Antimicrobial activity-specific to Gram-negative bacteria and immune modulation-mediated NF-kappa B and Sp1 of a medaka beta-defensin

Defensins are a group of cationic antimicrobial peptides which play an important role in the innate immune system by exerting their antimicrobial activity against pathogens. In this study, we cloned a novel beta-defensin cDNA from medaka (Oryzias latipes) by rapid amplification of cDNA ends (RACE) technique. The full-length cDNA consists of 480 bp, and the open reading frame (CRF) of 189 bp encodes a polypeptide of 63 amino acids (aa) with a predicted molecular weight of 7.44 kDa. Its genomic organization was analyzed, and Southern blot detection confirmed that only one copy of beta-defensin exists in the medaka HNI strain. RT-PCR, Western blot and immunohistochemistry detections showed that the beta-defensin transcript and protein could be detected in eyes, liver, kidney, blood, spleen and gill, and obviously prevalent expression was found in eyes. Antimicrobial activity of the medaka beta-defensin was evaluated, and the antibacterial activity-specific to Gram-negative bacteria was revealed. Furthermore, the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, was demonstrated to be able to induce about 13-fol up-regulation of the beta-defensin within first 12 h. In addition, promoter and promoter mutagenesis analysis were performed in the medaka beta-defensin. A proximal 100 base pair(bp) sequence (+26 to -73)and the next 1700 bp sequence (-73 to -1755) were demonstrated to be responsible for the basal promoter activity and for the transcription regulation. Three nuclear factor kappa B (NF-kappa B) cis-elements and a Sp1 cis-element were revealed by mutagenesis analysis to exist in the 5' flanking sequence, and they were confirmed to be responsible for the up-regulation of medaka beta-defensin stimulated by LPS. And, the Sp1 cis-element was further revealed to be related to the basal promoter activity, and transcriptional factor II D (TFIID) was found to be in charge of the gene transcription initiation. All the obtained data suggested that the novel medaka beta-defensin should have antimicrobial activity-specific to Gram-negative bacteria, and the antibacterial immune function should be modulated by NF-kappa B and Sp1. (C) 2008 Elsevier Ltd. All rights reserved.

The DUB/USP17 deubiquitinating enzymes: A gene family within a tandemly repeated sequence, is also embedded within the copy number variable Beta-defensin cluste

Background: The DUB/USP17 subfamily of deubiquitinating enzymes were originally identified as immediate early genes induced in response to cytokine stimulation in mice (DUB-1, DUB-1A, DUB-2, DUB-2A). Subsequently we have identified a number of human family members and shown that one of these (DUB-3) is also cytokine inducible. We originally showed that constitutive expression of DUB-3 can block cell proliferation and more recently we have demonstrated that this is due to its regulation of the ubiquitination and activity of the 'CAAX' box protease RCE1.

Results: Here we demonstrate that the human DUB/USP17 family members are found on both chromosome 4p16.1, within a block of tandem repeats, and on chromosome 8p23.1, embedded within the copy number variable betadefensin cluster. In addition, we show that the multiple genes observed in humans and other distantly related mammals have arisen due to the independent expansion of an ancestral sequence within each species. However, it is also apparent when sequences from humans and the more closely related chimpanzee are compared, that duplication events have taken place prior to these species separating.

Conclusions: The observation that the DUB/USP17 genes, which can influence cell growth and survival, have evolved from an unstable ancestral sequence which has undergone multiple and varied duplications in the species examined marks this as a unique family. In addition, their presence within the beta-defensin repeat raises the question whether they may contribute to the influence of this repeat on immune related conditions.

Green tea extract and its major constituent, epigallocatechin-3-gallate, induce epithelial beta-defensin secretion and prevent beta-defensin degradation by Porphyromonas gingivalis

Background and Objective: Antimicrobial peptides, such as beta-defensins, secreted by gingival epithelial cells, are thought to play a major role in preventing periodontal diseases. In the present study, we investigated the ability of green tea polyphenols to induce human beta-defensin (hBD) secretion in gingival epithelial cells and to protect hBDs from proteolytic degradation by Porphyromonas gingivalis.Material and Methods: Gingival epithelial cells were treated with various amounts (25-200 mu g/mL) of green tea extract or epigallocatechin-3-gallate (EGCG). The secretion of hBD1 and hBD2 was measured using ELISAs, and gene expression was quantified by real-time PCR. The treatments were also carried out in the presence of specific kinase inhibitors to identify the signaling pathways involved in hBD secretion. The ability of green tea extract and EGCG to prevent hBD degradation by proteases of P. gingivalis present in a bacterial culture supernatant was evaluated by ELISA.Results: The secretion of hBD1 and hBD2 was up-regulated, in a dose-dependent manner, following the stimulation of gingival epithelial cells with a green tea extract or EGCG. Expression of the hBD gene in gingival epithelial cells treated with green tea polyphenols was also increased. EGCG-induced secretion of hBD1 and hBD2 appeared to involve extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase. Lastly, green tea extract and EGCG prevented the degradation of recombinant hBD1 and hBD2 by a culture supernatant of P. gingivalis.Conclusion: Green tea extract and EGCG, through their ability to induce hBD secretion by epithelial cells and to protect hBDs from proteolytic degradation by P. gingivalis, have the potential to strengthen the epithelial antimicrobial barrier. Future clinical studies will indicate whether these polyphenols represent a valuable therapeutic agent for treating/preventing periodontal diseases.

Synergistic Anti-Inflammatory Activity of the Antimicrobial Peptides Human Beta-Defensin-3 (hBD-3) and Cathelicidin (LL-37) in a Three-Dimensional Co-Culture Model of Gingival Epithelial Cells and Fibroblasts

Given the spread of antibiotic resistance in bacterial pathogens, antimicrobial peptides that can also modulate the immune response may be a novel approach for effectively controlling periodontal infections. In the present study, we used a three-dimensional (3D) co-culture model of gingival epithelial cells and fibroblasts stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) to investigate the anti-inflammatory properties of human beta-defensin-3 (hBD-3) and cathelicidin (LL-37) and to determine whether these antimicrobial peptides can act in synergy. The 3D co-culture model composed of gingival fibroblasts embedded in a collagen matrix overlaid with gingival epithelial cells had a synergistic effect with respect to the secretion of IL-6 and IL-8 in response to LPS stimulation compared to fibroblasts and epithelial cells alone. The 3D co-culture model was stimulated with non-cytotoxic concentrations of hBD-3 (10 and 20 mu M) and LL-37 (0.1 and 0.2 mu M) individually and in combination in the presence of A. actinomycetemcomitans LPS. A multiplex ELISA assay was used to quantify the secretion of 41 different cytokines. hBD-3 and LL-37 acted in synergy to reduce the secretion of GRO-alpha, G-CSF, IP-10, IL-6, and MCP-1, but only had an additive effect on reducing the secretion of IL-8 in response to A. actinomycetemcomitans LPS stimulation. The present study showed that hBD-3 acted in synergy with LL-37 to reduce the secretion of cytokines by an LPS-stimulated 3D model of gingival mucosa. This combination of antimicrobial peptides thus shows promising potential as an adjunctive therapy for treating inflammatory periodontitis.

Human beta-defensin 2 and protease activated receptor-2 expression in patients with chronic periodontitis

Objective: Some previous studies have shown that gingipains, trypsin-like proteases produced by Porphyromonas gingivalis, up-regulate human beta defensin-2 (HBD-2) mRNA expression through protease-activated receptor-2 (PAR(2)) in gingival epithelial cells. This study aimed at investigating salivary HBD-2 levels and crevicular PAR(2) mRNA expression in human chronic periodontitis and evaluating whether periodontal treatment affected this process. Methods: Salivary and gingival crevicular fluid (GCF) samples were collected from periodontally healthy (control) and chronic periodontitis patients at baseline and 50 days after nonsurgical periodontal treatment. Salivary HBD-2, and GCF TNF-alpha levels were analysed by ELISA, and PAR(2) mRNA at the GCF was evaluated by RT-PCR. Results: P. gingivalis was significantly (p < 0.05) more prevalent in patients with chronic periodontitis when compared to controls. This prevalence decreased after periodontal therapy (p < 0.0001). The control group showed statistically significant lower levels of HBD-2, TNF-alpha, and PAR(2) expression when compared to the chronic periodontitis group. In addition, periodontal treatment significantly reduced PAR(2) expression and HBD-2 levels in chronic periodontitis patients (p < 0.001). Conclusions: Our results suggest that salivary HBD-2 levels and PAR(2) mRNA expression from GCF are higher in subjects with chronic periodontitis than in healthy subjects, and that periodontal treatment decreases both HBD-2 levels and PAR(2) expression. (C) 2012 Elsevier Ltd. All rights reserved.

Human beta-defensin-2 and psoriasin are overexpressed in lesions of acne inversa

BACKGROUND: Acne inversa is a chronic inflammatory disorder of apocrine gland-bearing skin. The role of the innate immune system in the pathogenesis of the disease is controversial. OBJECTIVES: We investigated the expression of antimicrobial peptide/proteins in acne inversa. METHODS: Tissue samples were obtained from patients with acne inversa and compared with normal-appearing skin. The expression of psoriasin and human beta-defensin (hBD)-2 on messenger RNA and protein level was analyzed. RESULTS: Both messenger RNA and protein levels of psoriasin and hBD-2 were significantly increased in acne inversa. Macrophages expressing hBD-2 were found in the dermis. LIMITATIONS: Small sample size is a limitation. CONCLUSIONS: Antimicrobial peptide/proteins are overexpressed in acne inversa lesions as compared with normal-appearing skin. The site of the major expression depends on the particular antimicrobial peptide/protein. Psoriasin is overexpressed in epidermal keratinocytes whereas hBD-2 is produced mainly by dermal macrophages, leaving a relative deficiency of hBD-2 in the epidermis of acne inversa lesions.

Comparison of multiplex ligation-dependent probe amplification and real-time PCR accuracy for gene copy number quantification using the beta-defensin locus

The reliable quantification of gene copy number variations is a precondition for future investigations regarding their functional relevance. To date, there is no generally accepted gold standard method for copy number quantification, and methods in current use have given inconsistent results in selected cohorts. In this study, we compare two methods for copy number quantification. beta-defensin gene copy numbers were determined in parallel in 80 genomic DNA samples by real-time PCR and multiplex ligation-dependent probe amplification (MLPA). The pyrosequencing-based paralog ratio test (PPRT) was used as a standard of comparison in 79 out of 80 samples. Realtime PCR and MPLA results confirmed concordant DEFB4, DEFB103A, and DEFB104A copy numbers within samples. These two methods showed identical results in 32 out of 80 samples; 29 of these 32 samples comprised four or fewer copies. The coefficient of variation of MLPA is lower compared with PCR. In addition, the consistency between MLPA and PPRT is higher than either PCR/MLPA or PCR/PPRT consistency. In summary, these results suggest that MLPA is superior to real-time PCR in beta-defensin copy number quantification.

Copy number variation of the Beta-defensin genes in europeans: no supporting evidence for association with lung function, chronic obstructive pulmonary disease or asthma

Lung function measures are heritable, predict mortality and are relevant in diagnosis of chronic obstructive pulmonary disease (COPD). COPD and asthma are diseases of the airways with major public health impacts and each have a heritable component. Genome-wide association studies of SNPs have revealed novel genetic associations with both diseases but only account for a small proportion of the heritability. Complex copy number variation may account for some of the missing heritability. A well-characterised genomic region of complex copy number variation contains beta-defensin genes (DEFB103, DEFB104 and DEFB4), which have a role in the innate immune response. Previous studies have implicated these and related genes as being associated with asthma or COPD. We hypothesised that copy number variation of these genes may play a role in lung function in the general population and in COPD and asthma risk. We undertook copy number typing of this locus in 1149 adult and 689 children using a paralogue ratio test and investigated association with COPD, asthma and lung function. Replication of findings was assessed in a larger independent sample of COPD cases and smoking controls. We found evidence for an association of beta-defensin copy number with COPD in the adult cohort (OR = 1.4, 95%CI:1.02-1.92, P = 0.039) but this finding, and findings from a previous study, were not replicated in a larger follow-up sample(OR = 0.89, 95%CI:0.72-1.07, P = 0.217). No robust evidence of association with asthma in children was observed. We found no evidence for association between beta-defensin copy number and lung function in the general populations. Our findings suggest that previous reports of association of beta-defensin copy number with COPD should be viewed with caution. Suboptimal measurement of copy number can lead to spurious associations. Further beta-defensin copy number measurement in larger sample sizes of COPD cases and children with asthma are needed.

8p23 beta-defensin copy number determination by single-locus pseudogene-based paralog ratio tests risk bias due to low-frequency sequence variations.

BACKGROUND The copy number variation (CNV) in beta-defensin genes (DEFB) on human chromosome 8p23 has been proposed to contribute to the phenotypic differences in inflammatory diseases. However, determination of exact DEFB CN is a major challenge in association studies. Quantitative real-time PCR (qPCR), paralog ratio tests (PRT) and multiplex ligation-dependent probe amplification (MLPA) have been extensively used to determine DEFB CN in different laboratories, but inter-method inconsistencies were observed frequently. In this study we asked which one is superior among the three methods for DEFB CN determination. RESULTS We developed a clustering approach for MLPA and PRT to statistically correlate data from a single experiment. Then we compared qPCR, a newly designed PRT and MLPA for DEFB CN determination in 285 DNA samples. We found MLPA had the best convergence and clustering results of the raw data and the highest call rate. In addition, the concordance rates between MLPA or PRT and qPCR (32.12% and 37.99%, respectively) were unacceptably low with underestimated CN by qPCR. Concordance rate between MLPA and PRT (90.52%) was high but PRT systematically underestimated CN by one in a subset of samples. In these samples a sequence variant which caused complete PCR dropout of the respective DEFB cluster copies was found in one primer binding site of one of the targeted paralogous pseudogenes. CONCLUSION MLPA is superior to PRT and even more to qPCR for DEFB CN determination. Although the applied PRT provides in most cases reliable results, such a test is particularly sensitive to low-frequency sequence variations preferably accumulating in loci like pseudogenes which are most likely not under selective pressure. In the light of the superior performance of multiplex assays, the drawbacks of such single PRTs could be overcome by combining more test markers.

Inactivation of Human beta-Defensins 2 and 3 by Elastolytic Cathepsins

beta-Defensins are antimicrobial peptides that contribute to the innate immune responses of eukaryotes. At least three defensins, human beta-defensins 1, 2, and 3 (HBD-1, -2, and -3), are produced by epithelial cells lining the respiratory tract and are active toward Gram-positive (HBD-3) and Gram-negative (HBD-1, -2, and -3) bacteria. It has been postulated that the antimicrobial activity of defensins is compromised by changes in airway surface liquid composition in lungs of patients with cystic fibrosis (CF), therefore contributing to the bacterial colonization of the lung by Pseudomonas and other bacteria in CF. In this report we demonstrate that HBD-2 and HBD-3 are susceptible to degradation and inactivation by the cysteine proteases cathepsins B, L, and S. In addition, we show that all three cathepsins are present and active in CF bronchoalveolar lavage. Incubation of HBD-2 and -3 with CF bronchoalveolar lavage leads to their degradation, which can be completely (HBD-2) or partially (HBD-3) inhibited by a cathepsin inhibitor. These results suggest that beta-defensins are susceptible to degradation and inactivation by host proteases, which may be important in the regulation of beta-defensin activity. In chronic lung diseases associated with infection, overexpression of cathepsins may lead to increased degradation of HBD-2 and -3, thereby favoring bacterial infection and colonization.

Sequence analysis of a 212 kb defensin gene cluster on ECA 27q17

Defensins are a family of evolutionary ancient antimicrobial peptides consisting of three sub-families: alpha-, beta- and theta-defensins. This investigation was focused on the genomic characterization of equine beta-defensins and the investigation of the potential clustering of beta-defensin genes in the equine genome. Six genomic BAC clones were isolated from the CHORI-241 library and one of these was mapped by FISH to ECA 27q17. This location was confirmed by RH-mapping. The contiguous 212 kb sequence of this clone was determined. Sequence analysis revealed the identification of ten pseudogenes and nine genes, six of which were highly homologous to human beta-defensin DEFB4. Clustering of the beta-defensin genes was confirmed and the order of the genes on the analyzed BAC was related to the corresponding defensin cluster on HSA 8. The knowledge about the sequence and the genomic structure of the equine beta-defensin genes will improve the classification of different paralogous defensin genes and is a prerequisite for subsequent functional studies. Additionally, the first alpha-defensin-like sequence outside the groups of primates, lagomorphs and rodents (glires) was identified.