Innate immunity is the first line of defense against microorganisms in vertebrates and acts by providing an initial barrier to microorganisms and triggering adaptive immune responses. Peptides such as beta-defensins are an important component of this defense, providing a broad spectrum of antimicrobial activity against bacteria, fungi, mycobacteria and several enveloped viruses. Beta-defensins are small cationic peptides that vary in their expression patterns and spectrum of pathogen specificity. Disruptions in beta-defensin function have been implicated in human diseases, including cystic fibrosis, and a fuller understanding of the variety, function and evolution of human beta-defensins might form the basis for novel therapies. Here we use a combination of laboratory and computational techniques to characterize the main human beta-defensin locus on chromosome 8p22-p23.

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.

β-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 β-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 β-defensins are essential for successful sperm maturation, and their disruption leads to alteration in intracellular calcium, inappropriate spontaneous acrosome reaction and profound male infertility.

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.

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 β-galactosidase (β-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 β-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.

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-1β-mediated mechanism, independent of effects on claudin-1. Induction of keratinocyte expression of the antimicrobial/host defense peptide human β-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.

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.

A major boost to the cystic fibrosis disease research was given by the generation of various mouse models using gene targeting in embryonal stem cells. Moreover, the introduction of the same mutation on different inbred strains generating congenic strains facilitated the search for modifier genes. From the original CftrTgH(neoim)Hgu CF mouse model we have generated using strict brother x sister mating two inbred CftrTgH(neoim)Hgu mouse lines (CF/1 and CF/3). Thereafter, the insertional mutation was introgressed from CF/3 into three inbred backgrounds (C57BL/6, BALB/c, DBA/2J) generating congenic animals. In every backcross cycle germline transmission of the insertional mutation was monitored by direct probing the insertion via Southern RFLP. In order to bypass this time consuming procedure we devised an alternative PCR based protocol whereby mouse strains are differentiated at the Cftr locus by Cftr intragenic microsatellite genotypes that are tightly linked to the disrupted locus.

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.

The neuropeptides pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are implicated in the photic entrainment of circadian rhythms in the suprachiasmatic nuclei (SCN). We now report that mice carrying a null mutation of the VPAC(2) receptor for VIP and PACAP (Vipr2(-/-)) are incapable of sustaining normal circadian rhythms of rest/activity behavior. These mice also fail to exhibit circadian expression of the core clock genes mPer1, mPer2, and mCry1 and the clock-controlled gene arginine vasopressin (AVP) in the SCN. Moreover, the mutants fail to show acute induction of mPer1 and mPer2 by nocturnal illumination. This study highlights the role of intercellular neuropeptidergic signaling in maintenance of circadian function within the SCN.

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-1β 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.

Adult-derived bone marrow stem cells are capable of reconstituting the haematopoietic system. However there is ongoing debate in the literature as to whether bone marrow derived cells have the ability to populate other tissues and express tissue specific markers. The airway has been an organ of major interest and was one of the first where this was demonstrated. We have previously demonstrated that the mouse airway can be repopulated by side population bone marrow transplanted cells. Here we investigate the frequency and phenotypic nature of these bone marrow derived cells.

Beta-defensins are antimicrobial peptides with an essential role in the innate immune response. In addition beta-defensins can also chemoattract cells involved in adaptive immunity. Until now, based on evidence from dendritic cell stimulation, human beta defensin-3 (hBD3) was considered pro-inflammatory. We present evidence here that hBD3 lacks pro-inflammatory activity in human and mouse primary Mphi. In addition, in the presence of LPS, hBD3 and the murine orthologue Defb14 (but not hBD2), effectively inhibit TNF-alpha and IL-6 accumulation implying an anti-inflammatory function. hBD3 also inhibits CD40/IFN-gamma stimulation of Mphi and in vivo, hBD3 significantly reduces the LPS-induced TNF-alpha level in serum. Recent work has revealed that hBD3 binds melanocortin receptors but we provide evidence that these are not involved in hBD3 immunomodulatory activity. This implies a dual role for hBD3 in antimicrobial activity and resolution of inflammation.

β-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 β-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.

We have examined the evolution of the genes at the major human beta-defensin locus and the orthologous loci in a range of other primates and mouse. For the first time these data allow us to examine selective episodes in the more recent evolutionary history of this locus as well as the ancient past. We have used a combination of maximum likelihood based tests and a maximum parsimony based sliding window approach to give a detailed view of the varying modes of selection operating at this locus.

Human β-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 β-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-β 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-β. Mice expressing a transgene encoding hBD3 had elevated basal levels of Interferon-β, 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.

Human β-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.