Expression and regulation of the human beta-defensins hBD-1 and hBD-2 in intestinal epithelium

The intestinal epithelium forms a physical barrier to limit access of enteric microbes to the host and contributes to innate host defense by producing effector molecules against luminal microbes. To further define the role of the intestinal epithelium in antimicrobial host defense, we analyzed the expression, regulation, and production of two antimicrobial peptides, human defensins hBD-1 and hBD-2, by human intestinal epithelial cells in vitro and in vivo. The human colon epithelial cell lines HT-29 and Caco-2 constitutively express hBD-1 mRNA and protein but not hBD-2. However, hBD-2 expression is rapidly induced by IL-1alpha stimulation or infection of those cells with enteroinvasive bacteria. Moreover, hBD-2 functions as a NF-kappaB target gene in the intestinal epithelium as blocking NF-kappaB activation inhibits the up-regulated expression of hBD-2 in response to IL-1alpha stimulation or bacterial infection. Caco-2 cells produce two hBD-1 isoforms and a hBD-2 peptide larger in size than previously described hBD-2 isoforms. Paralleling the in vitro findings, human fetal intestinal xenografts constitutively express hBD-1, but not hBD-2, and hBD-2 expression, but not hBD-1, is up-regulated in xenografts infected intraluminally with Salmonella. hBD-1 is expressed by the epithelium of normal human colon and small intestine, with a similar pattern of expression in inflamed colon. In contrast, there is little hBD-2 expression by the epithelium of normal colon, but abundant hBD-2 expression by the epithelium of inflamed colon. hBD-1 and hBD-2 may be integral components of epithelial innate immunity in the intestine, with each occupying a distinct functional niche in intestinal mucosal defense.     

Rhinovirus increases human beta-defensin-2 and -3 mRNA expression in cultured bronchial epithelial cells

Human beta-defensins (hBDs) are antimicrobial peptides that play important roles in host defense against infection, inflammation and immunity. Previous studies showed that micro-organisms and proinflammatory mediators regulate the expression of these peptides in airway epithelial cells. The aim of the present study was to investigate the modulation of expression of hBDs in cultured primary bronchial epithelial cells (PBEC) by rhinovirus-16 (RV16), a respiratory virus responsible for the common cold and associated with asthma exacerbations. RV16 was found to induce expression of hBD-2 and -3 mRNA in PBEC, but did not affect hBD-1 mRNA. Viral replication appeared essential for rhinovirus-induced beta-defensin mRNA expression, since UV-inactivated rhinovirus did not increase expression of hBD-2 and hBD-3 mRNA. Exposure to synthetic double-stranded RNA (dsRNA) molecule polyinosinic:polycytidylic acid had a similar effect as RV16 on mRNA expression of these peptides in PBEC. In line with this, PBEC were found to express TLR3, a Toll-like receptor involved in recognition of dsRNA. This study shows that rhinovirus infection of PBEC leads to increased hBD-2 and hBD-3 mRNA expression, which may play a role in both the uncomplicated common cold and in virus-associated exacerbations of asthma.     

Regulation of human beta-defensin-2 in gingival epithelial cells: the involvement of mitogen-activated protein kinase pathways, but not the NF-kappaB transcription factor family.

Stratified epithelia of the oral cavity are continually exposed to bacterial challenge that is initially resisted by neutrophils and epithelial factors, including antimicrobial peptides of the beta-defensin family. Previous work has shown that multiple signaling pathways are involved in human beta-defensin (hBD)-2 mRNA regulation in human gingival epithelial cells stimulated with a periodontal bacterium, Fusobacterium nucleatum, and other stimulants. The goal of this study was to further characterize these pathways. The role of NF-kappaB in hBD-2 regulation was investigated initially due to its importance in inflammation and infection. Nuclear translocation of p65 and NF-kappaB activation was seen in human gingival epithelial cells stimulated with F. nucleatum cell wall extract, indicating possible involvement of NF-kappaB in hBD-2 regulation. However, hBD-2 induction by F. nucleatum was not blocked by pretreatment with two NF-kappaB inhibitors, pyrrolidine dithiocarbamate and the proteasome inhibitor, MG132. To investigate alternative modes of hBD-2 regulation, we explored involvement of mitogen-activated protein kinase pathways. F. nucleatum activated p38 and c-Jun NH(2)-terminal kinase (JNK) pathways, whereas it had little effect on p44/42. Furthermore, inhibition of p38 and JNK partially blocked hBD-2 mRNA induction by F. nucleatum, and the combination of two inhibitors completely blocked expression. Our results suggest that NF-kappaB is neither essential nor sufficient for hBD-2 induction, and that hBD-2 regulation by F. nucleatum is via p38 and JNK, while phorbol ester induces hBD-2 via the p44/42 extracellular signal-regulated kinase pathway. Studies of hBD-2 regulation provide insight into how its expression may be enhanced to control infection locally within the mucosa and thereby reduce microbial invasion into the underlying tissue.     

Protease-activated receptor signaling increases epithelial antimicrobial peptide expression

Epithelial tissues provide both a physical barrier and an antimicrobial barrier. Antimicrobial peptides of the human beta-defensin (hBD) family are part of the innate immune responses that play a role in mucosal defense. hBDs are made in epithelia including oral epithelium where the bacterial load is particularly great. hBD-2 and hBD-3 are up-regulated in response to bacterial stimuli. Previous studies show that hBD-2 expression in human gingival epithelial cells (GEC) is stimulated by both nonpathogenic and pathogenic bacteria, including Porphyromonas gingivalis, a Gram-negative pathogen associated with periodontitis. Present evidence suggests that hBD-2 expression in GEC uses several signaling pathways, including an NF-kappaB-mediated pathway but without apparent LPS-TLR4 signaling. Protease-activated receptors (PAR) are G-protein-coupled receptors that mediate cellular responses to extracellular proteinases. P. gingivalis secretes multiple proteases that contribute to its virulence mechanisms. To determine whether PAR signaling is used in hBD-2 induction, GEC were stimulated with wild-type P. gingivalis or mutants lacking one or more proteases. hBD-2 mRNA expression was reduced in GEC stimulated with single protease mutants (11-67% compared with wild type), strongly reduced in double mutants (0.1-16%), and restored to wild-type levels (93%) in mutant with restored protease activity. Stimulation by wild type was partially blocked by inhibitors of phospholipase C, a main signaling pathway for PARs. Expression of hBD-3 was unaffected. Peptide agonist of PAR-2, but not PAR-1 activator, also induced hBD-2 in GEC. Thus, P. gingivalis proteases are directly involved in regulation of hBD-2 in cultured GEC, and this induction partially uses the PAR-2 receptor and signaling pathway.     

IL-4 differentially regulates eotaxin and MCP-4 in lung epithelium and circulating mononuclear cells

To investigate the mechanisms of eosinophil recruitment in allergic airway inflammation, we examined the effects of interleukin (IL)-4, a Th2-type cytokine, on eotaxin and monocyte chemoattractant protein-4 (MCP-4) expression in human peripheral blood mononuclear cells (PBMCs; n = 10), in human lower airway mononuclear cells (n = 5), in the human lung epithelial cell lines A549 and BEAS-2B, and in human cultured airway epithelial cells. IL-4 inhibited eotaxin and MCP-4 mRNA expression induced by IL-1 beta and tumor necrosis factor-alpha in PBMCs but did not significantly inhibit expression in epithelial cells. Eotaxin and MCP-4 mRNA expression was not significantly induced by proinflammatory cytokines in lower airway mononuclear cells. IL-1 beta-induced eotaxin and MCP-4 protein production was also inhibited by IL-4 in PBMCs, whereas IL-4 enhanced eotaxin protein production in A549 cells. In contrast, dexamethasone inhibited eotaxin and MCP-4 expression in both PBMCs and epithelial cells. The divergent effects of IL-4 on eotaxin and MCP-4 expression between PBMCs and epithelial cells may create chemokine concentration gradients between the subepithelial layer and the capillary spaces that may promote the recruitment of eosinophils to the airway in Th2-type responses.     

Induction of human beta-defensin-2 mRNA expression by Helicobacter pylori in human gastric cell line MKN45 cells on cag pathogenicity island.

Helicobacter pylori is an etiological agent of gastritis, peptic ulcer, and gastric cancer. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide which belongs to one of the most important host defense systems against bacterial infection in several epithelial tissues. We studied the effect of H. pylori on the expression of hBD-2 mRNA in MKN45 gastric mucosal cells. H. pylori, but not culture filtrate, increased the hBD-2 mRNA level in MKN45 cells; the inductive effect of H. pylori was not detected with Intestine 407 cells. Among H. pylori strains, strain OHPC0002, which lacks a cag Pathogenicity Island (PAI), did not induce hBD-2 mRNA in MKN45 cells. These results suggested that H. pylori cag PAI is critical for the induction of hBD-2 mRNA in MKN45 cells. Exposure of MKN45 cells to Salmonella typhimurium, S. enteritidis, S. typhi, and S. dublin, but not Escherichia coli ML35, also resulted in induction of hBD-2 mRNA.     

Human beta defensin 2 promotes intestinal wound healing in vitro

Limiting microbial threats, maintenance and re-establishment of the mucosal barrier are vital for intestinal homeostasis. Antimicrobial peptides have been recognized as essential defence molecules and decreased expression of these peptides has been attributed to chronic inflammation of the human intestinal mucosa. Recently, pluripotent properties, including stimulation of proliferation and migration have been suggested for a number of antimicrobial peptides. However, it is currently unknown, whether the human beta-defensin 2 (hBD-2) in addition to its known antimicrobial properties has further effects on healing and protection of the intestinal epithelial barrier. Caco-2 and HT-29 cells were stimulated with 0.1-10 microg/ml hBD-2 for 6-72 h. Effects on cell viability and apoptosis were monitored and proliferation was quantified by bromo-deoxyuridine incorporation. Migration was quantified in wounding assays and characterized by immunohistochemistry. Expression of mucins was determined by quantitative PCR and slot-blot analysis. Furthermore, anti-apoptotic capacities of hBD-2 were studied. Over a broad range of concentrations and stimulation periods, hBD-2 was well tolerated by IECs and did not induce apoptosis. hBD-2 significantly increased migration but not proliferation of intestinal epithelial cells. Furthermore, hBD-2 induced cell line specific the expression of mucins 2 and 3 and ameliorated TNF-related apoptosis-inducing ligand (TRAIL) induced apoptosis. In addition to its known antimicrobial properties, hBD-2 might have further protective effects on the intestinal epithelium. Results of this in vitro study suggest, that hBD-2 expression may play a dual role in vivo, i.e. in impaired intestinal barrier function observed in patients with inflammatory bowel disease.     

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 µM) and LL-37 (0.1 and 0.2 µM) 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.     

Novel anti-bacterial activities of β-defensin 1 in human platelets: suppression of pathogen growth and signaling of neutrophil extracellular trap formation

Human β-defensins (hBD) are antimicrobial peptides that curb microbial activity. Although hBD's are primarily expressed by epithelial cells, we show that human platelets express hBD-1 that has both predicted and novel antibacterial activities. We observed that activated platelets surround Staphylococcus aureus (S. aureus), forcing the pathogens into clusters that have a reduced growth rate compared to S. aureus alone. Given the microbicidal activity of β-defensins, we determined whether hBD family members were present in platelets and found mRNA and protein for hBD-1. We also established that hBD-1 protein resided in extragranular cytoplasmic compartments of platelets. Consistent with this localization pattern, agonists that elicit granular secretion by platelets did not readily induce hBD-1 release. Nevertheless, platelets released hBD-1 when they were stimulated by α-toxin, a S. aureus product that permeabilizes target cells. Platelet-derived hBD-1 significantly impaired the growth of clinical strains of S. aureus. hBD-1 also induced robust neutrophil extracellular trap (NET) formation by target polymorphonuclear leukocytes (PMNs), which is a novel antimicrobial function of β-defensins that was not previously identified. Taken together, these data demonstrate that hBD-1 is a previously-unrecognized component of platelets that displays classic antimicrobial activity and, in addition, signals PMNs to extrude DNA lattices that capture and kill bacteria.     

Expression of lipocortins in human bronchial epithelial cells: effects of IL-1beta , TNF-alpha, LPS and dexamethasone

In this study, we investigated the expression of lipocortin I and II (annexin I and I in the human bronchial epithelium, both in vivo and in vitro. A clear expression of lipocortin I and II protein was found in the epithelium in sections of bronchial tissue. In cultured human bronchial epithelial cells we demonstrated the expression of lipocortin I and II mRNA and protein using Northern blotting, FACScan analysis and ELISA. No induction of lipocortin I or II mRNA or protein was observed after incubation with dexamethasone. Stimulation of bronchial epithelial cells with IL-1beta, TNF-alpha or LPS for 24 h did not affect the lipocortin I or II mRNA or protein expression, although PGE(2) and 6-keto-PGF(1alpha) production was significantly increased. This IL-1beta- and LPS-mediated increase in eicosanoids could be reduced by dexamethasone, but was not accompanied by an increase in lipocortin I or II expression. In human bronchial epithelial cells this particular glucocorticoid action is not mediated through lipocortin I or II induction.     

Distinct signaling pathways leading to the induction of human β-defensin 2 by stimulating an electrolyticaly-generated acid functional water and double strand RNA in oral epithelial cells

Abstract

Defensins, a major family of cationic antimicrobial peptides, play important roles in innate immunity. In the present study, we investigated whether double-stranded RNA (dsRNA), a by-product of RNA virus replication, can induce human β-defensins-2 (hBD-2) expression in oral epithelial cells (OECs). We also examined the hBD-2-inducible activity of acid-electrolyzed functional water (FW). The results indicated that both dsRNA- and FW-induced hBD-2 expression in OECs. The induction efficiency was much higher for FW than for dsRNA. FW-induced production of hBD-2 was clearly observed by immunofluorescence staining. A luciferase assay was performed with 1.2?kb of the 5′-untranslated region (5′-UTR) of the hBD-2 gene. The results indicated that the nuclear factor-kappa B (NF-κB)-binding site proximal to the translation initiation site was indispensable for dsRNA-stimulated hBD-2 expression, but not in the case of FW. Moreover, FW-stimulated hBD-2 expression did not depend on NF-κB activity; instead, FW inhibited NF-κB activity. Pretreatment of the cells with specific inhibitors against NF-κB further confirmed NF-κB-independent hBD-2 induction by FW. In analogy to the results for intestinal epithelial cells (IECs), the dsRNA signal, but not FW, was sensed by toll-like receptor 3 (TLR3) in OECs. These results suggested that hBD-2 expression induced by dsRNA and FW is regulated by distinct mechanisms in OECs.     

Extracellular HIV-1 Tat induces human beta-defensin-2 production via NF-kappaB/AP-1 dependent pathways in human B cells

Defensins, a family of antimicrobial peptides, are one of the first lines of host defense. Human beta-defensins (hBD) such as hBD-2 and -3 have anti-HIV activity. Previous studies have shown that HIV-1 virion can induce the expression of hBD, although the exact components of HIV-1 virion that are responsible for hBD expression have not yet been elucidated. In this study, we examined the effect of HIV-1 Tat on the expression of hBD in B cells. Stimulation of B cells with HIV-1 Tat protein significantly increased the mRNA and protein levels of hBD-2. HIV-1 Tat also induced the activation of a reporter gene for hBD-2 in a dose-dependent manner in B cells. Pretreatment of B cells with a JNK inhibitor suppressed HIV-1 Tat-induced hBD-2 expression. Pretreatment of B cells with AP-1 inhibitors or NF-κB inhibitors led to a decrease in HIV-1 Tat-induced protein and mRNA expression of hBD-2. Taken together, our results indicate that HIV-1 Tat can up-regulate the expression of hBD-2 via JNK-NF-κB/AP-1-dependent pathways in human B cells.