A mechanistic evaluation of human beta defensin 2 mediated protection of human skin barrier in vitro.

The human skin barrier, a biological imperative, is impaired in inflammatory skin diseases such as atopic dermatitis (AD). Staphylococcus aureus is associated with AD lesions and contributes to pathological inflammation and further barrier impairment. S. aureus secretes extracellular proteases, such as V8 (or 'SspA'), which cleave extracellular proteins to reduce skin barrier. Previous studies demonstrated that the host defence peptide human beta-defensin 2 (HBD2) prevented V8-mediated damage. Here, the mechanism of HBD2-mediated barrier protection in vitro is examined. Application of exogenous HBD2 provided protection against V8, irrespective of timeline of application or native peptide folding, raising the prospect of simple peptide analogues as therapeutics. HBD2 treatment, in context of V8-mediated damage, modulated the proteomic/secretomic profiles of HaCaT cells, altering levels of specific extracellular matrix proteins, potentially recovering V8 damage. However, HBD2 alone did not substantially modulate cellular proteomic/secretomics profiles in the absence of damage, suggesting possible therapeutic targeting of lesion damage sites only. HBD2 did not show any direct protease inhibition or induce expression of known antiproteases, did not alter keratinocyte migration or proliferation, or form protective nanonet structures. These data validate the barrier-protective properties of HBD2 in vitro and establish key protein datasets for further targeted mechanistic analyses.

The Dichotomous Responses Driven by ß-Defensins.

Defensins are short, rapidly evolving, cationic antimicrobial host defence peptides with a repertoire of functions, still incompletely realised, that extends beyond direct microbial killing. They are released or secreted at epithelial surfaces, and in some cases, from immune cells in response to infection and inflammation. Defensins have been described as endogenous alarmins, alerting the body to danger and responding to inflammatory signals by promoting both local innate and adaptive systemic immune responses. However, there is now increasing evidence that they exert variable control on the response to danger; creating a dichotomous response that can suppress inflammation in some circumstances but exacerbate the response to danger and damage in others and, at higher levels, lead to a cytotoxic effect. Focussing in this review on human ß-defensins, we discuss the evidence for their functions as proinflammatory, immune activators amplifying the response to infection or damage signals and/or as mediators of resolution of damage, contributing to a return to homeostasis. Finally, we consider their involvement in the development of autoimmune diseases.

Cathelicidins and the Onset of Labour.

Preterm birth, defined as delivery before 37 weeks of gestation, is the leading cause of neonatal mortality and morbidity. Infection and inflammation are frequent antecedents of spontaneous preterm birth. Cathelicidin, an antimicrobial host defence peptide, is induced by infection and inflammation and although expressed in the reproductive tract and fetal tissues, its role in the pathogenesis of spontaneous preterm birth is unknown. Here we demonstrate that cathelicidin expression is increased at RNA and protein level in the mouse uterus in a model of inflammation-induced labour, where ultrasound guided intrauterine injection of lipopolysaccharide (LPS) at E17 stimulates preterm delivery within 24 hours. Cathelicidin-deficient (Camp-/-) mice are less susceptible to preterm delivery than wild type mice following intrauterine injection of 1 µg of LPS, and this is accompanied by a decrease in circulating IL-6, an inflammatory mediator implicated in the onset of labour. We also show that the proportion of cathelicidin expressing cells in the myometrium is higher in samples obtained from women in labour at term than pre-labour. Together, these data suggest that cathelicidin has roles in mediating pro-inflammatory responses in a murine model of inflammation-induced labour, and in human term labour.

Cathelicidin is a "fire alarm", generating protective NLRP3-dependent airway epithelial cell inflammatory responses during infection with Pseudomonas aeruginosa.

Pulmonary infections are a major global cause of morbidity, exacerbated by an increasing threat from antibiotic-resistant pathogens. In this context, therapeutic interventions aimed at protectively modulating host responses, to enhance defence against infection, take on ever greater significance. Pseudomonas aeruginosa is an important multidrug-resistant, opportunistic respiratory pathogen, the clearance of which can be enhanced in vivo by the innate immune modulatory properties of antimicrobial host defence peptides from the cathelicidin family, including human LL-37. Initially described primarily as bactericidal agents, cathelicidins are now recognised as multifunctional antimicrobial immunomodulators, modifying host responses to pathogens, but the key mechanisms involved in these protective functions are not yet defined. We demonstrate that P. aeruginosa infection of airway epithelial cells promotes extensive infected cell internalisation of LL-37, in a manner that is dependent upon epithelial cell interaction with live bacteria, but does not require bacterial Type 3 Secretion System (T3SS). Internalised LL-37 acts as a second signal to induce inflammasome activation in airway epithelial cells, which, in contrast to myeloid cells, are relatively unresponsive to P. aeruginosa. We demonstrate that this is mechanistically dependent upon cathepsin B release, and NLRP3-dependent activation of caspase 1. These result in LL-37-mediated release of IL-1ß and IL-18 in a manner that is synergistic with P. aeruginosa infection, and can induce caspase 1-dependent death of infected epithelial cells, and promote neutrophil chemotaxis. We propose that cathelicidin can therefore act as a second signal, required by P. aeruginosa infected epithelial cells to promote an inflammasome-mediated altruistic cell death of infection-compromised epithelial cells and act as a "fire alarm" to enhance rapid escalation of protective inflammatory responses to an uncontrolled infection. Understanding this novel modulatory role for cathelicidins, has the potential to inform development of novel therapeutic strategies to antibiotic-resistant pathogens, harnessing innate immunity as a complementation or alternative to current interventions.

Discovery and mechanisms of host defense to oncogenesis: targeting the ß-defensin-1 peptide as a natural tumor inhibitor.

Human beta-defensin-1 (hBD-1) is one of a number of small cationic host-defense peptides. Besides its well-known broad-spectrum antimicrobial function, hBD-1 has recently been identified as a chromosome 8p tumor-suppressor gene. The role of hBD-1 in modulating the host immune response to oncogenesis, associated with cell signaling and potential therapeutic applications, has become increasingly appreciated over time. In this study, multiple approaches were used to illustrate hBD-1 anti-tumor activities. Results demonstrate that hBD-1 peptide alters human epidermal growth factor receptor 2 (HER2) signal transduction and represses retroviral-mediated transgene expression in cancer cells. Loss of orthologous murine defense-1 (mBD1) in mice enhances nickel sulfate-induced leiomyosarcoma and causes mouse kidney cells to exhibit increased susceptibility to HPV-16 E6/7-induced neoplastic transformation. Furthermore, for the first time, a novel function of the urine-derived hBD-1 peptide was discovered to suppress bladder cancer growth and this may lead to future applications in the treatment of malignancy.

Human ß-defensin 3 increases the TLR9-dependent response to bacterial DNA.

Human ß-defensin 3 (hBD3) is a cationic antimicrobial peptide with potent bactericidal activity in vitro. HBD3 is produced in response to pathogen challenge and can modulate immune responses. The amplified recognition of self-DNA by human plasmacytoid dendritic cells has been previously reported, but we show here that hBD3 preferentially enhances the response to bacterial DNA in mouse Flt-3 induced dendritic cells (FLDCs) and in human peripheral blood mononuclear cells. We show the effect is mediated through TLR9 and although hBD3 significantly increases the cellular uptake of both E. coli and self-DNA in mouse FLDCs, only the response to bacterial DNA is enhanced. Liposome transfection also increases uptake of bacterial DNA and amplifies the TLR9-dependent response. In contrast to hBD3, lipofection of self-DNA enhances inflammatory signaling, but the response is predominantly TLR9-independent. Together, these data show that hBD3 has a role in the innate immune-mediated response to pathogen DNA, increasing inflammatory signaling and promoting activation of the adaptive immune system via antigen presenting cells including dendritic cells. Therefore, our data identify an additional immunomodulatory role for this copy-number variable defensin, of relevance to host defence against infection and indicate a potential for the inclusion of HBD3 in pathogen DNA-based vaccines.

IL-1ß-Induced Protection of Keratinocytes against Staphylococcus aureus-Secreted Proteases Is Mediated by Human ß-Defensin 2.

Atopic dermatitis (AD) is a common chronic inflammatory skin disease that results in significant morbidity. A hallmark of AD is disruption of the critical barrier function of upper epidermal layers, causatively linked to environmental stimuli, genetics, and infection, and a critical current target for the development of new therapeutic and prophylactic interventions. Staphylococcus aureus is an AD-associated pathogen producing virulence factors that induce skin barrier disruption in vivo and contribute to AD pathogenesis. We show, using immortalized and primary keratinocytes, that S. aureus protease SspA/V8 is the dominant secreted factor (in laboratory and AD clinical strains of S. aureus) inducing barrier integrity impairment and tight junction damage. V8-induced integrity damage was inhibited by an IL-1ß-mediated mechanism, independent of effects on claudin-1. Induction of keratinocyte expression of the antimicrobial/host defense peptide human ß-defensin 2 (hBD2) was found to be the mechanism underpinning this protective effect. Endogenous hBD2 expression was required and sufficient for protection against V8 protease-mediated integrity damage, and exogenous application of hBD2 was protective. This modulatory property of hBD2, unrelated to antibacterial effects, gives new significance to the defective induction of hBD2 in the barrier-defective skin lesions of AD and indicates therapeutic potential.

Correction: Human ß-D-3 Exacerbates MDA5 but Suppresses TLR3 Responses to the Viral Molecular Pattern Mimic Polyinosinic:Polycytidylic Acid.

[This corrects the article DOI: 10.1371/journal.pgen.1005673.].

Human ß-Defensin 3 [corrected] Exacerbates MDA5 but Suppresses TLR3 Responses to the Viral Molecular Pattern Mimic Polyinosinic:Polycytidylic Acid.

Human ß-defensin 3 (hBD3) is a cationic host defence peptide and is part of the innate immune response. HBD3 is present on a highly copy number variable block of six ß-defensin genes, and increased copy number is associated with the autoimmune disease psoriasis. It is not known how this increase influences disease development, but psoriasis is a T cell-mediated disease and activation of the innate immune system is required for the initial trigger that leads to the amplification stage. We investigated the effect of hBD3 on the response of primary macrophages to various TLR agonists. HBD3 exacerbated the production of type I Interferon-ß in response to the viral ligand mimic polyinosinic:polycytidylic acid (polyI:C) in both human and mouse primary cells, although production of the chemokine CXCL10 was suppressed. Compared to polyI:C alone, mice injected with both hBD3 peptide and polyI:C also showed an enhanced increase in Interferon-ß. Mice expressing a transgene encoding hBD3 had elevated basal levels of Interferon-ß, and challenge with polyI:C further increased this response. HBD3 peptide increased uptake of polyI:C by macrophages, however the cellular response and localisation of polyI:C in cells treated contemporaneously with hBD3 or cationic liposome differed. Immunohistochemistry showed that hBD3 and polyI:C do not co-localise, but in the presence of hBD3 less polyI:C localises to the early endosome. Using bone marrow derived macrophages from knockout mice we demonstrate that hBD3 suppresses the polyI:C-induced TLR3 response mediated by TICAM1 (TRIF), while exacerbating the cytoplasmic response through MDA5 (IFIH1) and MAVS (IPS1/CARDIF). Thus, hBD3, a highly copy number variable gene in human, influences cellular responses to the viral mimic polyI:C implying that copy number may have a significant phenotypic effect on the response to viral infection and development of autoimmunity in humans.

Novel phenotype of mouse spermatozoa following deletion of nine ß-defensin genes.

ß-defensin peptides are a large family of antimicrobial peptides. Although they kill microbes in vitro and interact with immune cells, the precise role of these genes in vivo remains uncertain. Despite their inducible presence at mucosal surfaces, their main site of expression is the epididymis. Recent evidence suggests that a major function of these peptides is in sperm maturation. In addition to previous work suggesting this, work at the MRC Human Genetics Unit, Edinburgh, has shown that homozygous deletion of a cluster of nine ß-defensin genes in the mouse results in profound male sterility. The spermatozoa derived from the mutants had reduced motility and increased fragility. Epididymal spermatozoa isolated from the cauda region of the homozygous mutants demonstrated precocious capacitation and increased spontaneous acrosome reactions compared with those from wild-types. Despite this, these mutant spermatozoa had reduced ability to bind to the zona pellucida of oocytes. Ultrastructural examination revealed a disintegration of the microtubule structure of mutant-derived spermatozoa isolated from the epididymal cauda region, but not from the caput. Consistent with premature acrosome reaction and hyperactivation, spermatozoa from mutant animals had significantly increased intracellular calcium content. This work demonstrates that in vivo ß-defensins are essential for successful sperm maturation, and that their disruption alters intracellular calcium levels, which most likely leads to premature activation and spontaneous acrosome reactions that result in hyperactivation and loss of microtubule structure of the axoneme. Determining which of the nine genes are responsible for the phenotype and the relevance to human sperm function is important for future work on male infertility.

The RNA-editing enzyme ADAR1 controls innate immune responses to RNA.

The ADAR RNA-editing enzymes deaminate adenosine bases to inosines in cellular RNAs. Aberrant interferon expression occurs in patients in whom ADAR1 mutations cause Aicardi-Goutières syndrome (AGS) or dystonia arising from striatal neurodegeneration. Adar1 mutant mouse embryos show aberrant interferon induction and die by embryonic day E12.5. We demonstrate that Adar1 embryonic lethality is rescued to live birth in Adar1; Mavs double mutants in which the antiviral interferon induction response to cytoplasmic double-stranded RNA (dsRNA) is prevented. Aberrant immune responses in Adar1 mutant mouse embryo fibroblasts are dramatically reduced by restoring the expression of editing-active cytoplasmic ADARs. We propose that inosine in cellular RNA inhibits antiviral inflammatory and interferon responses by altering RLR interactions. Transfecting dsRNA oligonucleotides containing inosine-uracil base pairs into Adar1 mutant mouse embryo fibroblasts reduces the aberrant innate immune response. ADAR1 mutations causing AGS affect the activity of the interferon-inducible cytoplasmic isoform more severely than the nuclear isoform.

Importance of ß-defensins in sperm function.

Recent work in humans and mouse has confirmed the involvement of the host defence ß-defensin peptides in male fertility. We discuss here the work that has implicated ß-defensins in sperm function including the identification of the epididymis as the predominant site of expression of the peptides and the in vivo consequences of mutation and deletion. The potential dual role of these peptides in the regulation of infection and control of sperm maturation is compelling and may combine their antimicrobial activity with the ability of these molecules to interact with cell membrane receptors and modulate ion transport.

Development of five digits is controlled by a bipartite long-range cis-regulator.

Conservation within intergenic DNA often highlights regulatory elements that control gene expression from a long range. How conservation within a single element relates to regulatory information and how internal composition relates to function is unknown. Here, we examine the structural features of the highly conserved ZRS (also called MFCS1) cis-regulator responsible for the spatiotemporal control of Shh in the limb bud. By systematically dissecting the ZRS, both in transgenic assays and within in the endogenous locus, we show that the ZRS is, in effect, composed of two distinct domains of activity: one domain directs spatiotemporal activity but functions predominantly from a short range, whereas a second domain is required to promote long-range activity. We show further that these two domains encode activities that are highly integrated and that the second domain is crucial in promoting the chromosomal conformational changes correlated with gene activity. During limb bud development, these activities encoded by the ZRS are interpreted differently by the fore limbs and the hind limbs; in the absence of the second domain there is no Shh activity in the fore limb, and in the hind limb low levels of Shh lead to a variant digit pattern ranging from two to four digits. Hence, in the embryo, the second domain stabilises the developmental programme providing a buffer for SHH morphogen activity and this ensures that five digits form in both sets of limbs.

Partial deletion of chromosome 8 ß-defensin cluster confers sperm dysfunction and infertility in male mice.

ß-defensin peptides are a family of antimicrobial peptides present at mucosal surfaces, with the main site of expression under normal conditions in the male reproductive tract. Although they kill microbes in vitro and interact with immune cells, the precise role of these genes in vivo remains uncertain. We show here that homozygous deletion of a cluster of nine ß-defensin genes (DefbΔ9) in the mouse results in male sterility. The sperm derived from the mutants have reduced motility and increased fragility. Epididymal sperm isolated from the cauda should require capacitation to induce the acrosome reaction but sperm from the mutants demonstrate precocious capacitation and increased spontaneous acrosome reaction compared to wild-types but have reduced ability to bind the zona pellucida of oocytes. Ultrastructural examination reveals a defect in microtubule structure of the axoneme with increased disintegration in mutant derived sperm present in the epididymis cauda region, but not in caput region or testes. Consistent with premature acrosome reaction, sperm from mutant animals have significantly increased intracellular calcium content. Thus we demonstrate in vivo that ß-defensins are essential for successful sperm maturation, and their disruption leads to alteration in intracellular calcium, inappropriate spontaneous acrosome reaction and profound male infertility.

Enzymatic removal of ribonucleotides from DNA is essential for mammalian genome integrity and development.

The presence of ribonucleotides in genomic DNA is undesirable given their increased susceptibility to hydrolysis. Ribonuclease (RNase) H enzymes that recognize and process such embedded ribonucleotides are present in all domains of life. However, in unicellular organisms such as budding yeast, they are not required for viability or even efficient cellular proliferation, while in humans, RNase H2 hypomorphic mutations cause the neuroinflammatory disorder Aicardi-Goutières syndrome. Here, we report that RNase H2 is an essential enzyme in mice, required for embryonic growth from gastrulation onward. RNase H2 null embryos accumulate large numbers of single (or di-) ribonucleotides embedded in their genomic DNA (>1,000,000 per cell), resulting in genome instability and a p53-dependent DNA-damage response. Our findings establish RNase H2 as a key mammalian genome surveillance enzyme required for ribonucleotide removal and demonstrate that ribonucleotides are the most commonly occurring endogenous nucleotide base lesion in replicating cells.

ß-Defensins: multifunctional modulators of infection, inflammation and more?

Defensins comprise one of the largest groups of host defence peptides, present throughout evolution, in fungi and flowering plants as well as in invertebrates and vertebrates. These cysteine-rich, cationic peptides have a common ability to kill a broad range of microorganisms including bacteria, yeast and viruses. As such, they are a strong component of the arsenal that is an organism's innate immunity. It is becoming increasingly clear, however, that antimicrobial action is only one of the numerous roles of these multifunctional peptides. In recent years, the functions of defensins in immunomodulation have been widely investigated, and their involvement in other processes (such as fertility) is becoming evident. This review addresses recent advances in the immunomodulatory activity of ß-defensins as well as the involvement of ß-defensins in fertility, development, wound healing and cancer.

Rare corneal clones in mice suggest an age-related decrease of stem cell activity and support the limbal epithelial stem cell hypothesis.

The anterior ocular surface comprises the cornea, conjunctiva and a narrow intermediate region called the limbus. It is widely accepted that the corneal epithelium is maintained by stem cells but different hypotheses propose that the stem cells that maintain the mouse corneal epithelium during normal homeostasis are located either in the basal limbal epithelium or throughout the basal corneal epithelium. There are no specific markers to help test these alternatives and new methods are required to distinguish between them. We observed that KRT5(LacZ/-) transgenic mice produced rare ß-galactosidase (ß-gal)-positive radial stripes in the corneal epithelium. These stripes are likely to be clonal lineages of cells derived from stem cells, so they provide a lineage marker for actively proliferating stem cells. The distributions of the ß-gal-positive radial stripes suggested they extended centripetally from the limbus, supporting the limbal epithelial stem cell (LESC) hypothesis. Stripe frequency declined between 15 and 30 weeks, which predicts a reduction in stem cell function with age. Pax6(+/-), KRT5(LacZ/-) corneas had small patches rather than stripes, which confirms that corneal maintenance is abnormal in Pax6(+/-) mice.

Human ß-defensin 3 affects the activity of pro-inflammatory pathways associated with MyD88 and TRIF.

ß-Defensins are cationic host defense peptides that form an amphipathic structure stabilized by three intramolecular disulfide bonds. They are key players in innate and adaptive immunity and have recently been shown to limit the production of pro-inflammatory cytokines in TLR4-stimulated macrophages. In the present study, we investigate the mechanism underlying the anti-inflammatory effect of human ß-defensin 3 (hBD3). We show that the canonical structure of hBD3 is required for this immunosuppressive effect and that hBD3 rapidly associates with and enters macrophages. Examination of the global effect of hBD3 on transcription in TLR4-stimulated macrophages shows that hBD3 inhibits the transcription of pro-inflammatory genes. Among the altered genes there is significant enrichment of groups involved in the positive regulation of NF-κB including components of Toll-like receptor signaling pathways. We confirm these observations by showing corresponding decreases in protein levels of pro-inflammatory cytokines and cell surface molecules. In addition, we show that hBD3 reduces NF-κB signaling in cells transfected with MyD88 or TRIF and that hBD3 inhibits the TLR4 response in both MyD88- and TRIF-deficient macrophages. Taken together these findings suggest that the mechanism of hBD3 anti-inflammatory activity involves specific targeting of TLR signaling pathways resulting in transcriptional repression of pro-inflammatory genes.

Binding a heparin derived disaccharide to defensin inspired peptides: insights to antimicrobial inhibition from gas-phase measurements.

Due to the ubiquitous presence of polysaccharide moieties on bacterial surfaces, it is hypothesised that a peptide-saccharide interaction plays a key role during the recognition of invading microorganisms by beta-defensins. We have employed different gas-phase methods to investigate these interactions. This manuscript describes: an MS-based titration assay measuring the gas-phase binding of ten beta-defensin related peptides to a sulfated disaccharide derived from heparin (HDD); ion mobility-mass spectrometry-determined collision cross sections of 3 peptides (both free and binding HDD); and results from molecular modelling with the aim of reconciling some of our experimental observations. We observe a clear qualitative correlation between the antimicrobial activity of several beta-defensins and related peptides and their gas-phase binding to a heparin-derived disaccharide (HDD). Four of the ten peptides show >100 micromolar K(d) values with HDD, and no bacteriocidal activity, illustrating that HDD binding correlates with peptide antimicrobial activity. For five of the remaining six peptides, bacteriocidal activity was re-measured with HDD present. For the peptides containing intramolecular disulfide bonds in two out of five, bacteriocidal activity was reduced approximately 10-fold; for the remaining three peptides, which lack intramolecular disulfide bonds, HDD addition had little effect on bacteriocidal activity. The latter results are suggested to arise from the greater degree of flexibility imparted by the removal of disulfide bonds giving the peptides the ability to envelope HDD and assume a "defensin-like" fold. Thus gas-phase analysis is put forward as a powerful tool for assessing the properties of antimicrobial peptides providing valuable insights in the mechanism of antimicrobial inhibition.