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Background and aims: Ulcerative colitis (UC) is an acute and chronic inflammatory disease of the large bowel with unknown aetiology. The immune response against normal commensal microorganisms is believed to drive inflammatory processes associated with UC. Therefore, modulation of bacterial communities on the gut mucosa, through the use of probiotics and prebiotics, may be used to modify the disease state. Methods: A synbiotic was developed for use in UC patients combining a probiotic, Bifidobacterium longum, isolated from healthy rectal epithelium, and a prebiotic (Synergy 1), a preferential inulin-oligofructose growth substrate for the probiotic strain. Treatment was employed in a double blinded randomised controlled trial using 18 patients with active UC for a period of one month. Clinical status was scored and rectal biopsies were collected before and after treatment, and transcription levels of epithelium related immune markers were measured. Results: Sigmoidoscopy scores (scale 0–6) were reduced in the test group (start 4.5 (1.4), end 3.1 (2.5)) compared with placebo (start 2.6 (2.1), end 3.2 (2.2)) (p = 0.06). mRNA levels for human beta defensins 2, 3, and 4, which are strongly upregulated in active UC, were significantly reduced in the test group after treatment (p = 0.016, 0.038, and 0.008, respectively). Tumour necrosis factor α and interleukin 1α, which are inflammatory cytokines that drive inflammation and induce defensin expression, were also significantly reduced after treatment (p = 0.018 and 0.023, respectively). Biopsies in the test group had reduced inflammation and regeneration of epithelial tissue. Conclusions: Short term synbiotic treatment of active UC resulted in improvement of the full clinical appearance of chronic inflammation in patients receiving this therapy.

Throughout life, there is an aging of the immune system that causes impairment of its defense capability. Prevention or delay of this deterioration is considered crucial to maintain general health and increase longevity. We evaluated whether dietary supplementation with Lactobacillus delbrueckii subsp. bulgaricus 8481 could enhance the immune response in the elderly. This multi-center, double-blind, and placebo controlled study enrolled 61 elderly volunteers who were randomly assigned to receive either placebo or probiotics. Each capsule of probiotics contained at least 3 × 10 7   L. delbrueckii subsp. bulgaricus 8481. Individuals in the study were administered three capsules per day for 6 months. Blood samples were obtained at baseline (time 0), end of month 3, and month 6. We characterized cell subpopulations, measured cytokines by flow cytometry, quantified T cell receptor excision circle (TREC) by real-time PCR (RT-PCR), and determined human β-defensin-2 (hBD-2) concentrations and human cytomegalovirus (CMV) titers by enzyme-linked immunosorbent assay (ELISA). Elderly responded to the intake of probiotic with an increase in the percentage of NK cells, an improvement in the parameters defining the immune risk profile (IRP), and an increase in the T cell subsets that are less differentiated. The probiotic group also showed decreased concentrations of the pro-inflammatory cytokine IL-8 but increased antimicrobial peptide hBD-2. These effects disappeared within 6 months of stopping the probiotic intake. Immunomodulation induced by L. delbrueckii subsp. bulgaricus 8481 could favor the maintenance of an adequate immune response, mainly by slowing the aging of the T cell subpopulations and increasing the number of immature T cells which are potential responders to new antigens.

The antimicrobial peptides (AMP) produced by intestinal epithelial cells (IEC) play crucial roles in the regulation of intestinal homeostasis by controlling microbiota. Gut microbiota has been shown to promote IEC expression of RegIIIγ and certain defensins. However, the mechanisms involved are still not completely understood. In this report, we found that IEC expression levels of RegIIIγ and β-defensins 1, 3, and 4 were lower in G protein-coupled receptor (GPR)43−/− mice compared to that of wild-type (WT) mice. Oral feeding with short-chain fatty acids (SCFA) promoted IEC production of RegIIIγ and defensins in mice. Furthermore, SCFA induced RegIIIγ and β-defensins in intestinal epithelial enteroids generated from WT but not GPR43−/− mice. Mechanistically, SCFA activated mTOR and STAT3 in IEC, and knockdown of mTOR and STAT3 impaired SCFA induction of AMP production. Our studies thus demonstrated that microbiota metabolites SCFA promoted IEC RegIIIγ and β-defensins in a GPR43-dependent manner. The data thereby provide a novel pathway by which microbiota regulates IEC expression of AMP and intestinal homeostasis.

Failure to predict and understand the causes of preterm birth, the leading cause of neonatal morbidity and mortality, have limited effective interventions and therapeutics. From a cohort of 2000 pregnant women, we performed a nested case control study on 107 well-phenotyped cases of spontaneous preterm birth (sPTB) and 432 women delivering at term. Using innovative Bayesian modeling of cervicovaginal microbiota, seven bacterial taxa were significantly associated with increased risk of sPTB, with a stronger effect in African American women. However, higher vaginal levels of β-defensin-2 lowered the risk of sPTB associated with cervicovaginal microbiota in an ethnicity-dependent manner. Surprisingly, even in Lactobacillus spp. dominated cervicovaginal microbiota, low β-defensin-2 was associated with increased risk of sPTB. These findings hold promise for diagnostics to accurately identify women at risk for sPTB early in pregnancy. Therapeutic strategies could include immune modulators and microbiome-based therapeutics to reduce this significant health burden. Here, Elovitz et al. investigate associations between cervicovaginal microbiota (CVM) and spontaneous preterm birth (sPTB) in a large cohort of African American and non-African American women, and find that CVM and local immune response early in pregnancy are associated with sPTB in an ethnicity-dependent manner.

Although vitamin D deficiency is a common feature among patients presenting with active tuberculosis, the full scope of vitamin D action during Mycobacterium tuberculosis (Mtb) infection is poorly understood. As macrophages are the primary site of Mtb infection and are sites of vitamin D signaling, we have used these cells to understand the molecular mechanisms underlying modulation of the immune response by the hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D). We found that the virulent Mtb strain H37Rv elicits a broad host transcriptional response. Transcriptome profiling also revealed that the profile of target genes regulated by 1,25D is substantially altered by infection, and that 1,25D generally boosts infection-stimulated cytokine/chemokine responses. We further focused on the role of 1,25D- and infection-induced interleukin 1β (IL-1β) expression in response to infection. 1,25D enhanced IL-1β expression via a direct transcriptional mechanism. Secretion of IL-1β from infected cells required the NLRP3/caspase-1 inflammasome. The impact of IL-1β production was investigated in a novel model wherein infected macrophages were co-cultured with primary human small airway epithelial cells. Co-culture significantly prolonged survival of infected macrophages, and 1,25D/infection-induced IL-1β secretion from macrophages reduced mycobacterial burden by stimulating the anti-mycobacterial capacity of co-cultured lung epithelial cells. These effects were independent of 1,25D-stimulated autophagy in macrophages but dependent upon epithelial IL1R1 signaling and IL-1β-driven epithelial production of the antimicrobial peptide DEFB4/HBD2. These data provide evidence that the anti-microbial actions of vitamin D extend beyond the macrophage by modulating paracrine signaling, reinforcing its role in innate immune regulation in humans.

Rainbow trout β-defensin 3 ( rt Defb3) is identified as a small cationic antimicrobial peptide, but its recombinant expression and functional characterization have not been reported. In this study, the mature rt Defb3 Komagataella phaffii coding sequence was cloned from trout liver cDNA and heterologously expressed in GS115 using the pPIC9K with an α-factor secretion signal. Expression condition was optimized by adjusting methanol concentration (0.5–1.25%), induction temperature (26–30 °C), and induction duration (24–168 h). Optimized induction with 1.0% methanol at 30 ℃ for 96 h produced ~ 7 mg/mL of secreted peptide, which was purified to > 90% purity using one-step Ni-IDA affinity chromatography. The identity and purity of the recombinant rt Defb3 were confirmed by Tricine-SDS-PAGE, HPLC (~ 97% purity), and MALDI-TOF mass spectrometry. Functional assays revealed potent broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. These findings demonstrate, for the first time, the successful production of active rt Defb3 in Komagataella phaffii , establishing a scalable expression platform for fish β-defensins. The recombinant rt Defb3 peptide holds promise as a natural antimicrobial agent for aquaculture and potential therapeutic applications, addressing the urgent demand for alternatives to conventional antibiotics. Key points • First successful recombinant production of rainbow trout β-defensin 3 in Komagataella phaffii • via optimized induction strategy. • High-yield secretory expression (~ 7 mg/mL) with > 90% purity confirmed by HPLC and biochemical analysis. • Recombinant rtDefb3 displayed potent broad-spectrum antibacterial activity, offering a repeatable and scalable system and functional validation. Graphical Abstract Heterologous expression of rainbow trout β-defensin 3 ( rt Defb3) in Komagataella phaffii enabled efficient secretion, one-step purification, and functional validation. The recombinant peptide displayed broad-spectrum antimicrobial activity, highlighting its potential as a natural alternative to antibiotics in aquaculture and biomedical applications.

Bovine male fertility in animals has a direct impact on the productivity of dairy herds. The epididymal sperm maturations involve extensive sperm surface modifications to gain the fertilizing ability, especially by absorptions of the plethora of biomolecules, including glycoprotein beta-defensins (BDs), enzymes, organic ions, protein, and phospholipids. Defensins are broad-range nonspecific antimicrobial peptides that exhibit strong relations with innate and adaptive immunity, but their roles in male fertility are relatively recently identified. In the course of evolution, BD genes give rise to different clusters with specific functions, especially reproductive functions, by undergoing duplications and nonsynonymous mutations. BD polymorphisms have been reported with milk compositions, disease resistance, and antimicrobial activities. However, in recent decades, the link of BD polymorphisms with fertility has emerged as an appealing improvement of reproductive performance such as sperm motility, membrane integrity, cervical mucus penetration, evading of uterus immunosurveillance, oviduct cell attachment, and egg recognition. The reproductive-specific glycosylated BD class-A BDs (CA-BDs) have shown age- and sex-specific expressions in male reproductive organs, signifying their physiological pleiotropism, especially in the sperm maturation and sperm transport in the female reproductive tract. By considering adult male reproductive organ-specific BD expressions, importance in sperm functionalities, and bioinformatic analysis, we have selected two bovine BBD126 and BBD129 genes as novel potential biomarkers of bovine male fertility. Despite the importance of BDs, however, genomic characterization of most BD genes across most livestock and nonmodel organisms remains predictive/incomplete. The current review discusses our understanding of BD pleiotropic functions, polymorphism, and genomic structural attributes concerning the fertilizability of the male gamete in dairy animals. Graphical Abstract (A) Sperm epididymis maturation and antimicrobial activity—BDs are secreted by epithelial cells of epididymis which facilitate or prepare sperm for fertilizable and protect sperm from reproductive microbial infection. (B) Epididymis maturation for immune protection of male MRT and FRT—BDs are rich in glycosylation which provide a thick coat on outer plasma membrane masking important proteins/antigens from immune recognition of MRT and FRT. (C) Epididymis sperm motility—BDs interact with sperm plasma membrane ion channels and regulate calcium ion which help in sperm motility. (D) FRT sperm-cervical mucus penetration—the negative charge of BDs help sperm to pass cervical mucus by creating a repulsion force between negatively charged mucin protein of cervix. (E) Cervix-uterine immunomodulator and immune-protectant—BDs mask sperm surface antigen which help sperm to evade FRT immune surveillance. (F) FRT sperm-oviductal epithelial cells binding (sperm reservoir formation)—the carbohydrates present at the end terminal of glycosylated BDs help sperm to interact with oviduct epithelial cells lead to sperm reservoir formation. (G) FRT sperm capacitation and unshielding of hidden proteins—the removal of BDs from sperm surface lead to change in sperm membrane and capacitation. (H) egg–sperm interactions—unshielding of BDs leads to unmasking of hidden protein that is very essential for the egg recognition and penetration.

Type I interferons (IFN-α and IFN-β) are important for protection against many viral infections, whereas type II interferon (IFN-γ) is essential for host defense against some bacterial and parasitic pathogens. Study of IFN responses in human leprosy revealed an inverse correlation between IFN-β and IFN-γ gene expression programs. IFN-γ and its downstream vitamin D—dependent antimicrobial genes were preferentially expressed in self-healing tuberculoid lesions and mediated antimicrobial activity against the pathogen Mycobacterium leprae in vitro. In contrast, IFN-β and its downstream genes, including interleukin-10 (IL-10), were induced in monocytes by M. leprae in vitro and preferentially expressed in disseminated and progressive lepromatous lesions. The IFN-γ—induced macrophage vitamin D—dependent antimicrobial peptide response was inhibited by IFN-β and by IL-10, suggesting that the differential production of IFNs contributes to protection versus pathogenesis in some human bacterial infections.

The last decade led to the discovery and characterization of several human beta-defensins. Analysis of genomic information indicates that the number of beta-defensin-like molecules encoded by the human genome may number in the tens. Growing interest in beta-defensins steadily enhances our knowledge about various aspects of their gene location, expression patterns and the transcription factors involved in their regulation in vivo. The hallmark property of beta-defensins, their antimicrobial activity, is clearly only the tip of the iceberg in the extensive network of inter-relations within the immune system in which these peptides function. Structural studies of beta-defensins provide the molecular basis for a better understanding of their properties, functions and their potential for practical applications. In this review, we present some recent advances in the studies of human beta-defensins, with an emphasis on possible correlations between their structural and functional properties.

Biliary atresia (BA) is a neonatal cholangiopathy that often progresses to cirrhosis despite timely Kasai portoenterostomy (KPE), and prognostic biomarkers remain undefined. Given its role in adult cholestasis, we evaluated human beta-defensin-1 (hBD1) in murine and human BA for associations with disease progression and outcome. This study analyzed hepatic expression of hBD1 and TGF-ß by qRT-PCR in BA at KPE ( n  = 36) and liver transplantation (LT, n  = 44), and compared with normal ( n  = 15) and cholestatic disease controls ( n  = 36). Serum hBD1 was measured by ELISA in BA ( n  = 23) and healthy infants ( n  = 11). Murine BD1 was assessed in a Rhesus rotavirus (RRV) BA model. BD1 was found to be upregulated in murine and human BA, with higher expression at LT than at KPE. Hepatic hBD1 correlated with TGF-ß (R 2 = 0.21), Ishak fibrosis score (R 2 = 0.36), and serum bile acids (R 2 = 0.23). Serum hBD1 was elevated in BA versus controls. Elevated hBD1 at KPE predicted persistent jaundice and reduced native liver survival (X 2 = 9.5), with ROC analysis showing good discrimination for failure of jaundice clearance at 3 months post-KPE (AUC 0.81 for liver and 0.87 for serum). Thus, hBD1 may serve as a negative predictor of jaundice clearance and native liver survival at the time of KPE.