumor necrosis factor-α transcript levels decreased significantly, suggesting that Toll-like receptor 4 suppression repressed DEFA5 production. Furthermore, among the chloride channels, DEFA5 was found to significantly increase the transcript levels of cystic fibrosis transmembrane conductance regulators. These results indicate that DEFA5 plays a significant role in the mechanism of chloride channel-mediated diarrhea induced by EGFR-TKIs. Therefore, we successfully elucidated the potential host action of DEFA5 in cancer therapy for the first time.

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

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.

Are Synovial Inflammatory Markers Increased in Patients Who Have Aseptic Total Hip Arthroplasty Dislocation Indicated for Revision?

BACKGROUND: Previous studies have speculated on elevated synovial inflammatory markers in patients undergoing surgical revision for total hip arthroplasty (THA) dislocation. However, this assumption is based on small patient series and a full investigation according to International Consensus Meeting (ICM) criteria has not yet been performed. METHODS: Patients who had aseptic THA dislocation indicated for revision surgery were identified retrospectively. Only patients who had available diagnostic workup according to ICM 2018 criteria, including preoperative and intraoperative parameters, were included. For comparison, we analyzed a matched cohort of patients indicated for aseptic THA revision for other conditions. The 2 cohorts each consisted of 55 patients and were not different regarding age, sex, BMI, or implant fixation. RESULTS: There was no difference in synovial white blood cell count (2,238 ± 2,544 versus 2,533 ± 3,448 c/µL; P = .601), alpha-defensin quotient (0.14 ± 0.11 versus 0.19 ± 0.28; P = .207), or polymorphonuclear neutrophil percentage (% PMN) (36.7 ± 22.6 versus 31.3 ± 24.5%; P = .312) between the groups. In the dislocation cohort, 20% of patients had a synovial white blood cell count of 3,000 c/µL or higher, compared with 18% in the control cohort. However, all patients in the dislocation cohort were below the cutoff for alpha-defensin or % PMN. CONCLUSION: In patients who have aseptic THA dislocation, synovial inflammatory markers are not elevated compared with patients undergoing aseptic revision for other complications. A detailed preoperative analysis of synovial inflammatory markers using ICM criteria appears critical in patients who have a THA dislocation to exclude periprosthetic joint infection. LEVEL OF EVIDENCE: Level III, retrospective, comparative study.

A top-down proteomic approach reveals a salivary protein profile able to classify Parkinson's disease with respect to Alzheimer's disease patients and to healthy controls.

Parkinson's disease (PD) is a complex neurodegenerative disease with motor and non-motor symptoms. Diagnosis is complicated by lack of reliable biomarkers. To individuate peptides and/or proteins with diagnostic potential for early diagnosis, severity and discrimination from similar pathologies, the salivary proteome in 36 PD patients was investigated in comparison with 36 healthy controls (HC) and 35 Alzheimer's disease (AD) patients. A top-down platform based on HPLC-ESI-IT-MS allowed characterizing and quantifying intact peptides, small proteins and their PTMs (overall 51). The three groups showed significantly different protein profiles, PD showed the highest levels of cystatin SA and antileukoproteinase and the lowest of cystatin SN and some statherin proteoforms. HC exhibited the lowest abundance of thymosin ß4, short S100A9, cystatin A, and dimeric cystatin B. AD patients showed the highest abundance of α-defensins and short oxidized S100A9. Moreover, different proteoforms of the same protein, as S-cysteinylated and S-glutathionylated cystatin B, showed opposite trends in the two pathological groups. Statherin, cystatins SA and SN classified accurately PD from HC and AD subjects. α-defensins, histatin 1, oxidized S100A9, and P-B fragments were the best classifying factors between PD and AD patients. Interestingly statherin and thymosin ß4 correlated with defective olfactory functions in PD patients. All these outcomes highlighted implications of specific proteoforms involved in the innate-immune response and inflammation regulation at oral and systemic level, suggesting a possible panel of molecular and clinical markers suitable to recognize subjects affected by PD.

Application of enzyme-linked immunosorbent assay to detect antimicrobial peptides in human intestinal lumen.

Intestinal transplantation is the definitive treatment for intestinal failure. However, tissue rejection and graft-versus-host disease are relatively common complications, necessitating aggressive immunosuppression that can itself pose further complications. Tracking intraluminal markers in ileal effluent from standard ileostomies may present a noninvasive and sensitive way to detect developing pathology within the intestinal graft. This would be an improvement compared to current assessments, which are limited by poor sensitivity and specificity, contributing to under or over-immunosuppression, respectively, and by the need for invasive biopsies. Herein, we report an approach to reproducibly analyze ileal fluid obtained through stoma sampling for antimicrobial peptide/protein concentrations, reasoning that these molecules may provide an assessment of intestinal homeostasis and levels of intestinal inflammation over time. Concentrations of lysozyme (LYZ), myeloperoxidase (MPO), calprotectin (S100A8/A9) and ß-defensin 2 (DEFB2) were assessed using adaptations of commercially available enzyme-linked immunosorbent assays (ELISAs). The concentration of α-defensin 5 (DEFA5) was assessed using a newly developed sandwich ELISA. Our data support that with proper preparation of ileal effluent specimens, precise and replicable determination of antimicrobial peptide/protein concentrations can be achieved for each of these target molecules via ELISA. This approach may prove to be reliable as a clinically useful assessment of intestinal homeostasis over time for patients with ileostomies.

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

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-1ß, 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.

Alpha-defensins inhibit ERK/STAT3 signaling during monocyte-macrophage differentiation and impede macrophage function.

Alpha-1-antitrypsin deficiency (AATD) is a genetic disorder associated with a 5-tenfold decrease in lung levels of alpha-1-antitrypsin (AAT) and an increased risk for obstructive lung disease. α-defensins are cationic broad-spectrum cytotoxic and pro-inflammatory peptides found in the azurophilic granules of neutrophils. The concentration of α-defensins is less than 30 nM in the bronchoalveolar lavage fluid of healthy controls but is up to 6 µM in AATD individuals with significant lung function impairment. Alveolar macrophages are generally classified into pro-inflammatory (M1) or anti-inflammatory (M2) subsets that play distinct roles in the initiation and resolution of inflammation. Therefore, monocyte-macrophage differentiation should be tightly controlled to maintain lung integrity. In this study, we determined the effect of α-defensins on monocyte-macrophage differentiation and identified the molecular mechanism of this effect. The results of this study demonstrate that 2.5 µM of α-defensins inhibit the phosphorylation of ERK1/2 and STAT3 and suppress the expression of M2 macrophage markers, CD163 and CD206. In addition, a scratch assay shows that the high concentration of α-defensins inhibits cell movement by ~ 50%, and the phagocytosis assay using flow cytometry shows that α-defensins significantly reduce the bacterial phagocytosis rate of monocyte-derived macrophages (MDMs). To examine whether exogenous AAT is able to alleviate the inhibitory effect of α-defensins on macrophage function, we incubated MDMs with AAT prior to α-defensin treatment and demonstrate that AAT improves the migratory ability and phagocytic ability of MDMs compared with MDMs incubated only with α-defensins. Taken together, this study suggests that a high concentration of α-defensins inhibits the activation of ERK/STAT3 signaling, negatively regulates the expression of M2 macrophage markers, and impairs innate immune function of macrophages.

FOXO inhibition rescues α-defensin expression in human intestinal organoids.

To mediate critical host-microbe interactions in the human small intestine, Paneth cells constitutively produce abundant levels of α-defensins and other antimicrobials. We report that the expression profile of these antimicrobials is dramatically askew in human small intestinal organoids (enteroids) as compared to that in paired tissue from which they are derived, with a reduction of α-defensins to nearly undetectable levels. Murine enteroids, however, recapitulate the expression profile of Paneth cell α-defensins seen in tissue. WNT/TCF signaling has been found to be instrumental in the regulation of α-defensins, yet in human enteroids exogenous stimulation of WNT signaling appears insufficient to rescue α-defensin expression. By stark contrast, forkhead box O (FOXO) inhibitor AS1842856 induced the expression of α-defensin mRNA in enteroids by >100,000-fold, restoring DEFA5 and DEFA6 to levels comparable to those found in primary human tissue. These results newly identify FOXO signaling as a pathway of biological and potentially therapeutic relevance for the regulation of human Paneth cell α-defensins in health and disease.