Β-defensin Research Articles

Natural antimicrobial production by the amnion

The purpose of this study was to determine the expression of natural antimicrobials in primary cultured amnion epithelial cells and to examine their regulation by interleukin-1 beta (IL-1beta). Primary amnion epithelial cells were cultured from samples that were obtained at prelabor cesarean section (n = 12) and stimulated with IL-1beta. Natural antimicrobial messenger RNA expression was determined by real-time quantitative polymerase chain reaction, and protein was measured by enzyme-linked immunosorbent assay. Data was analyzed by analysis of variance. Primary amnion epithelial cells express messenger RNA for human beta defensin (HBD) 1 to 3, secretory leukocyte protease inhibitor and elafin, but not HBD4. IL-1beta 10 ng/mL stimulates HBD2 messenger RNA in a biphasic pattern, with a 51-fold increase at 6 hours and a 67-fold at 12 hours (P < .001). HBD2 protein production is significantly increased by 24 hours (P < .05). The amnion produces potent natural antimicrobials that may help protect the pregnancy from infection. HBD2 production is dramatically upregulated by the labor-associated inflammatory cytokine IL-1beta.

P803 Different antimicrobial activity of beta defensins and colonic biopsy extracts against aerobic and anaerobic bacterial strains of the gastrointestinal tract

P803 Different antimicrobial activity of beta defensins and colonic biopsy extracts against aerobic and anaerobic bacterial strains of the gastrointestinal tract

Differential Expression of Human Beta Defensin 2 and Human Beta Defensin 3 in Human Middle Ear Cholesteatoma

The purpose of this study was to investigate the differential expressions of human beta defensin (hBD) 2 and hBD-3 in human middle ear cholesteatoma epithelium. The expressions of hBD-2 and hBD-3 were analyzed by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical staining. Samples were obtained from 10 patients who underwent middle ear surgery for middle ear cholesteatoma. Real-time RT-PCR and Western blot analysis showed that the messenger RNAs and proteins of hBD-2 and hBD-3 were higher in the cholesteatoma epithelium than in normal external auditory canal skin. In cholesteatoma epithelium, hBD-2 and hBD-3 activities were present in the upper granular layer and in the prickle cell layer, but in the normal skin they were poorly expressed in all layers. Increased expressions of hBD-2 and hBD-3 in cholesteatoma epithelium suggest that cholesteatoma, a chronic inflammatory state of middle ear keratinocytes, may induce an innate immune response. That the induction of hBD-2 was found to be more intense than that of hBD-3 in cholesteatoma epithelium implies that hBD-2 is the major effector in terms of chronic epithelial inflammatory responses.

Changes in vaginal morphology, steroid receptor and natural antimicrobial content following treatment with low-dose mifepristone

Background We have previously shown that the antigestagen mifepristone is contraceptive when given in a daily dose of 5 mg, po. Epidemiological studies suggest that gestagen-only contraceptives may increase the risk of transmission of human immunodeficiency virus (HIV) due to effects on the vaginal defenses to infection. We investigate the effects of mifepristone on vaginal thickness, steroid receptor and natural antimicrobial content and pharmacokinetics of mifepristone. Methods In a pilot study, eight women were given mifepristone 5 mg/day for an average of 33 days. Ovarian function was assessed by measurement of estradiol and progesterone in blood and their metabolites in urine and by serial ultrasound of their ovaries. Vaginal biopsies were collected before (late proliferative) and after taking mifepristone. Results All subjects showed a similar pattern of descending serum concentrations of mifepristone. The elimination phase half-life was 18±5.1 h (mean±SD). Mean C max measured at 1 h was 641.7 nmol/L (range, 502–740 nmol/L). All eight women reported amenorrhea for the duration of treatment and seven of eight women showed biochemical and ultrasound evidence of anovulation. There was no significant change in vaginal thickness following treatment [342±40 μm pretreatment, 303±69 μm posttreatment (mean±SEM); p>.05]. Estrogen (ERα, ERβ) and androgen receptor were expressed in both vaginal epithelium and subepithelial stroma, whereas progesterone receptor was expressed predominantly in the subepithelial stroma. There was no change in receptor content and distribution following mifepristone treatment. Natural antimicrobial mRNA [secretory leukocyte protease inhibitor, human beta defensins mRNA (HBD1, HBD2, HBD3, HBD5), granulysin and elafin] was extracted from the vaginal tissues, and the content was unaffected by mifepristone treatment. Conclusion The absence of changes in vaginal thickness, steroid receptor and natural antimicrobial content and its distribution in this preliminary study suggests that in contrast to other estrogen-free contraceptives, mifepristone is unlikely to be associated with the increased risk of transmission of HIV and other sexually transmitted infections.

Expression of Human Beta Defensin 4 in Genetically Modified Keratinocytes Enhances Antimicrobial Activity

Defensins are cationic peptides of the innate host defense system with antimicrobial activity against many of the microorganisms commonly found in burn units. Beta defensins are variably expressed in the epithelia of skin and other organs. Human beta defensin 4 reportedly has antimicrobial activity against Pseudomonas aeruginosa and is not normally expressed in intact skin. Genetic modification was used to ectopically express human beta defensin 4 in cultured primary epidermal keratinocytes. Keratinocytes expressing human beta defensin 4 showed significantly elevated antimicrobial activity against clinically-isolated P. aeruginosa compared with controls. These results suggest that genetic modification of keratinocytes can increase their resistance to microbial contamination. Bioengineered skin replacements containing human beta defensin 4-modified keratinocytes may be useful for transplantation to contaminated burn wounds.

Characterization and functions of beta defensins in the epididymis

Characterization and functions of beta defensins in the epididymis

The Rat Epididymal Transcriptome: Comparison of Segmental Gene Expression in the Rat and Mouse Epididymides1

Regional differences along the epididymis are essential for the establishment of the luminal environment required for sperm maturation. In the current study, 19 morphologically distinct segments of the rat epididymis were identified by microdissection. Total RNA was isolated from each segment and subjected to microarray analysis. Segmental analysis of epididymal gene expression identified more than 16,000 expressed qualifiers, whereas profiling of RNA from whole rat epididymis identified approximately 12,000 expressed qualifiers. Screening a panel of normal rat tissues identified both epididymal-selective and epididymal-specific transcripts. In addition, more than 3500 qualifiers were shown to be present and differentially upregulated or downregulated by more than fourfold between any two segments. The present study complements our previous segment-dependent analysis of gene expression in the mouse epididymis and allows for comparative analyses between datasets. A total of 492 genes was shown to be present on both the MOE430 (mouse) and RAE230_2 (rat) microarrays, expressed in the epididymis of both species, and differentially expressed by more than fourfold in between segments in each species. Moreover, in-depth quantitative RT-PCR analysis of 36 members of the beta defensin gene family showed highly conserved patterns of expression along the lengths of the mouse and rat epididymides. These analyses elucidate global gene expression patterns along the length of the rat epididymis and provide a novel evaluation of conserved and nonconserved gene expression patterns in the epididymides of the two species. Furthermore, these data provide a powerful resource for the research community for future studies of biological factors that mediate sperm maturation and storage.

Association of β-Defensin 1 Single Nucleotide Polymorphisms with Atopic Dermatitis

Background: Atopic dermatitis (AD) is a chronic multifactorial allergic disease with unclear etiology. The antimicrobial human β-defensin 1 is chemotactic for dendritic cells, which are important regulators of allergic immune responses. In an attempt to identify useful markers that could predict susceptibility to AD, we investigated single nucleotide polymorphisms (SNPs) of the β-defensin 1 gene (DEFB1) with potential functional consequences. Methods: Four SNPs of the DEFB1 gene were genotyped either by real-time polymerase chain reaction or polymerase chain reaction-restriction fragment length polymorphisms in 59 patients with AD and 151 controls from the Mexican population. Correlation analyses were carried out between genetic, environmental and clinical variables in AD patients. Results: The genotypes associated with susceptibility to AD and no other allergy were 692 GG (OR = 3.21, 95% CI 1.37–7.34) and 1654 AA (OR = 17.37, 95% CI 1.62–860.83). The allele 668 C is a risk factor for AD (OR = 2.23, 95% CI 1.22–4.01) and the allele A in site 1836 correlates with earlier age at onset (Spearman’s ρ = 0.232; p = 0.03). The prolonged duration of breastfeeding correlates with earlier age at onset as well as with the severity of AD. Conclusions: The DEFB1 gene is probably involved in the incidence and development of AD, but additional functional studies will be necessary to understand the biological role of these SNPs.

Protective effect of recombinant beta-defensin-2 on acute lung injury induced by sepsis in rats

To explore the protective effect of recombinant beta-defensin-2 (BD2) on acute lung injury induced by sepsis in rats. Recombinant adenovirus carrying an expression cassette of rat BD2 (rBD2) or control adenovirus carrying empty vector was administered intratracheally to Sprague-Dawley rats 48 h before acute lung injury, which was induced by cecal ligation and double puncture (CLP). Histological changes of the lungs and 7-day survival rate were determined. The alveolar damage, interstitial edema, and infiltration of neutrophils were reduced and the survival rate was increased significantly in BD2 group than those in control group (P < 0.05). The CFU of abdominal bacteria was comparable to that in the control rats (P > 0.05). The administration of recombinant BD2 protects lung injury and improves the survival of rats with induced sepsis.

The non‐typeable Haemophilus influenzae Sap transporter provides a mechanism of antimicrobial peptide resistance and SapD‐dependent potassium acquisition

We have shown that non-typeable Haemophilus influenzae (NTHI) resists killing by antimicrobial peptides (APs). A mutant defective in expression of the sap (sensitivity to antimicrobial peptides) gene cluster product SapA is sensitive to killing by APs and is significantly attenuated in its ability to survive in a chinchilla model of otitis media compared with the parent strain. In NTHI, SapA is believed to function as the periplasmic solute binding protein of an ABC transporter. Here, we demonstrated that recombinant chinchilla beta defensin-1 specifically interacted with recombinant SapA and that AP exposure increased expression of the sap operon. We further demonstrated that the putative Sap transporter ATPase protein, SapD, was required for AP resistance as well as potassium uptake in NTHI strain 86-028NP. Loss of SapD additionally abrogated NTHI survival in vivo. Complementation of the sapD mutation restored the ability to grow in potassium-limited medium, resistance to AP-mediated killing and survival in vivo. Collectively, these data support a mechanism of Sap system-mediated resistance to APs that depends on Sap-dependent transport of APs and a Sap-dependent restoration of potassium homeostasis. Thus, NTHI required a functional Sap system to mediate bacterial survival and pathogenesis in vivo.

Mechanism of HBD-3 deficiency in atopic dermatitis

Extrinsic atopic dermatitis (EAD) and intrinsic atopic dermatitis (IAD) patients suffer from recurrent bacterial and viral infections. In this study, we demonstrate significantly decreased expression of human beta defensin (HBD)-3, a potent antimicrobial peptide (AMP), in lesional skin of both IAD ( p < 0.01) and EAD patients ( p < 0.01), as compared to psoriasis patients. Using primary keratinocytes from EAD and IAD patients, we determined that the deficiency in HBD-3 expression is an acquired rather than a constitutive defect. Furthermore, we demonstrate the down-regulatory effect of IL-4, IL-10, and IL-13 – which are over-expressed in the skin of AD patients – on HBD-3 expression in keratinocytes. Additionally, treatment of EAD skin explants with antibodies against IL-4, IL-10, and IL-13 augmented the expression of HBD-3. These studies suggest that neutralizing the Th2 cytokine milieu in AD skin may augment the innate immune response against bacterial and viral pathogens.

PROTECTION AGAINST PSEUDOMONAS AERUGINOSA PNEUMONIA AND SEPSIS-INDUCED LUNG INJURY BY OVEREXPRESSION of β-DEFENSIN-2 IN RATS

Beta-defensin-2 (BD-2), a small cationic antimicrobial peptide, was first described to be an inducible defensin at the epithelial surfaces. In vitro studies have demonstrated that it may play a pivotal role in the anti-inflammatory immune response in addition to its antimicrobial activity. The purpose of this study was to evaluate the effect of overexpression of BD-2 on lung injury to crudely investigate whether the function of BD-2 in the lung attributed to both antimicrobial action and modulation of the immune response. Recombinant adenovirus carrying an expression cassette of rat BD-2 or control adenovirus carrying empty vector was administered intratracheally to Sprague-Dawley rats 48 h before performing acute lung injury, which was induced either by Pseudomonas aeruginosa infection or by cecal ligation and double puncture (2CLP). In vivo antimicrobial activity of BD-2, histological changes of the lungs in both infectious and 2CLP models, pulmonary intracellular adhesion molecule-1 protein level, as well as the 7-day survival rate in the latter model were determined. Amounts of the P. aeruginosa in the lung with BD-2 overexpression were significantly lower compared with that in controls (2.87+/-0.76x10(4) colony-forming units [CFU]/mL vs. 2.49+/-0.74x10(6) CFU/mL, P<0.05). Overexpression of BD-2 reduced alveolar damage, interstitial edema, and infiltration of neutrophils in both models. Furthermore, in the 2CLP model, recombinant BD-2 not only significantly decreased protein levels of intracellular adhesion molecule-1 in lung tissue at 24, 36, and 72 h after 2CLP (P<0.05), but also significantly improved the survival of rats (P<0.05). The CFU of abdominal bacteria was comparable to that in the control rats (P>0.05). Therefore, overexpression of BD-2 protects against P. aeruginosa pneumonia and 2CLP-induced lung injury based on its antimicrobial and anti-inflammatory activities, respectively. Modulating the expression level of BD-2 may serve as an approach to attenuate lung injury.

Mycobacterium bovis-mediated induction of human β-defensin-2 in epithelial cells is controlled by intracellular calcium and p38MAPK

Induction of human beta defensin-2 (HBD-2) by mycobacteria has been reported. However, the molecular mechanism(s) by which mycobacteria up-regulates HBD-2 gene expression in epithelial cells remains poorly understood. In this work, we provide evidence that the induction of HBD-2 mRNA in response to Mycobacterium bovis bacillus Calmette-Guerin (BCG) was inhibited in a dose-dependent manner by pretreatment with a cell-permeable BAPTA-AM, which chelates intracellular calcium. Our data also demonstrate that HBD-2 mRNA induction by M. bovis in A549 lung epithelial cells requires activation of calmodulin. Interestingly, HBD-2 mRNA expression in response to M. bovis BCG was attenuated by pretreatment with SB203580 (an inhibitor of p38 mitogen-activated protein kinase [MAPK]), but not by an inhibitor of extracellular signal-regulated kinase (ERK): PD98059. Furthermore, we found that a second p38 MAPK inhibitor (SB202190) significantly blocked M. bovis BCG-mediated HBD-2 induction in A549 lung epithelial cells. Together, these data suggest that M. bovis BCG induces HBD-2 mRNA expression in A549 lung epithelial cells at least in part mediated through intracellular calcium flux as well as activation of signaling protein of p38MAPK, but not ERK.

Defensins HNP1 and HBD2 Stimulation of Wound-Associated Responses in Human Conjunctival Fibroblasts

To study the responses of human conjunctival fibroblasts (HCFs) to stimulation by human neutrophil defensin 1 (HNP1) and beta defensin2 (HBD2). Defensin-stimulated gene expression in primary cultures of HCFs was analyzed by real-time PCR after exposure to various concentrations of HNP1 or HBD2. Gene and protein expression for selected collagens, matrix metalloproteinases, and tissue inhibitors of metalloproteinases were determined by real-time PCR and ELISA analysis. Activation of p42/44 mitogen-activated protein (MAP) and Akt was analyzed by Western blot. HCFs did not express significant levels of the genes for HNP1 or HBD1-3. However, HNP1 and HBD2 stimulated HCF proliferation, the activation of p42/44 MAP kinase, and Akt kinase in a dose-dependent manner. HNP1 and HBD2 were not found to be chemotoxic for HCFs. It was demonstrated with the use of U0126 and wortmannin that the activation of p42/44 MAP kinase and Akt was responsible for the increased HCF proliferation observed under HNP1 and HBD2 stimulation. HNP1 stimulated the expression of the genes for collagen I, III, VI, and VIII. In addition, it reduced the secretion of collagen I protein but increased its intracellular retention. HNP1 and HBD2 upregulated the transcription and translation of MMP1. Small increases were observed in MMP14 gene expression after HNP1 stimulation and MMP2 gene expression after HBD2 stimulation. The results of this study suggest that HNP1 and HBD2 have a potential role in the biosynthetic and tissue remodeling responses of conjunctival fibroblasts.

DEFB1 gene polymorphisms and increased risk of HIV-1 infection in Brazilian children

In our study we analysed three single nucleotide polymorphisms (SNPs) in the 5' untranslated region (UTR) of the DEFB1 gene, namely -52(G/A) -44(C/G) and -20(G/A), in three groups of northeastern Brazilian children in order to assess their role in HIV-1 infection. Our results allowed us to hypothesize that the SNPs located in the 5' UTR of the DEFB1 gene can be employed as a marker of risk for HIV-1 infection.

A New Look at Crohn's Disease

Crohn's disease (CD), a chronic inflammatory disease of the intestinal mucosa is usually located in the small intestine (ileum) and or in the colon. Ileal CD has been linked to a mutation in the NOD2 gene, a bacterial recognition protein. A disturbed antimicrobial defense as provided by an arsenal of different epithelial defensins seems to be a critical factor in disease pathogenesis. Defensins are antimicrobial peptides and in the ileum are mainly expressed in Paneth cells (PCs), epithelial cells that also express NOD2. In the colon, defensins are expressed by enterocytes or metaplastic PCs. Ileal CD patients are characterized by a reduced antibacterial activity and a specific reduction of ileal PC defensins. In ileal Crohn's patients displaying a NOD2 mutation, this decrease is even more pronounced. In contrast, CD of the colon is characterized by an impaired induction of beta defensins in enterocytes. In conclusion, the regional localizations of CD, either ileal or colonic disease, can be linked to different defects in defensin expression. In line with these new findings, we predict that future therapeutic strategies aimed at restoring the host-microbe balance at the intestinal mucosa may prove superior to those that broadly suppress inflammation and adaptive immunity.

Effect of human beta defensin 2 on Staphylococcus aureus infection: in vivo study with transgenic mice

To investigate the effect of human beta defensin 2 (HBD-2) on Staphylococcus aureus infection. A minigene of HBD-2 containing pCMV promoter, full length of HBD-2 cDNA, and BGH polyA tail was generated by PCR amplification and introduced into the fertilized oocytes of C57/ICR hybridized mouse by microinjection. After gestation of 3 - 4 weeks, immunohistochemistry was used to detect the expression of HBD-2 peptide in different tissues of the transgenic young mice. Staphylococcus aureus was cultured and injected intraperitoneally to wild type mice and transgenic mice to observe their surviving status. PCR showed that the HBD-2 fragment had been successfully integrated into the chromosome of the mice. A widespread expression of HBD-2 gene was found in many tissues of the transgenic mice: trachea, lung, intestine, esophagus, testis, spleen, skin, endothelium, brain, etc. Four of the 7 transgenic mice survived the Staphylococcus aureus infection, and 10 wild type mice all died within 24 hours. HBD-2 may play an important role ion the host defense against Staphylococcus aureus infection.

Differential Expression of Ovine Innate Immune Genes by Preterm and Neonatal Lung Epithelia Infected with Respiratory Syncytial Virus

Preterm infants have increased susceptibility to severe manifestations of respiratory syncytial virus (RSV) infection. The cause(s) for this age-dependent vulnerability is/are not well-defined, but alterations in innate immune products have been implicated. In sheep, RSV disease severity has similar age-dependent characteristics and sheep have several related innate molecules for study during pulmonary infection including surfactant protein A (SP-A), surfactant protein D (SP-D), sheep beta defensin 1 (SBD1), monocyte chemotactic protein 1 (MCP1), and Toll-like receptor 4 (TLR4). However, the in vivo cellular gene expression as a response to RSV infection is poorly understood. In this study, the effect of RSV infection on expression of these innate immune genes was determined for bovine RSV-infected (bRSV+ fluorescence) epithelial cells, adjacent cells lacking bRSV antigen (adjoining cells lacking fluorescence), and control cells from non-infected lung using laser capture microdissection (LCM) and real-time RT-PCR. Control lambs had increased expression of innate immune molecules in full term (term) compared to preterm epithelia with statistical significance in SBD1, SP-D, and TLR4 mRNA. Infected cells (bRSV+ fluorescent cells) had consistently higher mRNA levels of SP-A (preterm and term), MCP1 (preterm and term), and SP-D (preterm). Interestingly, bRSV- cells of infected term lambs had significantly reduced SP-D mRNA expression compared to bRSV+ and control epithelia, suggesting that RSV infected cells may regulate the adjacent epithelial SP-D expression. This study defines specific innate immune components (e.g., SBD1, SP-D, and TLR4) that have differential age-dependent expression in the airway epithelia. Furthermore, cellular bRSV infection enhanced certain innate immune components while suppressing adjacent cellular SP-D expression in term animals. These in vivo gene expression results provide a framework for future studies on age-dependent susceptibility to RSV and RSV pathogenesis.

Antimicrobial Activity of Inducible Human β Defensin-2 Against Mycoplasma pneumoniae

Defensins in innate immunity are known to play critical roles to protect the host from infection by invasive microbes, including Gram-positive and -negative bacteria. However, little is known about the interactions between defensins and mycoplasmas. Human beta defensin (hBD)-2 and hBD-3, but not hBD-1, were found to exert strikingly antimicrobial activity against Mycoplasma pneumoniae. To elucidate the role of defensins in M. pneumoniae infection, a human pulmonary squamous cell line EBC-1 was stimulated with M. pneumoniae and interleukin (IL)-1beta. hBD-2 was markedly upregulated by IL-1beta as well as M. pneumoniae, but neither hBD-1 nor hBD-3 was apparently upregulated. Thus, the results suggest that inducible hBD-2 would play a critical role in the protection of M. pneumoniae infection.

Expression of Human Beta Defensin 4 in Genetically Modified Keratinocytes Enhances Antimicrobial Activity

Defensins are cationic peptides of the innate host defense system with antimicrobial activity against many of the microorganisms commonly found in burn units. Beta defensins are variably expressed in the epithelia of skin and other organs. Human beta defensin 4 reportedly has antimicrobial activity against Pseudomonas aeruginosa and is not normally expressed in intact skin. Genetic modification was used to ectopically express human beta defensin 4 in cultured primary epidermal keratinocytes. Keratinocytes expressing human beta defensin 4 showed significantly elevated antimicrobial activity against clinically-isolated P. aeruginosa compared with controls. These results suggest that genetic modification of keratinocytes can increase their resistance to microbial contamination. Bioengineered skin replacements containing human beta defensin 4-modified keratinocytes may be useful for transplantation to contaminated burn wounds.