Human β-defensin 3 increases the TLR9-dependent response to bacterial DNA.
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ABSTRACT:
SUBMITTER: McGlasson SL
PROVIDER: S-EPMC5412915 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Coordination of the primary defense mechanisms against pathogens relies on the appropriate expression of pathogen recognition receptors (PRRs) triggering the early release of effector molecules of the innate immune system. To analyze the impact of this system on the counteraction of infections of the mammary gland (mastitis), we characterized the bovine gene encoding the key PRR Toll-like receptor 9 (TLR9) and mapped its precise position on chromosome BTA22. The sequence information was used to establish real-time PCR quantification assays to measure the mRNA abundances of TLR9, TLR2, and TLR4 together with those of beta-defensin 5 (BNBD5), an early bactericidal effector molecule of the innate system, in healthy and infected mammary glands. Mastitis strongly increased (4- to 13-fold) the mRNA abundances of all of these genes except TLR9. Slight subclinical infections already caused a substantial increase in the copy numbers, though they did so the least for TLR9. Induction was not systemic, since mRNA abundance was low in uninfected control quarters of the udder but high in the severely infected quarters of the same animal. The number of TLR2 copies correlated well with those of TLR4, indicating coordinated regulation of these two PRRs during infection of the udder. Their coordinated regulation explains our unexpected observation that pure Staphylococcus aureus infections caused a strong increase also in TLR4 mRNA abundance. In situ hybridizations revealed that BNBD5 is expressed predominantly in the mammary epithelial cells (MEC) of the infected gland. Our data therefore suggest a significant contribution of the innate immune system to counteract mastitis and attribute a prominent effector function to the MEC.
| S-EPMC321333 | biostudies-literature
Project description:The Toll-like receptor (TLR) family consists of phylogenetically conserved transmembrane proteins, which function as mediators of innate immunity for recognition of pathogen-derived ligands and subsequent cell activation via the Toll/IL-1R signal pathway. Here, we show that human TLR9 (hTLR9) expression in human immune cells correlates with responsiveness to bacterial deoxycytidylate-phosphate-deoxyguanylate (CpG)-DNA. Notably "gain of function" to immunostimulatory CpG-DNA is achieved by expressing TLR9 in human nonresponder cells. Transfection of either human or murine TLR9 conferred responsiveness in a CD14- and MD2-independent manner, yet required species-specific CpG-DNA motifs for initiation of the Toll/IL-1R signal pathway via MyD88. The optimal CpG motif for hTLR9 was GTCGTT, whereas the optimal murine sequence was GACGTT. Overall, these data suggest that hTLR9 conveys CpG-DNA responsiveness to human cells by directly engaging immunostimulating CpG-DNA.
| S-EPMC55404 | biostudies-literature
Project description:Viral-bacterial co-infections are associated with severe exacerbations of COPD. Epithelial antimicrobial peptides, including human β-defensin-2 (HBD-2), are integral to innate host defenses. In this study, we examined how co-infection of airway epithelial cells with rhinovirus and Pseudomonas aeruginosa modulates HBD-2 expression, and whether these responses are attenuated by cigarette smoke and in epithelial cells obtained by bronchial brushings from smokers with normal lung function or from COPD patients. When human airway epithelial cells from normal lungs were infected with rhinovirus, Pseudomonas aeruginosa, or the combination, co-infection with rhinovirus and bacteria resulted in synergistic induction of HBD-2 (p<0.05). The combination of virus and flagellin replicated this synergistic increase (p<0.05), and synergy was not seen using a flagella-deficient mutant Pseudomonas (p<0.05). The effects of Pseudomonas aeruginosa were mediated via interactions of flagellin with TLR5. The effects of HRV-16 depended upon viral replication but did not appear to be mediated via the intracellular RNA helicases, retinoic acid-inducible gene-I or melanoma differentiation-associated gene-5. Cigarette smoke extract significantly decreased HBD-2 production in response to co-infection. Attenuated production was also observed following co-infection of cells obtained from healthy smokers or COPD patients compared to healthy controls (p<0.05). We conclude that co-exposure to HRV-16 and Pseudomonas aeruginosa induces synergistic production of HBD-2 from epithelial cells and that this synergistic induction of HBD-2 is reduced in COPD patients. This may contribute to the more severe exacerbations these patients experience in response to viral-bacterial co-infections.
| S-EPMC5425185 | biostudies-literature
Project description:Neisseria meningitidis is a gram-negative bacterium that often asymptomatically colonizes the human nasopharyngeal tract. These bacteria cross the epithelial barrier can cause life-threatening sepsis and/or meningitis. Antimicrobial peptides are one of the first lines of defense against invading bacterial pathogens. Human beta-defensin 2 (hBD2) is an antimicrobial peptide with broad antibacterial activity, although its mechanism of action is poorly understood. Here, we investigated the effect of hBD2 on N. meningitidis. We showed that hBD2 binds to and kills actively growing meningococcal cells. The lethal effect was evident after 2 h incubation with the peptide, which suggests a slow killing mechanism. Further, the membrane integrity was not changed during hBD2 treatment. Incubation with lethal doses of hBD2 decreased the presence of diplococci; the number and size of bacterial microcolonies/aggregates remained constant, indicating that planktonic bacteria may be more susceptible to the peptide. Meningococcal DNA bound hBD2 in mobility shift assays and inhibited the lethal effect of hBD2 in a dose-dependent manner both in suspension and biofilms, supporting the interaction between hBD2 and DNA. Taken together, the ability of meningococcal DNA to bind hBD2 opens the possibility that extracellular DNA due to bacterial lysis may be a means of N. meningitidis to evade immune defenses.
| S-EPMC8278289 | biostudies-literature
Project description:Previously, using the Illumina HumanHT-12 microarray we found that β-defensin 131 (DEFB131), an antimicrobial peptide, is upregulated in the human prostate epithelial cell line RWPE-1 upon stimulation with lipoteichoic acid (LTA; a gram-positive bacterial component), than that in the untreated RWPE-1 cells. In the current study, we aimed to investigate the role of DEFB131 in RWPE-1 cells during bacterial infection. We examined the intracellular signaling pathways and nuclear responses in RWPE-1 cells that contribute to DEFB131 gene induction upon stimulation with LTA. Chromatin immunoprecipitation was performed to determine whether NF-κB directly binds to the DEFB131 promoter after LTA stimulation in RWPE-1 cells. We found that DEFB131 expression was induced by LTA stimulation through TLR2 and p38MAPK/NF-κB activation, which was evident in the phosphorylation of both p38MAPK and IκBα. We also found that SB203580 and Bay11-7082, inhibitors of p38MAPK and NF-κB, respectively, suppressed LTA-induced DEFB131 expression. The chromatin immunoprecipitation assay showed that NF-κB directly binds to the DEFB131 promoter, suggesting that NF-κB is a direct regulator, and is necessary for LTA-induced DEFB131 expression in RWPE-1 cells. Interestingly, with DEFB131 overexpression in RWPE-1 cells, the accumulation of mRNA and protein secretion of cytokines (IL-1α, IL-1β, IL-6, and IL-12α) and chemokines (CCL20, CCL22, and CXCL8) were significantly enhanced. In addition, DEFB131-transfected RWPE-1 cells markedly induced chemotactic activity in THP-1 monocytes. We concluded that DEFB131 induces cytokine and chemokine upregulation through the TLR2/NF-κB signaling pathway in RWPE-1 cells during bacterial infection and promotes an innate immune response.
| S-EPMC4674080 | biostudies-literature
Project description:ObjectivesPrimary sclerosing cholangitis (PSC) is a chronic inflammatory disease of the bile ducts frequently associated with inflammatory bowel disease (IBD), suggesting an important role for the gut-liver axis. Defensins are small (3.5-4.5 kDa) anti-microbial peptides that contribute to innate immunity at mucosal surfaces and have been implicated in IBD. The aim of this study was to investigate copy number variation of the gene (DEFB4) encoding human β-defensin 2 (HBD2) and protein expression of HBD2 in PSC.MethodsUS and Italian PSC cases and unaffected controls (US PSC patients n=89, US controls n=87; Italian PSC patients n=46, Italian controls n=84) were used to estimate HBD2 gene copy number by both quantitative real-time PCR and paralog ratio test. Serum levels of HBD2 were measured by enzyme-linked immunosorbent assay and liver expression was analyzed by immunohistochemistry.ResultsMean serum levels of HBD2 were significantly greater in PSC (1,086±1,721 ng/μl) compared with primary biliary cholangitis (544±754 ng/μl), ulcerative colitis (417±506 ng/μl), and healthy controls (514±731 ng/μl) (P=0.02). However, no significant differences between the frequencies of high DEFB4 gene copy number, defined by >4 copies, and PSC were found in the US, Italian, or combined cohorts. Importantly, a high number of biliary ducts were found immunopositive in PSC samples compared with controls.ConclusionsOur data show that HBD2 serum levels and tissue expression are increased in PSC subjects, suggesting that this arm of innate immunity may be important in the etiopathogenesis of PSC.
| S-EPMC5387757 | biostudies-literature
Project description:Bacterial biofilms are associated with numerous human infections. The predominant protein expressed in enteric biofilms is the amyloid curli, which forms highly immunogenic complexes with DNA. Infection with curli-expressing bacteria or systemic exposure to purified curli-DNA complexes triggers autoimmunity via the generation of type I interferons (IFNs) and anti-double-stranded DNA antibodies. Here, we show that DNA complexed with amyloid curli powerfully stimulates Toll-like receptor 9 (TLR9) through a two-step mechanism. First, the cross beta-sheet structure of curli is bound by cell-surface Toll-like receptor 2 (TLR2), enabling internalization of the complex into endosomes. After internalization, the curli-DNA immune complex binds strongly to endosomal TLR9, inducing production of type I IFNs. Analysis of wild-type and TLR2-deficient macrophages showed that TLR2 is the major receptor that drives the internalization of curli-DNA complexes. Suppression of TLR2 internalization via endocytosis inhibitors led to a significant decrease in Ifnβ expression. Confocal microscopy analysis confirmed that the TLR2-bound curli was required for shuttling of DNA to endosomal TLR9. Structural analysis using small-angle X-ray scattering revealed that incorporation of DNA into curli fibrils resulted in the formation of ordered curli-DNA immune complexes. Curli organizes parallel, double-stranded DNA rods at an inter-DNA spacing that matches up well with the steric size of TLR9. We also found that production of anti-double-stranded DNA autoantibodies in response to curli-DNA was attenuated in TLR2- and TLR9-deficient mice and in mice deficient in both TLR2 and TLR9 compared to wild-type mice, suggesting that both innate immune receptors are critical for shaping the autoimmune adaptive immune response. We also detected significantly lower levels of interferon-stimulated gene expression in response to purified curli-DNA in TLR2 and TLR9 deficient mice compared to wild-type mice, confirming that TLR2 and TLR9 are required for the induction of type I IFNs. Finally, we showed that curli-DNA complexes, but not cellulose, were responsible elicitation of the immune responses to bacterial biofilms. This study defines the series of events that lead to the severe pro-autoimmune effects of amyloid-expressing bacteria and suggest a mechanism by which amyloid curli acts as a carrier to break immune tolerance to DNA, leading to the activation of TLR9, production of type I IFNs, and subsequent production of autoantibodies.
| S-EPMC5406031 | biostudies-literature
Project description:Human beta-defensins (hBDs) play an important role in the host defense against various microbes, showing different levels of antibacterial activity and salt resistance in vitro. It is of interest to investigate whether can chimeric hBD analogs enhanced antibacterial activity and salt resistance. In this study, we designed a chimeric human defensin, named H4, by combining sequences of human beta-defensin-3 (hBD-3) and human beta-defensin-4 (hBD-4), then evaluated its antibacterial activity, salt resistance, and cytotoxic effects. The result showed that the antibacterial activity of H4 against most tested strains, including Klebsiella pneumonia, Enterococcus faecalis, Staphyloccocus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, and Acinetobacter baumannii was significantly improved compared to that of hBD-3 and hBD-4. Notably, H4 exhibited significantly better antibacterial activity against multidrug resistant isolate A. baumannii MDR-ZJ06 than commonly used antibiotics. Chimeric H4 still showed more than 80% antibacterial activity at high salt concentration (150 μM), which proves its good salt tolerance. The cytotoxic effect assay showed that the toxicity of H4 to Hela, Vero, A549 cells and erythrocytes at a low dose (<10 μg/ml) was similar to that of hBD-3 and hBD-4. In conclusion, given its broad spectrum of antibacterial activity and high salt resistance, chimeric H4 could serve as a promising template for new therapeutic antimicrobial agents.
| S-EPMC8137984 | biostudies-literature
Project description:The mucosal epithelium secretes a variety of antimicrobial peptides that act as part of the innate immune system to protect against invading microbes. Here, we describe the functional properties of human defensin (HD) 5, the major antimicrobial peptide produced by Paneth cells in the ileum, in relation to its structure. The antimicrobial activity of HD-5 against Escherichia coli proved to be independent of its structure, whereas the unstructured peptide showed greatly reduced antimicrobial activity against Staphylococcus aureus. We find that HD-5 binds to the cell membrane of intestinal epithelial cells and induced secretion of the chemokine interleukin (IL)-8 in a concentration- and structure-dependent fashion. Incubation of HD-5 in the presence of tumor necrosis factor alpha further increased IL-8 secretion synergistically, suggesting that HD-5 may act as a regulator of the intestinal inflammatory response.
| S-EPMC1832120 | biostudies-literature
Project description:Mitochondrial DNA (mtDNA) contains unmethylated CpG motifs that exhibit immune stimulatory capacities. The aim of this study was to investigate whether mtDNA activates the Toll-like receptor 9 (TLR9)/nuclear factor-κB (NF-κB) pathway, thereby contributing to post-traumatic systemic inflammatory response syndrome (SIRS) and lung injury in rats. The effects of mtDNA on macrophage culture were examined in order to elucidate the putative cellular mechanisms. Rats and macrophage cultures were treated with phosphate-buffered saline, nuclear DNA, or mtDNA for 2, 4, 8 and 24 h. Histological analysis of lung tissue was undertaken following hematoxylin and eosin staining, and cytokine levels were assessed by ELISA. NF-κB and IκB-α phosphorylation levels, as well as TLR9 protein expression were determined by western blot analysis; NF-κB, IκB-α and TLR9 mRNA levels were analyzed by RT-PCR. A greater degree of inflammation and lung injury was observed in response to mtDNA. In addition, mtDNA increased serum tumor necrosis factor-α, interleukin (IL)-6 and IL-10 levels in vivo and increased their secretion by cultured macrophages (p<0.05). In lung tissue, mtDNA increased NF-κB, IκB-α and TLR9 mRNA levels (p<0.05); it also increased phosphorylated NF-κB p65 and TLR9 protein levels in the macrophage cultures. Thus, mtDNA may be part of the danger-associated molecular patterns, contributing to the initiation of sterile SIRS through the activation of the TLR9/NF-κB pathway and the induction of pro-inflammatory cytokine production.
| S-EPMC3976143 | biostudies-literature