Interactions of the Skin Pathogen Haemophilus ducreyi With the Human Host.

The obligate human pathogen Haemophilus ducreyi causes both cutaneous ulcers in children and sexually transmitted genital ulcers (chancroid) in adults. Pathogenesis is dependent on avoiding phagocytosis and exploiting the suppurative granuloma-like niche, which contains a myriad of innate immune cells and memory T cells. Despite this immune infiltrate, long-lived immune protection does not develop against repeated H. ducreyi infections-even with the same strain. Most of what we know about infectious skin diseases comes from naturally occurring infections and/or animal models; however, for H. ducreyi, this information comes from an experimental model of infection in human volunteers that was developed nearly three decades ago. The model mirrors the progression of natural disease and serves as a valuable tool to determine the composition of the immune cell infiltrate early in disease and to identify host and bacterial factors that are required for the establishment of infection and disease progression. Most recently, holistic investigation of the experimentally infected skin microenvironment using multiple "omics" techniques has revealed that non-canonical bacterial virulence factors, such as genes involved in central metabolism, may be relevant to disease progression. Thus, the immune system not only defends the host against H. ducreyi, but also dictates the nutrient availability for the invading bacteria, which must adapt their gene expression to exploit the inflammatory metabolic niche. These findings have broadened our view of the host-pathogen interaction network from considering only classical, effector-based virulence paradigms to include adaptations to the metabolic environment. How both host and bacterial factors interact to determine infection outcome is a current focus in the field. Here, we review what we have learned from experimental H. ducreyi infection about host-pathogen interactions, make comparisons to what is known for other skin pathogens, and discuss how novel technologies will deepen our understanding of this infection.

A potential contribution of antimicrobial peptide LL-37 to tissue fibrosis and vasculopathy in systemic sclerosis.

BACKGROUND: LL-37 is an antimicrobial peptide with pleiotropic effects on the immune system, angiogenesis and tissue remodelling. These are cardinal pathological events in systemic sclerosis (SSc). OBJECTIVES: To elucidate the potential role of LL-37 in SSc. METHODS: The expression of target molecules was evaluated by immunostaining and quantitative reverse-transcription real-time polymerase chain reaction in human and murine skin. The mechanisms regulating LL-37 expression in endothelial cells were examined by gene silencing and chromatin immunoprecipitation. Serum LL-37 levels were determined by enzyme-linked immunosorbent assay. RESULTS: In SSc lesional skin, LL-37 expression was increased in dermal fibroblasts, perivascular inflammatory cells, keratinocytes and, particularly, dermal small vessels. Expression positively correlated with interferon-α expression, possibly reflecting LL-37-dependent induction of interferon-α. In SSc animal models, bleomycin-treated skin exhibited the expression pattern of CRAMP, a murine homologue of LL-37, similar to that of LL-37 in SSc lesional skin. Furthermore, Fli1+/- mice showed upregulated expression of CRAMP in dermal small vessels. Fli1 binding to the CAMP (LL-37 gene) promoter and Fli1 deficiency-dependent induction of LL-37 were also confirmed in human dermal microvascular endothelial cells. In the analysis of sera, patients with SSc had serum LL-37 levels significantly higher than in healthy controls. Furthermore, serum LL-37 levels positively correlated with skin score and the activity of alveolitis and were significantly elevated in patients with digital ulcers compared with those without. CONCLUSIONS: LL-37 upregulation, induced by Fli1 deficiency at least in endothelial cells, potentially contributes to the development of skin sclerosis, interstitial lung disease and digital ulcers in SSc.

Amplifying renal immunity: the role of antimicrobial peptides in pyelonephritis.

Urinary tract infections (UTIs), including pyelonephritis, are among the most common and serious infections encountered in nephrology practice. UTI risk is increased in selected patient populations with renal and urinary tract disorders. As the prevalence of antibiotic-resistant uropathogens increases, novel and alternative treatment options will be needed to reduce UTI-associated morbidity. Discoveries over the past decade demonstrate a fundamental role for the innate immune system in protecting the urothelium from bacterial challenge. Antimicrobial peptides, an integral component of this urothelial innate immune system, demonstrate potent bactericidal activity toward uropathogens and might represent a novel class of UTI therapeutics. The urothelium of the bladder and the renal epithelium secrete antimicrobial peptides into the urinary stream. In the kidney, intercalated cells--a cell-type involved in acid-base homeostasis--have been shown to be an important source of antimicrobial peptides. Intercalated cells have therefore become the focus of new investigations to explore their function during pyelonephritis and their role in maintaining urinary tract sterility. This Review provides an overview of UTI pathogenesis in the upper and lower urinary tract. We describe the role of intercalated cells and the innate immune response in preventing UTI, specifically highlighting the role of antimicrobial peptides in maintaining urinary tract sterility.

Cathelicidin LL-37 induces time-resolved release of LTB4 and TXA2 by human macrophages and triggers eicosanoid generation in vivo.

In humans, LL-37 and eicosanoids are important mediators of inflammation and immune responses. Here we report that LL-37 promotes leukotriene B4 (LTB4) and thromboxane A2 (TXA2) generation by human monocyte-derived macrophages (HMDMs). LL-37 evokes calcium mobilization apparently via the P2X7 receptor (P2X7R), activation of ERK1/2 and p38 MAPKs, as well as cytosolic phospholipase A2 (cPLA2) and 5-lipoxygenase in HMDMs, leading to an early (1 h) release of LTB4. Similarly, TXA2 production at an early time involved the same signaling sequence along an LL-37-P2X7R-cPLA2-cyclooxygenase-1 (COX-1) axis. However, at later (6-8 h) time points, internalized LL-37 up-regulates COX-2 expression, promoting TXA2 production. Furthermore, intraperitoneal injection of mice with murine cathelicidin-related antimicrobial peptide (mCRAMP) induces significantly higher levels of LTB4 and TXA2 in mouse ascites rich in macrophages. Conversely, cathelicidin-deficient (Cnlp(-/-)) mice produce much less LTB4 and TXB2 in vivo in response to TNF-α compared with control mice. We conclude that LL-37 elicits a biphasic release of eicosanoids in macrophages with early, Ca(2+)-dependent formation of LTB4 and TXA2 followed by a late peak of TXA2, generated via induction of COX-2 by internalized LL-37, thus allowing eicosanoid production in a temporally controlled manner. Moreover, our findings provide evidence that LL-37 is an endogenous regulator of eicosanoid-dependent inflammatory responses in vivo.

Antimicrobial cathelicidin peptide LL-37 inhibits the LPS/ATP-induced pyroptosis of macrophages by dual mechanism.

Pyroptosis is a caspase-1 dependent cell death, associated with proinflammatory cytokine production, and is considered to play a crucial role in sepsis. Pyroptosis is induced by the two distinct stimuli, microbial PAMPs (pathogen associated molecular patterns) and endogenous DAMPs (damage associated molecular patterns). Importantly, cathelicidin-related AMPs (antimicrobial peptides) have a role in innate immune defense. Notably, human cathelicidin LL-37 exhibits the protective effect on the septic animal models. Thus, in this study, to elucidate the mechanism for the protective action of LL-37 on sepsis, we utilized LPS (lipopolysaccharide) and ATP (adenosine triphosphate) as a PAMP and a DAMP, respectively, and examined the effect of LL-37 on the LPS/ATP-induced pyroptosis of macrophage-like J774 cells. The data indicated that the stimulation of J774 cells with LPS and ATP induces the features of pyroptosis, including the expression of IL-1ß mRNA and protein, activation of caspase-1, inflammasome formation and cell death. Moreover, LL-37 inhibits the LPS/ATP-induced IL-1ß expression, caspase-1 activation, inflammasome formation, as well as cell death. Notably, LL-37 suppressed the LPS binding to target cells and ATP-induced/P2X7-mediated caspase-1 activation. Together these observations suggest that LL-37 potently inhibits the LPS/ATP-induced pyroptosis by both neutralizing the action of LPS and inhibiting the response of P2X7 to ATP. Thus, the present finding may provide a novel insight into the modulation of sepsis utilizing LL-37 with a dual action on the LPS binding and P2X7 activation.

Roles of cathelicidin-related antimicrobial peptide in murine osteoclastogenesis.

Cathelicidin-related antimicrobial peptide (CRAMP) not only kills bacteria but also binds to lipopolysaccharide (LPS) to neutralize its activity. CRAMP is highly expressed in bone marrow and its expression is reported to be up-regulated by inflammatory and infectious stimuli. Here, we examined the role of CRAMP in murine osteoclastogenesis. Osteoclasts were formed in co-cultures of osteoblasts and bone marrow cells in response to 1α,25-dihydroxyvitamin D3 [1α,25(OH)2 D3 ], prostaglandin E2 (PGE2 ), and Toll-like receptor (TLR) ligands such as LPS and flagellin through the induction of receptor activator of nuclear factor-κB ligand (RANKL) expression in osteoblasts. CRAMP inhibited the osteoclastogenesis in co-cultures treated with LPS and flagellin, but not in those treated with 1α,25(OH)2 D3 or PGE2 . Although bone marrow macrophages (BMMs) highly expressed formyl peptide receptor 2 (a receptor of CRAMP), CRAMP showed no inhibitory effect on osteoclastogenesis in BMM cultures treated with RANKL. CRAMP suppressed both LPS- and flagellin-induced RANKL expression in osteoblasts and tumour necrosis factor-α (TNF-α) expression in BMMs, suggesting that CRAMP neutralizes the actions of LPS and flagellin. LPS and flagellin enhanced the expression of CRAMP mRNA in osteoblasts. Extracellularly added CRAMP suppressed LPS- and flagellin-induced CRAMP expression. These results suggest that the production of CRAMP promoted by LPS and flagellin is inhibited by CRAMP released by osteoblasts through a feedback regulation. Even though CRAMP itself has no effect on osteoclastogenesis in mice, we propose that CRAMP is an osteoblast-derived protector in bacterial infection-induced osteoclastic bone resorption.

The human antimicrobial peptide LL-37, but not the mouse ortholog, mCRAMP, can stimulate signaling by poly(I:C) through a FPRL1-dependent pathway.

LL-37 is an antimicrobial peptide produced by human cells that can down-regulate the lipopolysaccharide-induced innate immune responses and up-regulate double-stranded (ds) RNA-induced innate responses through Toll-like receptor 3 (TLR3). The murine LL-37 ortholog, mCRAMP, also inhibited lipopolysaccharide-induced responses, but unlike LL-37, it inhibited viral-induced responses in mouse cells. A fluorescence polarization assay showed that LL-37 was able to bind dsRNA better than mCRAMP. In the human lung epithelial cell line BEAS-2B, LL-37, but not mCRAMP, colocalized with TLR3, and the colocalization was increased in the presence of dsRNA. The presence of poly(I:C) increased the accumulation of LL-37 in Rab5 endosomes. Signaling by cells induced with both LL-37 and poly(I:C) was sensitive to inhibitors that affect clathrin-independent trafficking, whereas signaling by poly(I:C) alone was not, suggesting that the LL-37-poly(I:C) complex trafficked to signaling endosomes by a different mechanism than poly(I:C) alone. siRNA knockdown of known LL-37 receptors identified that FPRL1 was responsible for TLR3 signaling induced by LL-37-poly(I:C). These results show that LL-37 and mCRAMP have different activities in TLR3 signaling and that LL-37 can redirect trafficking of poly(I:C) to effect signaling by TLR3 in early endosomes in a mechanism that involves FPRL1.

Susceptibility to vaccinia virus infection and spread in mice is determined by age at infection, allergen sensitization and mast cell status.

BACKGROUND: Patients, especially young children, with atopic dermatitis are at an increased risk of developing eczema vaccinatum, a severe reaction to the smallpox vaccine, either through direct vaccination or indirect contact with a person recently vaccinated. METHODS: Using a mouse model of infection, the severity of vaccinia-induced lesions was assessed from their appearance and viral DNA content. The response to vaccinia inoculation was assessed in young and adult mice, allergen-sensitized mice, and in mast cell-deficient mice. RESULTS: Young age, sensitization to an allergen prior to infection, and a mast cell deficit, accomplished by using mast cell-deficient mice, resulted in more severe viral lesions at the site of inoculation, according to lesion appearance and viral DNA content. All three factors combined demonstrated maximal susceptibility, characterized by the severity of primary lesions and the development of secondary (satellite) lesions, as occurs in eczema vaccinatum in humans. Resistance to the appearance of satellite lesions could be restored by adoptive transfer of bone marrow-derived mast cells from either wild-type or cathelicidin-related antimicrobial peptide-deficient mice. Primary lesions were more severe following the latter transfer, indicating that cathelicidin-related antimicrobial peptide does contribute to the protective activity of mast cells against infection. CONCLUSIONS: The combination of young age, allergen sensitization and a mast cell deficit resulted in the most severe lesions, including satellite lesions. Understanding the factors determining the relative resistance/sensitivity to vaccinia virus will aid in the development of strategies for preventing and treating adverse reactions which can occur after smallpox vaccination.

LL37 and hBD-3 elevate the ß-1,3-exoglucanase activity of Candida albicans Xog1p, resulting in reduced fungal adhesion to plastic.

The opportunistic fungus Candida albicans causes oral thrush and vaginal candidiasis, as well as candidaemia in immunocompromised patients including those undergoing cancer chemotherapy, organ transplant and those with AIDS. We previously found that the AMPs (antimicrobial peptides) LL37 and hBD-3 (human ß-defensin-3) inhibited C. albicans viability and its adhesion to plastic. For the present study, the mechanism by which LL37 and hBD-3 reduced C. albicans adhesion was investigated. After AMP treatment, C. albicans adhesion to plastic was reduced by up to ~60% and was dose-dependent. Our previous study indicated that LL37 might interact with the cell-wall ß-1,3-exoglucanase Xog1p, which is involved in cell-wall ß-glucan metabolism, and consequently the binding of LL37 or hBD-3 to Xog1p might cause the decrease in adhesion. For the present study, Xog1p(41-438)-6H, an N-terminally truncated, active, recombinant construct of Xog1p and Xog1p fragments were produced and used in pull-down assays and ELISA in vitro, which demonstrated that all constructs interacted with both AMPs. Enzymatic analyses showed that LL37 and hBD-3 enhanced the ß-1,3-exoglucanase activity of Xog1p(41-438)-6H approximately 2-fold. Therefore elevated Xog1p activity might compromise cell-wall integrity and decrease C. albicans adhesion. To test this hypothesis, C. albicans was treated with 1.3 µM Xog1p(41-438)-6H and C. albicans adhesion to plastic decreased 47.7%. Taken together, the evidence suggests that Xog1p is one of the LL37/hBD-3 targets, and elevated ß-1,3-exoglucanase activity reduces C. albicans adhesion to plastic.

Cathelicidin LL-37 increases lung epithelial cell stiffness, decreases transepithelial permeability, and prevents epithelial invasion by Pseudomonas aeruginosa.

In addition to its antibacterial activity, the cathelicidin-derived LL-37 peptide induces multiple immunomodulatory effects on host cells. Atomic force microscopy, F-actin staining with phalloidin, passage of FITC-conjugated dextran through a monolayer of lung epithelial cells, and assessment of bacterial outgrowth from cells subjected to Pseudomonas aeruginosa infection were used to determine LL-37's effect on epithelial cell mechanical properties, permeability, and bacteria uptake. A concentration-dependent increase in stiffness and F-actin content in the cortical region of A549 cells and primary human lung epithelial cells was observed after treatment with LL-37 (0.5-5 µM), sphingosine 1-phosphate (1 µM), or LPS (1 µg/ml) or infection with PAO1 bacteria. Other cationic peptides, such as RK-31, KR-20, or WLBU2, and the antibacterial cationic steroid CSA-13 did not reproduce the effect of LL-37. A549 cell pretreatment with WRW4, an antagonist of the transmembrane formyl peptide receptor-like 1 protein attenuated LL-37's ability to increase cell stiffness. The LL-37-mediated increase in cell stiffness was accompanied by a decrease in permeability and P. aeruginosa uptake by a confluent monolayer of polarized normal human bronchial epithelial cells. These results suggested that the antibacterial effect of LL-37 involves an LL-37-dependent increase in cell stiffness that prevents epithelial invasion by bacteria.

[Structure, function and molecular design strategies of antibacterial peptide SMAP-29: a review].

Antibacterial peptides are a family of host-defense peptides most of which are gene-encoded and produced by living organisms of all types. Antibacterial peptides are small molecular proteins with broad antimicrobial spectrum against bacteria, viruses, fungi and sometimes even as anticancer peptide. SMAP-29, a cathelicidin-like peptide derived from sheep myeloid, line alpha-helical Structure, exerts a powerful broad antimicrobial activity against different pathogens including Gram-positive and Gram-negative bacteria, fungi, viruses, parasites, spirochaetes, chlamydia and antiendotoxin activity, and particular antibacterial mechanism, rapidly to permeabilize membranes of susceptible organisms. This paper summarizes the lately research progress of SMAP-29 and Variants including the characteristics of structure, structure-activity relationships, mode of action, diverse biological functions, gene recombinant and expression. We put emphasis on the necessity of molecular design, and primary and secondary structure-based modification, to provides a strong foundation for further drug development and design of SMAP-29.

Antibacterial peptides and gastrointestinal diseases.

Defensins and cathelicidins are small cationic peptides produced by neutrophils and epithelial cells. They are highly expressed during infection. The role of constitutive and inducible antibacterial peptides has been extensively studied over the recent years; especially in the gastrointestinal (GI) tract, where the balance between the luminal bacteria and antibacterial peptides is crucial in the maintenance of a healthy GI tract. There are reports showing that the expressions of defensins and cathelicidins in the gut are dysregulated in various disease states. They could participate in the development of different disorders ranging from inflammation to cancer. Experimental findings showed that supplementation with animal cathelicidin promoted gastric ulcer healing in rats and suppressed tumorigenesis of gastric cancer in mice. Mouse cathelicidin could alleviate murine colitis by preserving mucus content and suppression of apoptosis. Other clinical applications for these antibacterial peptides are awaiting for further studies.

Uropathogenic Escherichia coli modulates immune responses and its curli fimbriae interact with the antimicrobial peptide LL-37.

Bacterial growth in multicellular communities, or biofilms, offers many potential advantages over single-cell growth, including resistance to antimicrobial factors. Here we describe the interaction between the biofilm-promoting components curli fimbriae and cellulose of uropathogenic E. coli and the endogenous antimicrobial defense in the urinary tract. We also demonstrate the impact of this interplay on the pathogenesis of urinary tract infections. Our results suggest that curli and cellulose exhibit differential and complementary functions. Both of these biofilm components were expressed by a high proportion of clinical E. coli isolates. Curli promoted adherence to epithelial cells and resistance against the human antimicrobial peptide LL-37, but also increased the induction of the proinflammatory cytokine IL-8. Cellulose production, on the other hand, reduced immune induction and hence delayed bacterial elimination from the kidneys. Interestingly, LL-37 inhibited curli formation by preventing the polymerization of the major curli subunit, CsgA. Thus, even relatively low concentrations of LL-37 inhibited curli-mediated biofilm formation in vitro. Taken together, our data demonstrate that biofilm components are involved in the pathogenesis of urinary tract infections by E. coli and can be a target of local immune defense mechanisms.

The human cathelicidin hCAP18/LL-37: a multifunctional peptide involved in mycobacterial infections.

Antimicrobial peptides are predominantly small cationic polypeptides that are classified together on the basis of these molecules to directly kill or inhibit the growth of microorganisms including mycobacteria, and to activate mechanisms of cellular and adaptive immunity. Various families of antimicrobial peptides have been identified, including the cathelicidins. The cathelicidin family is characterised by a conserved N-terminal cathelin domain and a variable C-terminal antimicrobial domain that can be released from the precursor protein after cleavage by proteinases. LL-37 is the C-terminal part of the only human cathelicidin identified to date called human cationic antimicrobial protein (hCAP18), which is mainly expressed by neutrophils and epithelial cells. The cathelicidin hCAP18/LL-37 is a multifunctional molecule that may mediate various host responses, including bactericidal action, chemotaxis, epithelial cell activation, angiogenesis, epithelial wound repair and activation of chemokine secretion. The antimicrobial peptide LL-37 is produced from human cells during infection of mycobacteria and exerts a microbicidal effect. The discussion will (1) describe recent work on the antimicrobial and immunomodulatory functions of the cathelicidin hCAP18/LL-37, (2) highlight the effectiveness of the cathelicidin hCAP18/LL-37 as a potent component in antimycobacterial immune responses and (3) summarise current progress in the understanding of the therapeutic application of hCAP18/LL-37 and its derivates antimicrobial peptides in mycobacterial infection.

Antimicrobial peptides in oral cancer.

There is increasing evidence that antimicrobial peptides (AMPs) are differentially regulated in cancers such as oral squamous cell carcinomas (OSCC). Data showing that AMPs influence the growth of tumor cells, exhibit direct cytotoxic activity towards cancer cells, function as a tumor suppressor gene or activate the adaptive immunity suggest that a dysregulation of AMPs may be associated with the development of cancer. There is no question that, with increasing resistance against conventional chemotherapy, novel anticancer agents are needed. It is interesting to speculate that natural AMP or synthetic derivatives can be used to develop novel strategies to fight cancer diseases and may represent a novel family of anticancer agents. However, future research is needed to employ the role of AMPs in cancer and to investigate their role as potential anticancer drugs.