Therapeutic S100A8/A9 blockade inhibits myocardial and systemic inflammation and mitigates sepsis-induced myocardial dysfunction.

BACKGROUND AND AIMS: The triggering factors of sepsis-induced myocardial dysfunction (SIMD) are poorly understood and are not addressed by current treatments. S100A8/A9 is a pro-inflammatory alarmin abundantly secreted by activated neutrophils during infection and inflammation. We investigated the efficacy of S100A8/A9 blockade as a potential new treatment in SIMD. METHODS: The relationship between plasma S100A8/A9 and cardiac dysfunction was assessed in a cohort of 62 patients with severe sepsis admitted to the intensive care unit of Linköping University Hospital, Sweden. We used S100A8/A9 blockade with the small-molecule inhibitor ABR-238901 and S100A9-/- mice for therapeutic and mechanistic studies on endotoxemia-induced cardiac dysfunction in mice. RESULTS: In sepsis patients, elevated plasma S100A8/A9 was associated with left-ventricular (LV) systolic dysfunction and increased SOFA score. In wild-type mice, 5 mg/kg of bacterial lipopolysaccharide (LPS) induced rapid plasma S100A8/A9 increase and acute LV dysfunction. Two ABR-238901 doses (30 mg/kg) administered intraperitoneally with a 6 h interval, starting directly after LPS or at a later time-point when LV dysfunction is fully established, efficiently prevented and reversed the phenotype, respectively. In contrast, dexamethasone did not improve cardiac function compared to PBS-treated endotoxemic controls. S100A8/A9 inhibition potently reduced systemic levels of inflammatory mediators, prevented upregulation of inflammatory genes and restored mitochondrial function in the myocardium. The S100A9-/- mice were protected against LPS-induced LV dysfunction to an extent comparable with pharmacologic S100A8/A9 blockade. The ABR-238901 treatment did not induce an additional improvement of LV function in the S100A9-/- mice, confirming target specificity. CONCLUSION: Elevated S100A8/A9 is associated with the development of LV dysfunction in severe sepsis patients and in a mouse model of endotoxemia. Pharmacological blockade of S100A8/A9 with ABR-238901 has potent anti-inflammatory effects, mitigates myocardial dysfunction and might represent a novel therapeutic strategy for patients with severe sepsis.

Osteopontin protects against pneumococcal infection in a murine model of allergic airway inflammation.

BACKGROUND: In atopic asthma, chronic Th2-biased inflammation is associated with an increased risk of pneumococcal infection. The anionic phosphoglycoprotein osteopontin (OPN) is highly expressed in asthma and has been ascribed several roles during inflammation. This study aimed to investigate whether OPN affects inflammation and vulnerability to pneumococcal infection in atopic asthma. METHODS: House dust mite (HDM) extract was used to induce allergic airway inflammation in both wild-type (Spp1+/+ ) and OPN knockout (Spp1-/- ) C57BL/6J mice, and the airway was then infected with Streptococcus pneumoniae. Parameters reflecting inflammation, tissue injury, and bacterial burden were measured. In addition, samples from humans with allergic asthma were analyzed. RESULTS: Both allergen challenge in individuals with allergic asthma and the intranasal instillation of HDM in mice resulted in increased OPN levels in bronchoalveolar lavage fluid (BALF). More immune cells (including alveolar macrophages, neutrophils, eosinophils, and lymphocytes) and higher levels of proinflammatory cytokines were found in Spp1-/- mice than in Spp1+/+ mice. Moreover, OPN-deficient mice exhibited increased levels of markers reflecting tissue injury. Upon infection with S. pneumoniae, Spp1+/+ mice with allergic airway inflammation had a significantly lower bacterial burden in both BALF and lung tissue than did Spp1-/- mice. Furthermore, Spp1-/- mice had higher levels of cytokines and immune cells in BALF than did Spp1+/+ mice. CONCLUSION: OPN reduces inflammation, decreases tissue injury, and reduces bacterial loads during concurrent pneumococcal infection and allergic airway inflammation in a murine model. These findings suggest that OPN significantly affects vulnerability to pneumococcal infection in atopic asthma.

DNA-fragmentation is a source of bactericidal activity against Pseudomonas aeruginosa.

Pseudomonas aeruginosa airway infection is common in cystic fibrosis (CF), a disease also characterized by abundant extracellular DNA (eDNA) in the airways. The eDNA is mainly derived from neutrophils accumulating in the airways and contributes to a high sputum viscosity. The altered environment in the lower airways also paves the way for chronic P. aeruginosa infection. Here, we show that mice with P. aeruginosa airway infection have increased survival and decreased bacterial load after topical treatment with DNase. Furthermore, DNA from the sputum of CF patients showed increased bactericidal activity after treatment with DNase ex vivo. Both degraded DNA of neutrophil extracellular traps (NETs) and genomic DNA degraded by serum, acquired bactericidal activity against P. aeruginosa In vitro, small synthetic DNA-fragments (<100 base pairs) but not large fragments nor genomic DNA, were bactericidal against Gram-negative but not Gram-positive bacteria. The addition of divalent cations reduced bacterial killing, suggesting that chelation of divalent cations by DNA results in destabilization of the lipopolysaccharide (LPS) envelope. This is a novel antibacterial strategy where fragmentation of eDNA and DNA-fragments can be used to treat P. aeruginosa airway infection.

The Nonantibiotic Macrolide EM703 Improves Survival in a Model of Quinolone-Treated Pseudomonas aeruginosa Airway Infection.

Macrolide antibiotics are used as anti-inflammatory agents, e.g., for prevention of exacerbations in chronic obstructive pulmonary disease and cystic fibrosis. Several studies have shown improved outcomes after the addition of macrolides to ß-lactam antibiotics for treatment of severe community-acquired pneumonia. However, a beneficial effect of macrolides in treating Gram-negative bacterial airway infections, e.g., those caused by Pseudomonas aeruginosa, remains to be shown. Macrolide antibiotics have significant side effects, in particular, motility-stimulating activity in the gastrointestinal tract and promotion of bacterial resistance. In this study, EM703, a modified macrolide lacking antibiotic and motility-stimulating activities but with retained anti-inflammatory properties, was used as an adjunct treatment for experimental P. aeruginosa lung infection, in combination with a conventional antibiotic. Airway infections in BALB/cJRj mice were induced by nasal instillation of P. aeruginosa; this was followed by treatment with the quinolone levofloxacin in the absence or presence of EM703. Survival, inflammatory responses, and cellular influx to the airways were monitored. Both pretreatment and simultaneous administration of EM703 dramatically improved survival in levofloxacin-treated mice with P. aeruginosa airway infections. In addition, EM703 reduced the levels of proinflammatory cytokines, increased the numbers of leukocytes in bronchoalveolar lavage fluid, and reduced the numbers of neutrophils present in lung tissue. In summary, the findings of this study show that the immunomodulatory properties of the modified macrolide EM703 can be important when treating Gram-negative pneumonia, as exemplified by P. aeruginosa infection in this study.

Extracellular Histones Induce Chemokine Production in Whole Blood Ex Vivo and Leukocyte Recruitment In Vivo.

The innate immune system relies to a great deal on the interaction of pattern recognition receptors with pathogen- or damage-associated molecular pattern molecules. Extracellular histones belong to the latter group and their release has been described to contribute to the induction of systemic inflammatory reactions. However, little is known about their functions in the early immune response to an invading pathogen. Here we show that extracellular histones specifically target monocytes in human blood and this evokes the mobilization of the chemotactic chemokines CXCL9 and CXCL10 from these cells. The chemokine induction involves the toll-like receptor 4/myeloid differentiation factor 2 complex on monocytes, and is under the control of interferon-γ. Consequently, subcutaneous challenge with extracellular histones results in elevated levels of CXCL10 in a murine air pouch model and an influx of leukocytes to the site of injection in a TLR4 dependent manner. When analyzing tissue biopsies from patients with necrotizing fasciitis caused by Streptococcus pyogenes, extracellular histone H4 and CXCL10 are immunostained in necrotic, but not healthy tissue. Collectively, these results show for the first time that extracellular histones have an important function as chemoattractants as their local release triggers the recruitment of immune cells to the site of infection.

Roflumilast Increases Bacterial Load and Dissemination in a Model of Pseudomononas Aeruginosa Airway Infection.

Exacerbations present a major clinical problem in many patients suffering from chronic obstructive pulmonary disease (COPD). Roflumilast, an inhibitor of phosphodiesterase 4, has shown beneficial effects in several clinical trials and is currently widely used to prevent exacerbations in severe COPD. Roflumilast has anti-inflammatory properties that may interfere with potentially important host defense functions, including cytotoxic properties of neutrophils at sites of inflammation. Since chronic bacterial infection is prevalent in severe COPD, Pseudomonas aeruginosa being a major pathogen, we hypothesized that this drug could impair host defense against P. aeruginosa. In this study, mice were pretreated with vehicle alone or roflumilast at doses of 5 mg/kg or 10 mg/kg, followed by instillation of P. aeruginosa in the airways. Bacterial load and dissemination, as well as inflammatory markers and immune cells, present in the airways were monitored. Roflumilast increased mortality, bacterial load, and dissemination in mice infected with P. aeruginosa. In addition, roflumilast-treated mice had significantly lower numbers of neutrophils in the bronchi, but not in the lung tissue airways, compared with untreated mice. Several proinflammatory cytokines decreased in roflumilast-treated mice but in neither the neutrophil-recruiting chemokine KC nor IL-6. These findings show that roflumilast treatment impairs host defense against P. aeruginosa in the airways, which may indicate that patients suffering from chronic bacterial infection of the airways could benefit from withholding of treatment with roflumilast.

NLF20: an antimicrobial peptide with therapeutic potential against invasive Pseudomonas aeruginosa infection.

OBJECTIVES: Increasing resistance to antibiotics makes antimicrobial peptides interesting as novel therapeutics. Here, we report on studies of the peptide NLF20 (NLFRKLTHRLFRRNFGYTLR), corresponding to an epitope of the D helix of heparin cofactor II (HCII), a plasma protein mediating bacterial clearance. METHODS: Peptide effects were evaluated by a combination of in vitro and in vivo methods, including antibacterial, anti-inflammatory and cytotoxicity assays, fluorescence and electron microscopy, and experimental models of endotoxin shock and Pseudomonas aeruginosa sepsis. RESULTS: The results showed that NLF20 displayed potent antimicrobial effects against the Gram-negative bacteria Escherichia coli and P. aeruginosa, the Gram-positive Bacillus subtilis and Staphylococcus aureus and the fungi Candida albicans and Candida parapsilosis. Importantly, this antimicrobial effect was retained in human blood, particularly for P. aeruginosa. Fluorescence and electron microscopy studies showed that the peptide exerted membrane-breaking effects. In an animal model of P. aeruginosa sepsis, NLF20 reduced bacterial levels, resulting in improved survival. Reduced mortality was also observed in experimental animal models of endotoxin shock, which was paralleled with modulated IFN-γ, IL-10 and coagulation responses. CONCLUSIONS: Together, these results indicate that functional epitopes of HCII may have therapeutic potential against bacterial infection.

Vertebrate TFPI-2 C-terminal peptides exert therapeutic applications against Gram-negative infections.

BACKGROUND: Tissue factor pathway inhibitor-2 (TFPI-2) is a serine protease inhibitor that exerts multiple physiological and patho-physiological activities involving the modulation of coagulation, angiogenesis, tumor invasion, and apoptosis. In previous studies we reported a novel role of human TFPI-2 in innate immunity by serving as a precursor for host defense peptides. Here we employed a number of TFPI-2 derived peptides from different vertebrate species and found that their antibacterial activity is evolutionary conserved although the amino acid sequence is not well conserved. We further studied the theraputic potential of one selected TFPI-2 derived peptide (mouse) in a murine sepsis model. RESULTS: Hydrophobicity and net charge of many peptides play a important role in their host defence to invading bacterial pathogens. In vertebrates, the C-terminal portion of TFPI-2 consists of a highly conserved cluster of positively charged amino acids which may point to an antimicrobial activity. Thus a number of selected C-terminal TFPI-2 derived peptides from different species were synthesized and it was found that all of them exert antimicrobial activity against E. coli and P. aeruginosa. The peptide-mediated killing of E. coli was enhanced in human plasma, suggesting an involvement of the classical pathway of the complement. Under in vitro conditions the peptides displayed anti-coagulant activity by modulating the intrinsic pathway of coagulation and in vivo treatment with the mouse derived VKG24 peptide protects mice from an otherwise lethal LPS shock model. CONCLUSIONS: Our results suggest that the evolutionary conserved C-terminal part of TFPI-2 is an interesting agent for the development of novel antimicrobial therapies.

Antimicrobial effects of helix D-derived peptides of human antithrombin III.

Antithrombin III (ATIII) is a key antiproteinase involved in blood coagulation. Previous investigations have shown that ATIII is degraded by Staphylococcus aureus V8 protease, leading to release of heparin binding fragments derived from its D helix. As heparin binding and antimicrobial activity of peptides frequently overlap, we here set out to explore possible antibacterial effects of intact and degraded ATIII. In contrast to intact ATIII, the results showed that extensive degradation of the molecule yielded fragments with antimicrobial activity. Correspondingly, the heparin-binding, helix D-derived, peptide FFFAKLNCRLYRKANKSSKLV (FFF21) of human ATIII, was found to be antimicrobial against particularly the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. Fluorescence microscopy and electron microscopy studies demonstrated that FFF21 binds to and permeabilizes bacterial membranes. Analogously, FFF21 was found to induce membrane leakage of model anionic liposomes. In vivo, FFF21 significantly reduced P. aeruginosa infection in mice. Additionally, FFF21 displayed anti-endotoxic effects in vitro. Taken together, our results suggest novel roles for ATIII-derived peptide fragments in host defense.

An antimicrobial helix A-derived peptide of heparin cofactor II blocks endotoxin responses in vivo.

Host defense peptides are key components of the innate immune system, providing multi-facetted responses to invading pathogens. Here, we describe that the peptide GKS26 (GKSRIQRLNILNAKFAFNLYRVLKDQ), corresponding to the A domain of heparin cofactor II (HCII), ameliorates experimental septic shock. The peptide displays antimicrobial effects through direct membrane disruption, also at physiological salt concentration and in the presence of plasma and serum. Biophysical investigations of model lipid membranes showed the antimicrobial action of GKS26 to be mirrored by peptide incorporation into, and disordering of, bacterial lipid membranes. GKS26 furthermore binds extensively to bacterial lipopolysaccharide (LPS), as well as its endotoxic lipid A moiety, and displays potent anti-inflammatory effects, both in vitro and in vivo. Thus, for mice challenged with ip injection of LPS, GKS26 suppresses pro-inflammatory cytokines, reduces vascular leakage and infiltration in lung tissue, and normalizes coagulation. Together, these findings suggest that GKS26 may be of interest for further investigations as therapeutic against severe infections and septic shock.

The TFPI-2 derived peptide EDC34 improves outcome of gram-negative sepsis.

Sepsis is characterized by a dysregulated host-pathogen response, leading to high cytokine levels, excessive coagulation and failure to eradicate invasive bacteria. Novel therapeutic strategies that address crucial pathogenetic steps during infection are urgently needed. Here, we describe novel bioactive roles and therapeutic anti-infective potential of the peptide EDC34, derived from the C-terminus of tissue factor pathway inhibitor-2 (TFPI-2). This peptide exerted direct bactericidal effects and boosted activation of the classical complement pathway including formation of antimicrobial C3a, but inhibited bacteria-induced activation of the contact system. Correspondingly, in mouse models of severe Escherichia coli and Pseudomonas aeruginosa infection, treatment with EDC34 reduced bacterial levels and lung damage. In combination with the antibiotic ceftazidime, the peptide significantly prolonged survival and reduced mortality in mice. The peptide's boosting effect on bacterial clearance paired with its inhibiting effect on excessive coagulation makes it a promising therapeutic candidate for invasive Gram-negative infections.

Proteolytic activation transforms heparin cofactor II into a host defense molecule.

The abundant serine proteinase inhibitor heparin cofactor II (HCII) has been proposed to inhibit extravascular thrombin. However, the exact physiological role of this plasma protein remains enigmatic. In this study, we demonstrate a previously unknown role for HCII in host defense. Proteolytic cleavage of the molecule induced a conformational change, thereby inducing endotoxin-binding and antimicrobial properties. Analyses employing representative peptide epitopes mapped these effects to helices A and D. Mice deficient in HCII showed increased susceptibility to invasive infection by Pseudomonas aeruginosa, along with a significantly increased cytokine response. Correspondingly, decreased levels of HCII were observed in wild-type animals challenged with bacteria or endotoxin. In humans, proteolytically cleaved HCII forms were detected during wounding and in association with bacteria. Thus, the protease-induced uncovering of cryptic epitopes in HCII, which transforms the molecule into a host defense factor, represents a previously unknown regulatory mechanism in HCII biology and innate immunity.

Importance of lipopolysaccharide aggregate disruption for the anti-endotoxic effects of heparin cofactor II peptides.

Lipid membrane and lipopolysaccharide (LPS) interactions were investigated for a series of amphiphilic and cationic peptides derived from human heparin cofactor II (HCII), using dual polarization interferometry, ellipsometry, circular dichroism (CD), cryoTEM, and z-potential measurements. Antimicrobial effects of these peptides were compared to their ability to disorder bacterial lipid membranes, while their capacity to block endotoxic effects of LPS was correlated to the binding of these peptides to LPS and its lipid A moiety, and to charge, secondary structure, and morphology of peptide/LPS complexes. While the peptide KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR) displayed potent antimicrobial and anti-endotoxic effects, its truncated variants KYE21 (KYEITTIHNLFRKLTHRLFRR) and NLF20 (NLFRKLTHRLFRRNFGYTLR) provide some clues on structure-activity relations, since KYE21 retains both the antimicrobial and anti-endotoxic effects of KYE28 (although both attenuated), while NLF20 retains the antimicrobial but only a fraction of the anti-endotoxic effect, hence locating the anti-endotoxic effects of KYE28 to its N-terminus. The antimicrobial effect, on the other hand, is primarily located at the C-terminus of KYE28. While displaying quite different endotoxic effects, these peptides bind to a similar extent to both LPS and lipid A, and also induce comparable LPS scavenging on model eukaryotic membranes. In contrast, fragmentation and densification of LPS aggregates, in turn dependent on the secondary structure in the peptide/LPS aggregates, correlate to the anti-endotoxic effect of these peptides, thus identifying peptide-induced packing transitions in LPS aggregates as key for anti-endotoxic functionality. This aspect therefore needs to be taken into account in the development of novel anti-endotoxic peptide therapeutics.

Effects of PEGylation on membrane and lipopolysaccharide interactions of host defense peptides.

Effects of poly(ethylene glycol) (PEG) conjugation on peptide interactions with lipid membranes and lipopolysaccharide (LPS) were investigated for KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), an antimicrobial and anti-inflammatory peptide derived from human heparin cofactor II. In particular, effects of PEG length and localization was investigated by ellipsometry, circular dichroism, nanoparticle tracking analysis, and fluorescence/electron microscopy. PEGylation of KYE28 reduces peptide binding to lipid membranes, an effect accentuated at increasing PEG length, but less sensitive to conjugation site. The reduced binding causes suppressed liposome leakage induction, as well as bacterial lysis. As a result of this, the antimicrobial effects of KYE28 is partially lost with increasing PEG length, but hemolysis also strongly suppressed and selecticity improved. Through this, conditions can be found, at which the PEGylated peptide displays simultaneously efficient antimicrobial affects and low hemolysis in blood. Importantly, PEGylation does not markedly affect the anti-inflammatory effects of KYE28. The combination of reduced toxicity, increased selectivity, and retained anti-inflammatory effect after PEGylation, as well as reduced scavenging by serum proteins, thus shows that PEG conjugation may offer opportunities in the development of effective and selective anti-inflammatory peptides.

The role of extracellular vesicle fusion with target cells in triggering systemic inflammation.

Extracellular vesicles (EVs) play a crucial role in intercellular communication by transferring bioactive molecules from donor to recipient cells. As a result, EV fusion leads to the modulation of cellular functions and has an impact on both physiological and pathological processes in the recipient cell. This study explores the impact of EV fusion on cellular responses to inflammatory signaling. Our findings reveal that fusion renders non-responsive cells susceptible to inflammatory signaling, as evidenced by increased NF-κB activation and the release of inflammatory mediators. Syntaxin-binding protein 1 is essential for the merge and activation of intracellular signaling. Subsequent analysis show that EVs transfer their functionally active receptors to target cells, making them prone to an otherwise unresponsive state. EVs in complex with their agonist, require no further stimulation of the target cells to trigger mobilization of NF-κB. While receptor antagonists were unable to inhibit NF-κB activation, blocking of the fusion between EVs and their target cells with heparin mitigated inflammation in mice challenged with EVs.

Lipopolysaccharide interactions of C-terminal peptides from human thrombin.

Interactions with bacterial lipopolysaccharide (LPS), both in aqueous solution and in lipid membranes, were investigated for a series of amphiphilic peptides derived from the C-terminal region of human thrombin, using ellipsometry, dual polarization interferometry, fluorescence spectroscopy, circular dichroism (CD), dynamic light scattering, and z-potential measurements. The ability of these peptides to block endotoxic effects caused by LPS, monitored through NO production in macrophages, was compared to peptide binding to LPS and its endotoxic component lipid A, and to size, charge, and secondary structure of peptide/LPS complexes. While the antiendotoxic peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE) displayed significant binding to both LPS and lipid A, so did two control peptides with either selected D-amino acid substitutions or with maintained composition but scrambled sequence, both displaying strongly attenuated antiendotoxic effects. Hence, the extent of LPS or lipid A binding is not the sole discriminant for the antiendotoxic effect of these peptides. In contrast, helix formation in peptide/LPS complexes correlates to the antiendotoxic effect of these peptides and is potentially linked to this functionality. Preferential binding to LPS over lipid membrane was furthermore demonstrated for these peptides and preferential binding to the lipid A moiety within LPS inferred.

Identifying biomarkers deciphering sepsis from trauma-induced sterile inflammation and trauma-induced sepsis.

Objective: The purpose of this study was to identify a panel of biomarkers for distinguishing early stage sepsis patients from non-infected trauma patients. Background: Accurate differentiation between trauma-induced sterile inflammation and real infective sepsis poses a complex life-threatening medical challenge because of their common symptoms albeit diverging clinical implications, namely different therapies. The timely and accurate identification of sepsis in trauma patients is therefore vital to ensure prompt and tailored medical interventions (provision of adequate antimicrobial agents and if possible eradication of infective foci) that can ultimately lead to improved therapeutic management and patient outcome. The adequate withholding of antimicrobials in trauma patients without sepsis is also important in aspects of both patient and environmental perspective. Methods: In this proof-of-concept study, we employed advanced technologies, including Matrix-Assisted Laser Desorption/Ionization (MALDI) and multiplex antibody arrays (MAA) to identify a panel of biomarkers distinguishing actual sepsis from trauma-induced sterile inflammation. Results: By comparing patient groups (controls, infected and non-infected trauma and septic shock patients under mechanical ventilation) at different time points, we uncovered distinct protein patterns associated with early trauma-induced sterile inflammation on the one hand and sepsis on the other hand. SYT13 and IL1F10 emerged as potential early sepsis biomarkers, while reduced levels of A2M were indicative of both trauma-induced inflammation and sepsis conditions. Additionally, higher levels of TREM1 were associated at a later stage in trauma patients. Furthermore, enrichment analyses revealed differences in the inflammatory response between trauma-induced inflammation and sepsis, with proteins related to complement and coagulation cascades being elevated whereas proteins relevant to focal adhesion were diminished in sepsis. Conclusions: Our findings, therefore, suggest that a combination of biomarkers is needed for the development of novel diagnostic approaches deciphering trauma-induced sterile inflammation from actual infective sepsis.

Proteolysis of human thrombin generates novel host defense peptides.

The coagulation system is characterized by the sequential and highly localized activation of a series of serine proteases, culminating in the conversion of fibrinogen into fibrin, and formation of a fibrin clot. Here we show that C-terminal peptides of thrombin, a key enzyme in the coagulation cascade, constitute a novel class of host defense peptides, released upon proteolysis of thrombin in vitro, and detected in human wounds in vivo. Under physiological conditions, these peptides exert antimicrobial effects against Gram-positive and Gram-negative bacteria, mediated by membrane lysis, as well as immunomodulatory functions, by inhibiting macrophage responses to bacterial lipopolysaccharide. In mice, they are protective against P. aeruginosa sepsis, as well as lipopolysaccharide-induced shock. Moreover, the thrombin-derived peptides exhibit helical structures upon binding to lipopolysaccharide and can also permeabilize liposomes, features typical of "classical" helical antimicrobial peptides. These findings provide a novel link between the coagulation system and host-defense peptides, two fundamental biological systems activated in response to injury and microbial invasion.

Antimicrobial activity of peptides derived from human ß-amyloid precursor protein.

Antimicrobial peptides are important effector molecules of the innate immune system. Here, we describe that peptides derived from the heparin-binding disulfide-constrained loop region of human ß-amyloid precursor protein are antimicrobial. The peptides investigated were linear and cyclic forms of NWCKRGRKQCKTHPH (NWC15) as well as the cyclic form comprising the C-terminal hydrophobic amino acid extension FVIPY (NWCKRGRKQCKTHPHFVIPY; NWC20c). Compared with the benchmark antimicrobial peptide LL-37, these peptides efficiently killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive Staphylococcus aureus and Bacillus subtilis, and the fungi Candida albicans and Candida parapsilosis. Correspondingly, fluorescence and electron microscopy demonstrated that the peptides caused defects in bacterial membranes. Analogously, the peptides permeabilised negatively charged liposomes. Despite their bactericidal effect, the peptides displayed very limited hemolytic activities within the concentration range investigated and exerted very small membrane permeabilising effects on human epithelial cells. The efficiency of the peptides with respect to bacterial killing and liposome membrane leakage was in the order NWC20c > NWC15c > NWC15l, which also correlated to the adsorption density for these peptides at the model lipid membrane. Thus, whereas the cationic sequence is a minimum determinant for antimicrobial action, a constrained loop-structure as well as a hydrophobic extension further contributes to membrane permeabilising activity of this region of amyloid precursor protein.

C-terminal peptides of tissue factor pathway inhibitor are novel host defense molecules.

Tissue factor pathway inhibitor (TFPI) inhibits tissue factor-induced coagulation, but may, via its C terminus, also modulate cell surface, heparin, and lipopolysaccharide interactions as well as participate in growth inhibition. Here we show that C-terminal TFPI peptide sequences are antimicrobial against the gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungi Candida albicans and Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen for the "classic" human antimicrobial peptide LL-37. The killing of E. coli, but not P. aeruginosa, by the C-terminal peptide GGLIKTKRKRKKQRVKIAYEEIFVKNM (GGL27), was enhanced in human plasma and largely abolished in heat-inactivated plasma, a phenomenon linked to generation of antimicrobial C3a and activation of the classic pathway of complement activation. Furthermore, GGL27 displayed anti-endotoxic effects in vitro and in vivo in a mouse model of LPS shock. Importantly, TFPI was found to be expressed in the basal layers of normal epidermis, and was markedly up-regulated in acute skin wounds as well as wound edges of chronic leg ulcers. Furthermore, C-terminal fragments of TFPI were associated with bacteria present in human chronic leg ulcers. These findings suggest a new role for TFPI in cutaneous defense against infections.