Differential regulation of beta-defensin expression in human skin by microbial stimuli

In response to infection, epithelia mount an innate immune response that includes the production of antimicrobial peptides. However, the pathways that connect infection and inflammation with the induction of antimicrobial peptides in epithelia are not understood. We analyzed the molecular links between infection and the expression of three antimicrobial peptides of the beta-defensin family, human beta-defensin (hBD)-1, hBD-2, and hBD-3 in the human epidermis. After exposure to microbe-derived molecules, both monocytes and lymphocytes stimulated the epidermal expression of hBD-1, hBD-2, and hBD-3. The induced expression of hBD-3 was mediated by transactivation of the epidermal growth factor receptor. The mechanisms of induction of hBD-1 and hBD-3 were distinct from each other and from the IL-1-dependent induction of hBD-2 expression. Thus during inflammation, epidermal expression of beta-defensins is mediated by at least three different mechanisms.     

Expression and activity of beta-defensins and LL-37 in the developing human lung

Immaturity of innate immunity contributes to the increased susceptibility of human neonates to infection. The lung is a major portal of entry for potential pathogens in the neonate, and human beta-defensins (HBDs) and LL-37 participate in pulmonary innate immunity. We hypothesized that these antimicrobial factors would be developmentally regulated, expressed by neonatal pulmonary tissues, and participate in neonatal innate immunity. We found HBD-2 to be the predominant beta-defensin in human neonatal lung. HBD-2 mRNA expression was developmentally regulated, induced by the proinflammatory factor IL-1beta, and decreased by dexamethasone. Additionally, HBD-2 abundance in neonatal tracheal aspirates increased as a function of gestational age. HBD-1 had a lower level of expression compared with HBD-2 and was induced by dexamethasone. HBD-3 and LL-37 messages were not detected in airway epithelial cultures. Additionally, each antimicrobial peptide exhibited a unique spectrum of antimicrobial activity and salt sensitivity against bacteria commonly causing sepsis in the neonate. Lower levels of HBD-2 may be one factor contributing to the increased susceptibility of premature infants to pulmonary infections.     

Induction of human beta-defensin-2 expression in human astrocytes by lipopolysaccharide and cytokines

Defensins are cationic peptides with broad-spectrum antimicrobial activity. They are members of a supergene family consisting of alpha and beta subtypes and each subtype is comprised of a number of different isoforms. For example, human alpha-defensin (HAD) has six isoforms, which are expressed by polymorphonuclear leukocytes and Paneth cells. In contrast, human beta-defensin (HBD) has two isoforms that are expressed by epithelial cells of the skin, gut, respiratory and urogenital tracts. Recently, HBD-1 was detected in human brain biopsy tissue. However, little is known about the expression of HBD-1 or HBD-2 in the CNS and whether neural cells can secrete these peptides. For the present study, human astrocyte, microglial, meningeal fibroblast and neuronal cultures were probed for the expression of HBD-1 and HBD-2 mRNA and protein. Each cell type was either maintained in tissue culture medium alone or in medium containing lipopolysaccharide (LPS) at concentrations ranging from 0.1 to 1 microgram/mL, interleukin-1 beta (IL-1beta) at 1-50 ng/mL, or tumor necrosis factor alpha (TNF-alpha) at the same concentrations. The expression of HBD-1 and HBD-2 mRNAs was monitored by RT-PCR. The cDNA products were sequenced to characterize the gene product. HBD-2 protein was detected by immunoblot, immunoprecipitation and immunocytochemistry. Results of these studies showed that HBD-1 mRNA was detected in all cell cultures except in those enriched for neurons. In contrast, HBD-2 mRNA was detected only in astrocyte cultures that were treated with LPS, IL-1beta or TNF-alpha. The detection of the respective proteins correlated positively with the mRNA results. As such, these data represent the first demonstration of HBD-2 expression by astrocytes and suggest that this peptide may play a role in host defense against bacterial CNS pathogenesis.     

Discovery of new human beta-defensins using a genomics-based approach

Epithelial beta-defensins are broad-spectrum cationic antimicrobial peptides that also act as chemokines for adaptive immune cells. In the human genome, all known defensin genes cluster to a <1 Mb region of chromosome 8p22-p23. To identify new defensin genes, the DNA sequence from a contig of large-insert genomic clones from the region containing human beta-defensin-2 (HBD-2) was analyzed for the presence of defensin genes. This sequence survey identified a novel beta-defensin, termed HBD-3. The HBD-3 gene contains two exons, is located 13 kb upstream from the HBD-2 gene, and it is transcribed in the same direction. A partial HBD-3 cDNA clone was amplified from cDNA derived from IL-1beta induced fetal lung tissue. The cDNA sequence encodes for a 67 amino acid peptide that is approximately 43% identical to HBD-2 and shares the beta-defensin six cysteine motif. By PCR analysis of two commercial cDNA panels, HBD-3 expression was detected in adult heart, skeletal muscle, placenta and in fetal thymus. From RT-PCR experiments, HBD-3 expression was observed in skin, esophagus, gingival keratinocytes, placenta and trachea. Furthermore, in fetal lung explants and gingival keratinocytes, HBD-3 mRNA expression was induced by IL-1beta. Additional sequence analysis identified the HE2 (human epididymis secretory protein) gene 17 kb upstream from the HBD-3 gene. One splice variant of this gene (HE2beta1) encodes a beta-defensin consensus cysteine motif, suggesting it represents a defensin gene product. HE2beta1 mRNA expression was detected in gingival keratinocytes and bronchial epithelia using RT-PCR analysis. The discovery of these novel beta-defensin genes may allow further understanding of the role of defensins in host immunity at mucosal surfaces.     

IL-4 and IL-13 negatively regulate TNF-alpha- and IFN-gamma-induced beta-defensin expression through STAT-6, suppressor of cytokine signaling (SOCS)-1, and SOCS-3

Human beta-defensins (HBDs) are a major class of antimicrobial peptides that play an important role in the innate immune response, however, the induction and regulation of these antimicrobial peptides is not well understood. We demonstrate here that stimulation of keratinocytes with TNF-alpha/IFN-gamma induces HBD-2 and HBD-3 by activating STAT-1 and NF-kappaB signaling. We further demonstrate that IL-4 and IL-13 activate STAT-6 and induce the suppressors of cytokine signaling (SOCS)-1 and -3. This interferes with STAT-1 and NF-kappaB signaling, thereby inhibiting TNF-alpha/IFN-gamma-mediated induction of HBD-2 and HBD-3. These data suggest that targeting the STAT-1-signaling pathway or suppressor of cytokine signaling expression enhances beta-defensin expression and represents a new therapeutic strategy for reduction of infection in human diseases associated with beta-defensin deficiency.     

Capacity of human beta-defensin expression in gene-transduced and cytokine-induced cells

The purpose of this study was to determine the capacity of cells transduced with human beta-defensins (HBDs) to express antimicrobial peptides, since sufficient expression level is required for effective antimicrobial activity. Retroviral vector pBabeNeo and lentiviral vector SIN18cPPTRhMLV (SIN18) carrying HBDs were utilized to transduce non-HBD-expressing cells such as fibroblasts or HBD-producing oral epithelial cells. We found that HBD-3 gene transfer to fibroblasts was possible not via retrovirus but by direct vector transfection. SIN18 had high transduction efficiencies (80.9-99.9%) and transduced cells expressed higher amounts of HBD-2 than those by pBabeNeo. Primary human gingival epithelial cells (HGECs) expressed greater amounts of HBD-2 than primary fibroblasts after lentiviral transduction. Additionally, HBD-2 secretion from transduced HGECs cells was further increased when stimulated with IL-1 or TNFalpha. Our data indicate that while HBD-2 expression is limited in primary fibroblasts, its expression in HGECs may be maximized by gene transduction plus cytokine induction.     

Cytokine milieu of atopic dermatitis,as compared to psoriasis,skin prevents induction of innate immune response genes

Atopic dermatitis (AD) and psoriasis are the two most common chronic skin diseases. However patients with AD, but not psoriasis, suffer from frequent skin infections. To understand the molecular basis for this phenomenon, skin biopsies from AD and psoriasis patients were analyzed using GeneChip microarrays. The expression of innate immune response genes, human beta defensin (HBD)-2, IL-8, and inducible NO synthetase (iNOS) was found to be decreased in AD, as compared with psoriasis, skin (HBD-2, p = 0.00021; IL-8, p = 0.044; iNOS, p = 0.016). Decreased expression of the novel antimicrobial peptide, HBD-3, was demonstrated at the mRNA level by real-time PCR (p = 0.0002) and at the protein level by immunohistochemistry (p = 0.0005). By real-time PCR, our data confirmed that AD, as compared with psoriasis, is associated with elevated skin production of Th2 cytokines and low levels of proinflammatory cytokines such as TNF-alpha, IFN-gamma, and IL-1beta. Because HBD-2, IL-8, and iNOS are known to be inhibited by Th2 cytokines, we examined the effects of IL-4 and IL-13 on HBD-3 expression in keratinocyte culture in vitro. We found that IL-13 and IL-4 inhibited TNF-alpha- and IFN-gamma-induced HBD-3 production. These studies indicate that decreased expression of a constellation of antimicrobial genes occurs as the result of local up-regulation of Th2 cytokines and the lack of elevated amounts of TNF-alpha and IFN-gamma under inflammatory conditions in AD skin. These observations could explain the increased susceptibility of AD skin to microorganisms, and suggest a new fundamental rule that may explain the mechanism for frequent infection in other Th2 cytokine-mediated diseases.     

Toll-like receptor-4 (TLR4) mediates human β-defensin-2 (HBD-2) induction in response to Chlamydia pneumoniae in mononuclear cells

Monocytes are pivotal effector cells of the innate immune system that are vital for recognizing and eliminating invasive microbial pathogens. When microbial products bind to pathogen-recognition receptors, monocytes are activated and release a broad array of cytokines and defensins that orchestrate the host innate and adaptive immune responses. The aim of the present study is to investigate whether Toll-like receptor-4 (TLR4) mediates human β-defensin-2 (HBD-2) induction in response to Chlamydia pneumoniae in mononuclear cells. We showed that TLR4 is expressed in U937 cells and monocytes infected with viable microorganisms in a time-dependent fashion, while heat-inactivated microorganisms induced a lesser expression, albeit still significant, of TLR4 compared with viable organisms; flow cytometric analysis, in particular, revealed a higher level of TLR4 expression at 48 and 72 h postinfection. In addition, U937 cells and monocytes responded to C. pneumoniae in a TLR4-dependent manner with induction of mRNA and protein of the antimicrobial peptide HBD-2. The treatment of cells with TLR4-neutralizing antibody resulted in a decrease in C. pneumoniae- induced HBD-2 production. This study reveals that TLRs not only recognize ligands but also the types of effector molecules induced, namely, antimicrobial peptides. An understanding of the importance of the TLR-mediated antimicrobial mechanisms may provide new avenues for the development of therapeutic regimens aimed at activating the body's own defenses by stimulating TLR-dependent pathways.     

Human beta-defensin-2.

Human beta-defensin-2 (HBD-2) is a cysteine-rich cationic low molecular weight antimicrobial peptide recently discovered in psoriatic lesional skin. It is produced by a number of epithelial cells and exhibits potent antimicrobial activity against Gram-negative bacteria and Candida, but not Gram-positive Staphylococcus aureus. HBD-2 represents the first human defensin that is produced following stimulation of epithelial cells by contact with microorganisms such as Pseudomonas aeruginosa or cytokines such as TNF-alpha and IL-1 beta. The HBD-2 gene and protein are locally expressed in keratinocytes associated with inflammatory skin lesions such as psoriasis as well as in the infected lung epithelia of patients with cystic fibrosis. It is intriguing to speculate that HBD-2 is a dynamic component of the local epithelial defense system of the skin and respiratory tract having a role to protect surfaces from infection, and providing a possible reason why skin and lung infections with Gram-negative bacteria are rather rare.     

Secretion of proinflammatory cytokines by normal human melanocytes in response to lipopolysaccharide

A large body of evidence suggests that epidermal melanocytes are an integral part of the skin immune system and can be considered immunocompetent cells. Recently, it has been reported that human melanocytes constitutively express Toll-like receptors and may be involved in the induction of several inflammatory cytokines. In the study the secretion of IL-1β, IL-6 and TNF-α by cultured normal melanocytes was investigated after stimulation with lipopolysaccharide. LPS increased the secretion of IL-1β in a dose-dependent manner. IL-1β stimulated release of IL-6 and TNF-α by melanocytes, whereas LPS activated production of TNF-α, but not of IL-6. These observations indicate that LPS can participate in the regulation of cytokine activity in normal human melanocytes and suggest that cytokines released by melanocytes could affect melanocytes themselves or/and other cells of the epidermis.     

Endotoxin responsiveness of human airway epithelia is limited by low expression of MD-2

The expression of inducible antimicrobial peptides, such as human beta-defensin-2 (HBD-2) by epithelia, comprises a component of innate pulmonary defenses. We hypothesized that HBD-2 induction in airway epithelia is linked to pattern recognition receptors such as the Toll-like receptors (TLRs). We found that primary cultures of well-differentiated human airway epithelia express the mRNA for TLR-4, but little or no MD-2 mRNA, and display little HBD-2 expression in response to treatment with purified endotoxin +/- LPS binding protein (LBP) and soluble CD14. Expression of endogenous MD-2 by transduction of airway epithelial cells with an adenoviral vector encoding MD-2 or extracellular addition of recombinant MD-2 both increased the responses of airway epithelia to endotoxin + LBP and sCD14 by >100-fold, as measured by NF-kappaB-luciferase activity and HBD-2 mRNA expression. MD-2 mRNA could be induced in airway epithelia by exposure of these cells to specific bacterial or host products (e.g., killed Haemophilus influenzae, the P6 outer membrane protein from H. influenzae, or TNF-alpha + IFN-gamma). These findings suggest that MD-2, either coexpressed with TLR-4 or secreted when produced in excess of TLR-4 from neighboring cells, is required for airway epithelia to respond sensitively to endotoxin. The regulation of MD-2 expression in airway epithelia and pulmonary macrophages may serve as a means to modify endotoxin responsiveness in the airway.     

Sphingosine Kinase-1 Is Required for Toll Mediated β-Defensin 2 Induction in Human Oral Keratinocytes

Background

Host defense against invading pathogens is triggered by various receptors including toll-like receptors (TLRs). Activation of TLRs is a pivotal step in the initiation of innate, inflammatory, and antimicrobial defense mechanisms. Human β-defensin 2 (HBD-2) is a cationic antimicrobial peptide secreted upon Gram-negative bacterial perturbation in many cells. Stimulation of various TLRs has been shown to induce HBD-2 in oral keratinocytes, yet the underlying cellular mechanisms of this induction are poorly understood.

Principal Findings

Here we demonstrate that HBD-2 induction is mediated by the Sphingosine kinase-1 (Sphk-1) and augmented by the inhibition of Glycogen Synthase Kinase-3β (GSK-3β) via the Phosphoinositide 3-kinase (PI3K) dependent pathway. HBD-2 secretion was dose dependently inhibited by a pharmacological inhibitor of Sphk-1. Interestingly, inhibition of GSK-3β by SB 216763 or by RNA interference, augmented HBD-2 induction. Overexpression of Sphk-1 with concomitant inhibition of GSK-3β enhanced the induction of β-defensin-2 in oral keratinocytes. Ectopic expression of constitutively active GSK-3β (S9A) abrogated HBD-2 whereas kinase inactive GSK-3β (R85A) induced higher amounts of HBD-2.

Conclusions/Significance

These data implicate Sphk-1 in HBD-2 regulation in oral keratinocytes which also involves the activation of PI3K, AKT, GSK-3β and ERK 1/2. Thus we reveal the intricate relationship and pathways of toll-signaling molecules regulating HBD-2 which may have therapeutic potential.     

Lipopolysaccharides from Bordetella pertussis and Bordetella parapertussis differently modulate human dendritic cell functions resulting in divergent prevalence of Th17-polarized responses

Bordetella pertussis and B. parapertussis are the etiological agents of pertussis, yet the former has a higher incidence and is the cause of a more severe disease, in part due to pertussis toxin. To identify other factors contributing to the different pathogenicity of the two species, we analyzed the capacity of structurally different lipooligosaccharide (LOS) from B. pertussis and LPS from B. parapertussis to influence immune functions regulated by dendritic cells. Either B. pertussis LOS and B. parapertussis LPS triggered TLR4 signaling and induced phenotypic maturation and IL-10, IL-12p40, IL-23, IL-6, and IL-1beta production in human monocyte-derived dendritic cells (MDDC). B. parapertussis LPS was a stronger inducer of all these activities as compared with B. pertussis LOS, with the notable exception of IL-1beta, which was equally produced. Only B. parapertussis LPS was able to induce IL-27 expression. In addition, although MDDC activation induced by B. parapertussis LPS was greatly dependent on soluble CD14, B. pertussis LOS activity was CD14-independent. The analysis of the intracellular pathways showed that B. parapertussis LPS and B. pertussis LOS equally induced IkappaBalpha and p38 MAPK phosphorylation, but B. pertussis LOS triggered ERK1/2 phosphorylation more rapidly and at higher levels than B. parapertussis LPS. Furthermore, B. pertussis LOS was unable to induce MyD88-independent gene induction, which was instead activated by B. parapertussis LPS, witnessed by STAT1 phosphorylation and induction of the IFN-dependent genes, IFN regulatory factor-1 and IFN-inducible protein-10. These differences resulted in a divergent regulation of Th cell responses, B. pertussis LOS MDDC driving a predominant Th17 polarization. Overall, the data observed reflect the different structure of the two LPS and the higher Th17 response induced by B. pertussis LOS may contribute to the severity of pertussis in humans.     

Structure and mapping of the human β-defensin HBD-2 gene and its expression at sites of inflammation

We cloned a second human β-defensin gene, HBD-2, and determined its gene structure and expression in inflamed tissue sections. The entire gene spanned about 2 kb with two small exons and one intron. Radiation hybrid studies confirmed the location on chromosome 8p, were consistent with the order HNP-1, HBD-1 and HBD-2, and located HBD-2 as the most centromeric of the genes. By three-color fluorescence in situ hybridization on both free chromatin fiber mapping and interphase mapping, HBD-1, HBD-2 and HNP-1 were mapped to chromosome 8p23. HBD-1 was within 40–100 kb of HNP-1, while HBD-2 was about 500–600 kb from HBD-1, with the most likely order HNP-1, HBD-1, HBD-2. The expression of HBD-2 was locally regulated by inflammation. HBD-2 mRNA was markedly increased in the epidermis surrounding inflamed regions, but not detectable in adjacent non-inflamed areas, a distribution that was confirmed at the peptide level by immunostaining with HBD-2 antibody. The HBD-2 gene is the first member of the human defensin family that is locally inducible by inflammation.