RESUMO
Transfusion-related acute lung injury (TRALI) is one of the leading causes of transfusion-related fatalities and is characterized by the onset of acute respiratory distress within 6 hours upon blood transfusion. Specific therapies are unavailable. Preexisting inflammation is a risk factor for TRALI and neutrophils (polymorphonuclear neutrophils [PMNs]) are considered to be the major pathogenic cells. Osteopontin (OPN) is a multifunctional protein expressed at sites of inflammation and, for example, is involved in pulmonary disorders, can regulate cellular migration, and can function as a PMN chemoattractant. We investigated whether OPN is involved in TRALI induction by promoting PMN recruitment to the lungs. Using a previously established murine TRALI model, we found that in contrast to wild-type (WT) mice, OPN knockout (KO) mice were resistant to antibody-mediated PMN-dependent TRALI induction. Administration of purified OPN to the OPN KO mice, however, restored the TRALI response and pulmonary PMN accumulation. Alternatively, blockade of OPN in WT mice using an anti-OPN antibody prevented the onset of TRALI induction. Using pulmonary immunohistochemistry, OPN could be specifically detected in the lungs of mice that suffered from TRALI. The OPN-mediated TRALI response seemed dependent on macrophages, likely the cellular source of OPN and OPN polymerization, and independent from the OPN receptor CD44, interleukin 6 (IL-6), and other PMN chemoattractants including macrophage inflammatory protein-2 (MIP-2). These data indicate that OPN is critically required for induction of antibody-mediated murine TRALI through localization to the lungs and stimulation of pulmonary PMN recruitment. This suggests that anti-OPN antibody therapy may be a potential therapeutic strategy to explore in TRALI patients.
Assuntos
Neutrófilos/patologia , Osteopontina/metabolismo , Lesão Pulmonar Aguda Relacionada à Transfusão/metabolismo , Lesão Pulmonar Aguda Relacionada à Transfusão/patologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Collagen VI is a ubiquitous extracellular matrix component that forms extensive microfibrillar networks in most connective tissues. In this study, we describe for the first time, to our knowledge, that the collagen VI von Willebrand factor type A-like domains exhibit a broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria in human skin infections in vivo. In silico sequence and structural analysis of VWA domains revealed that they contain cationic and amphipathic peptide sequence motifs, which might explain the antimicrobial nature of collagen VI. In vitro and in vivo studies show that these peptides exhibited significant antibacterial activity against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa through membrane disruption. Our findings shed new light on the role of collagen VI-derived peptides in innate host defense and provide templates for development of peptide-based antibacterial therapies.
Assuntos
Antibacterianos/imunologia , Colágeno Tipo VI/imunologia , Peptídeos/imunologia , Bactérias/imunologia , Infecções Bacterianas/imunologia , Humanos , Imunidade Inata/imunologia , Domínios Proteicos/imunologia , Pele/imunologia , Pele/microbiologia , Dermatopatias Bacterianas/imunologiaRESUMO
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.
Assuntos
Asma/complicações , Osteopontina/farmacologia , Infecções Pneumocócicas/prevenção & controle , Animais , Asma/induzido quimicamente , Asma/microbiologia , Carga Bacteriana/efeitos dos fármacos , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Modelos Animais de Doenças , Humanos , Inflamação/induzido quimicamente , Lesão Pulmonar/tratamento farmacológico , Lesão Pulmonar/prevenção & controle , Camundongos , Camundongos Knockout , Osteopontina/genética , Infecções Pneumocócicas/tratamento farmacológico , Substâncias Protetoras/farmacologia , Pyroglyphidae/imunologiaRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Líquido da Lavagem Broncoalveolar/química , Quelantes/farmacologia , DNA/farmacologia , Neutrófilos/química , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Cátions Bivalentes , Quelantes/química , Quelantes/isolamento & purificação , Fibrose Cística/tratamento farmacológico , Fibrose Cística/imunologia , Fibrose Cística/microbiologia , Fibrose Cística/patologia , DNA/química , DNA/isolamento & purificação , Fragmentação do DNA , Desoxirribonuclease I/química , Armadilhas Extracelulares/química , Armadilhas Extracelulares/imunologia , Humanos , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Ativação de Neutrófilo , Neutrófilos/imunologia , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/mortalidade , Pseudomonas aeruginosa/crescimento & desenvolvimento , Escarro/química , Escarro/citologia , Escarro/imunologiaRESUMO
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.
Assuntos
Antibacterianos/uso terapêutico , Eritromicina/análogos & derivados , Levofloxacino/uso terapêutico , Pneumopatias/tratamento farmacológico , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Animais , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/sangue , Quimioterapia Combinada , Eritromicina/uso terapêutico , Feminino , Trato Gastrointestinal/efeitos dos fármacos , Contagem de Leucócitos , Leucócitos/citologia , Pulmão/citologia , Pneumopatias/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/citologiaRESUMO
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.
Assuntos
Aminopiridinas/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Carga Bacteriana/efeitos dos fármacos , Benzamidas/farmacologia , Infecções por Pseudomonas/microbiologia , Doença Pulmonar Obstrutiva Crônica/microbiologia , Infecções Respiratórias/microbiologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Quimiocinas/metabolismo , Contagem de Colônia Microbiana , Ciclopropanos/farmacologia , Citocinas/metabolismo , Feminino , Contagem de Leucócitos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos , Infecções por Pseudomonas/patologia , Infecções Respiratórias/patologiaRESUMO
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.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Fungos/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Infecções por Pseudomonas/tratamento farmacológico , Sepse/tratamento farmacológico , Animais , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Microscopia Eletrônica , Microscopia de Fluorescência , Permeabilidade/efeitos dos fármacos , Choque Séptico/tratamento farmacológico , Análise de Sobrevida , Resultado do TratamentoRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Glicoproteínas/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Peptídeos/química , Peptídeos/farmacologia , Vertebrados/metabolismo , Sequência de Aminoácidos , Animais , Anticoagulantes/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/patogenicidade , Coagulação Sanguínea/efeitos dos fármacos , Citocinas/análise , Modelos Animais de Doenças , Escherichia coli/efeitos dos fármacos , Feminino , Glicoproteínas/química , Glicoproteínas/classificação , Bactérias Gram-Negativas/patogenicidade , Hemólise , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana/efeitos dos fármacos , Peptídeos/classificação , Filogenia , Plasma , Pseudomonas aeruginosa/efeitos dos fármacos , Sepse/tratamento farmacológico , Alinhamento de Sequência , Homologia de Sequência , Especificidade da Espécie , Vertebrados/classificaçãoRESUMO
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.
Assuntos
Cofator II da Heparina/imunologia , Cofator II da Heparina/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Western Blotting , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , ProteóliseRESUMO
The mechanisms underlying antimicrobial and anti-endotoxic effects were investigated for a series of structurally related peptides derived from the C-terminal region of S1 peptidases. For this purpose, results on bacterial killing were compared to those on peptide-induced liposome leakage, and to ellipsometry and dual polarization interferometry results on peptide binding to, and disordering of, supported lipid bilayers. Furthermore, the ability of these peptides to block endotoxic effects caused by bacterial lipopolysaccharide (LPS), monitored through NO production in macrophages, was compared to the binding of these peptides to LPS, and to secondary structure formation in the peptide/LPS complex. Bacteria killing, occurring through peptide-induced membrane lysis, was found to correlate with liposome rupture, and with the extent of peptide binding to the lipid membrane, no adsorption threshold for peptide insertion being observed. Membrane and LPS binding was found to depend on peptide net charge, illustrated by LPS binding increasing with increasing peptide charge, and peptides with net negative charge being unable to lyse membranes, kill bacteria, and block LPS-induced endotoxic effect. These effects were, however, also influenced by peptide hydrophobicity. LPS binding was furthermore demonstrated to be necessary, but not sufficient, for anti-endotoxic effect of these peptides. Circular dichroism spectroscopy showed that pronounced helix formation occurs in peptide/LPS complexes for all peptides displaying anti-endotoxic effect, hence potentially linked to this functionality. Similarly, ordered secondary structure formation was correlated to membrane binding, lysis, and antimicrobial activity of these peptides. Finally, preferential binding of these peptides to LPS over the lipid membrane was demonstrated.
Assuntos
Bactérias/efeitos dos fármacos , Lipopolissacarídeos/química , Peptídeo Hidrolases/química , Peptídeos/química , Adsorção , Animais , Anti-Infecciosos/farmacologia , Dicroísmo Circular , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Interferometria/métodos , Lipossomos/metabolismo , Macrófagos/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espectrofotometria/métodos , TemperaturaRESUMO
A pronounced membrane selectivity is demonstrated for short, hydrophilic, and highly charged antimicrobial peptides, end-tagged with aromatic amino acid stretches. The mechanisms underlying this were investigated by a method combination of fluorescence and CD spectroscopy, ellipsometry, and Langmuir balance measurements, as well as with functional assays on cell toxicity and antimicrobial effects. End-tagging with oligotryptophan promotes peptide-induced lysis of phospholipid liposomes, as well as membrane rupture and killing of bacteria and fungi. This antimicrobial potency is accompanied by limited toxicity for human epithelial cells and low hemolysis. The functional selectivity displayed correlates to a pronounced selectivity of such peptides for anionic lipid membranes, combined with a markedly reduced membrane activity in the presence of cholesterol. As exemplified for GRR10W4N (GRRPRPRPRPWWWW-NH(2)), potent liposome rupture occurs for anionic lipid systems (dioleoylphosphatidylethanolamine (DOPE)/dioleoylphosphatidylglycerol (DOPG) and Escherichia coli lipid extract) while that of zwitterionic dioleoylphosphatidylcholine (DOPC)/cholesterol is largely absent under the conditions investigated. This pronounced membrane selectivity is due to both a lower peptide binding to the zwitterionic membranes (z≈-8-10mV) than to the anionic ones (z≈-35-40mV), and a lower degree of membrane incorporation in the zwitterionic membranes, particularly in the presence of cholesterol. Replacing cholesterol with ergosterol, thus mimicking fungal membranes, results in an increased sensitivity for peptide-induced lysis, in analogy to the antifungal properties of such peptides. Finally, the generality of the high membrane selectivity for other peptides of this type is demonstrated.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Sequência de Aminoácidos , Bactérias/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Colesterol/química , Dicroísmo Circular , Ergosterol/química , Fungos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Bicamadas Lipídicas/química , Lipossomos/química , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Fosfolipídeos/química , Refratometria , Espectrometria de FluorescênciaRESUMO
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.
Assuntos
Anti-Infecciosos/imunologia , Lipoproteínas/química , Fragmentos de Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Anti-Infecciosos/química , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Bovinos , Ativação do Complemento , Complemento C3a/biossíntese , Complexo de Ataque à Membrana do Sistema Complemento/biossíntese , Feminino , Fungos/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Úlcera da Perna/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ratos , Pele/lesões , Pele/metabolismoRESUMO
Peptides of the C-terminal region of human thrombin are released upon proteolysis and identified in human wounds. In this study, we wanted to investigate minimal determinants, as well as structural features, governing the antimicrobial and immunomodulating activity of this peptide region. Sequential amino acid deletions of the peptide GKYGFYTHVFRLKKWIQKVIDQFGE (GKY25), as well as substitutions at strategic and structurally relevant positions, were followed by analyses of antimicrobial activity against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive bacterium Staphylococcus aureus, and the fungus Candida albicans. Furthermore, peptide effects on lipopolysaccharide (LPS)-, lipoteichoic acid-, or zymosan-induced macrophage activation were studied. The thrombin-derived peptides displayed length- and sequence-dependent antimicrobial as well as immunomodulating effects. A peptide length of at least 20 amino acids was required for effective anti-inflammatory effects in macrophage models, as well as optimal antimicrobial activity as judged by MIC assays. However, shorter (>12 amino acids) variants also displayed significant antimicrobial effects. A central K14 residue was important for optimal antimicrobial activity. Finally, one peptide variant, GKYGFYTHVFRLKKWIQKVI (GKY20) exhibiting improved selectivity, i.e., low toxicity and a preserved antimicrobial as well as anti-inflammatory effect, showed efficiency in mouse models of LPS shock and P. aeruginosa sepsis. The work defines structure-activity relationships of C-terminal host defense peptides of thrombin and delineates a strategy for selecting peptide epitopes of therapeutic interest.
Assuntos
Anti-Infecciosos/farmacologia , Fragmentos de Peptídeos/farmacologia , Trombina/farmacologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/fisiologia , Relação Estrutura-Atividade , Trombina/química , Trombina/fisiologia , CatelicidinasRESUMO
Thymic stromal lymphopoietin (TSLP) is an interleukin-7-like cytokine expressed by epithelial cells and reported to be involved in allergic diseases and atopic eczema. The presence of several predicted α-helical regions in TSPL, a structure characterizing many classical antimicrobial peptides (AMPs), prompted us to investigate whether TSLP exerts antimicrobial activities. Recombinant human TSLP exerted antimicrobial activity, particularly against Gram-negative bacteria. Using synthetic overlapping peptide 20-mers of TSLP, it was demonstrated that the antimicrobial effect is primarily mediated by the C-terminal region of the protein. MKK34 (MKKRRKRKVTTNKCLEQVSQLQGLWRRFNRPLLK), a peptide spanning a C-terminal α-helical region in TSLP, showed potent antimicrobial activities, in physiological salt conditions and in the presence of human plasma. Fluorescent studies of peptide-treated bacteria, electron microscopy and liposome leakage models showed that MKK34 exerted membrane-disrupting effects comparable to those of the classical AMP LL-37. Moreover, TSLP was degraded into multiple fragments by staphylococcal V8 proteinase. One major antimicrobial degradation fragment was found to encompass the C-terminal antimicrobial region defined by the MKK34 peptide. We here describe a novel antimicrobial role for TSLP. The antimicrobial activity is primarily mediated by the C-terminal part of the protein. In combination with the previously known cytokine function of TSLP, our result indicates dual functions of the molecule and a previously unknown role in host defense.
Assuntos
Anti-Infecciosos/farmacologia , Citocinas/farmacologia , Sequência de Aminoácidos , Anti-Infecciosos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Bactérias/metabolismo , Candida albicans/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Escherichia coli/efeitos dos fármacos , Humanos , Elastase de Leucócito/metabolismo , Lipossomos/metabolismo , Metaloendopeptidases/metabolismo , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Fragmentos de Peptídeos/farmacologia , Peptídeo Hidrolases/metabolismo , Permeabilidade/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Proteínas Recombinantes/farmacologia , Staphylococcus aureus/enzimologia , Staphylococcus epidermidis/efeitos dos fármacos , Catelicidinas , Linfopoietina do Estroma do TimoRESUMO
Extracellular histones are present in the airways because of cell death occurring during inflammation. They promote inflammation and cause tissue damage due to their cationic nature. The anionic phosphoglycoprotein osteopontin (OPN) is expressed at high levels during airway inflammation and has been ascribed both pro- and anti-inflammatory roles. In this study, it was hypothesized that OPN may neutralize the harmful activities of extracellular histones at the airway mucosal surface. In a model of histone-induced acute lung injury, OPN-/- mice showed increased inflammation and tissue injury, and succumbed within 24 h, whereas wild-type mice showed lower degrees of inflammation and no mortality. In lipopolysaccharide-induced acute lung injury, wild-type mice showed less inflammation and tissue injury than OPN-/- mice. In bronchoalveolar lavage fluid from ARDS patients, high levels of OPN and also histone-OPN complexes were detected. In addition, OPN bound to histones with high affinity in vitro, resulting in less cytotoxicity and reduced formation of tissue-damaging neutrophil extracellular traps (NETs). The interaction between OPN and histones was dependent on posttranslational modification of OPN, i.e., phosphorylation. The findings demonstrate a novel role for OPN, modulating the pro-inflammatory and cytotoxic properties of free histones.
Assuntos
Lesão Pulmonar Aguda/imunologia , Armadilhas Extracelulares/imunologia , Neutrófilos/imunologia , Osteopontina/metabolismo , Síndrome do Desconforto Respiratório/imunologia , Lesão Pulmonar Aguda/induzido quimicamente , Animais , Células Cultivadas , Citotoxicidade Imunológica , Modelos Animais de Doenças , Espaço Extracelular , Histonas/toxicidade , Humanos , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteopontina/genética , FosforilaçãoRESUMO
Severe infectious diseases are often characterized by an overwhelming and unbalanced systemic immune response to microbial infections. Human antithrombin (hAT) is a crucial coagulation inhibitor with anti-inflammatory activities. Here we identify three hAT-binding proteins (CD13, CD300f and LRP-1) on human monocytes that are involved in blocking the activity of nuclear factor-κB. We found that the modulating effect is primarily restricted to the less abundant ß-isoform (hßAT) of hAT that lacks N-glycosylation at position 135. Individuals with a mutation at this position have increased production of hßAT and analysis of their blood, which was stimulated ex vivo with lipopolysaccharide, showed a decreased inflammatory response. Similar findings were recorded when heterozygotic mice expressing hAT or hßAT were challenged with lipopolysaccharide or infected with Escherichia coli bacteria. Our results finally demonstrate that in a lethal E. coli infection model, survival rates increased when mice were treated with hßAT one hour and five hours after infection. The treatment also resulted in a reduction of the inflammatory response and less severe organ damage.
Assuntos
Antitrombinas/química , Antitrombinas/imunologia , Infecções Bacterianas/imunologia , Animais , Antitrombinas/sangue , Quimiocinas , Citocinas , Modelos Animais de Doenças , Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Humanos , Lipopolissacarídeos/efeitos adversos , Masculino , Camundongos , Camundongos Transgênicos , Monócitos , Mutação , NF-kappa B , Isoformas de Proteínas , Células RAW 264.7RESUMO
Tissue factor pathway inhibitor-2 (TFPI-2) has previously been characterized as an endogenous anticoagulant. TFPI-2 is expressed in the vast majority of cells, mainly secreted into the extracellular matrix. Recently we reported that EDC34, a C-terminal peptide derived from TFPI-2, exerts a broad antimicrobial activity. In the present study, we describe a previously unknown antimicrobial mode of action for the human TFPI-2 C-terminal peptide EDC34, mediated via binding to immunoglobulins of the classes IgG, IgA, IgE, and IgM. In particular the interaction of EDC34 with the Fc part of IgG is of importance since this boosts interaction between the immunoglobulin and complement factor C1q. Moreover, we find that the binding increases the C1q engagement of the antigen-antibody interaction, leading to enhanced activation of the classical complement pathway during bacterial infection. In experimental murine models of infection and endotoxin challenge, we show that TFPI-2 is up-regulated in several organs, including the lung. Correspondingly, TFPI-2-/- mice are more susceptible to pulmonary Pseudomonas aeruginosa bacterial infection. No anti-coagulant role of TFPI-2 was observed in these models in vivo. Furthermore, in vivo, the mouse TFPI-2-derived C-terminal peptide VKG24, a homolog to human EDC34 is protective against systemic Escherichia coli bacterial infection. Moreover, in sputum from cystic fibrosis patients TFPI-2 C-terminal fragments are generated and found associated with immunoglobulins. Together our data describe a previously unknown host defense mechanism and therapeutic importance of TFPI-2 against invading Gram-negative bacterial pathogens.
Assuntos
Anticorpos Antibacterianos/imunologia , Infecções por Escherichia coli/imunologia , Escherichia coli/imunologia , Glicoproteínas/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Animais , Anticorpos Antibacterianos/genética , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/patologia , Glicoproteínas/genética , Humanos , Camundongos , Camundongos Knockout , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/patologia , Regulação para Cima/imunologiaRESUMO
Coagulation, complement, and innate immunity are tightly interwoven and form an alliance that can be traced back to early eukaryotic evolution. Here we employed an ecoimmunological approach using Tissue Factor Pathway Inhibitor (TFPI)-1-derived peptides from the different classes of vertebrates (i.e. fish, reptile, bird, and mammals) and tested whether they can boost killing of various human bacterial pathogens in plasma. We found signs of species-specific conservation and diversification during evolution in these peptides that significantly impact their antibacterial activity. Though all peptides tested executed bactericidal activity in mammalian plasma (with the exception of rodents), no killing was observed in plasma from birds, reptiles, and fish, pointing to a crucial role for the classical pathway of the complement system. We also observed an interference of these peptides with the human intrinsic pathway of coagulation though, unlike complement activation, this mechanism appears not to be evolutionary conserved.
Assuntos
Coagulação Sanguínea , Proteínas do Sistema Complemento , Evolução Molecular , Imunidade Inata , Lipoproteínas/genética , Vertebrados , Animais , Atividade Bactericida do Sangue , HumanosRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen known for its immune evasive abilities amongst others by degradation of a large variety of host proteins. Here we show that digestion of thrombin by P. aeruginosa elastase leads to the release of the C-terminal thrombin-derived peptide FYT21, which inhibits pro-inflammatory responses to several pathogen-associated molecular patterns in vitro and in vivo by preventing toll-like receptor dimerization and subsequent activation of down-stream signalling pathways. Thus, P. aeruginosa 'hijacks' an endogenous anti-inflammatory peptide-based mechanism, thereby enabling modulation and circumvention of host responses.
Assuntos
Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Inflamação/patologia , Metaloendopeptidases/metabolismo , Peptídeos/metabolismo , Trombina/metabolismo , Animais , Membrana Celular/metabolismo , Citocinas/metabolismo , Humanos , Leucócitos/metabolismo , Leucócitos/ultraestrutura , Lipopolissacarídeos/metabolismo , Camundongos , Viabilidade Microbiana , NF-kappa B/metabolismo , Peptídeos/isolamento & purificação , Ligação Proteica , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição AP-1/metabolismoRESUMO
Bacterial infections of the respiratory tract contribute to exacerbations and disease progression in chronic obstructive pulmonary disease (COPD). There is also an increased risk of invasive pneumococcal disease in COPD. The underlying mechanisms are not fully understood but include impaired mucociliary clearance and structural remodeling of the airways. In addition, antimicrobial proteins that are constitutively expressed or induced during inflammatory conditions are an important part of the airway innate host defense. In the present study, we show that osteopontin (OPN), a multifunctional glycoprotein that is highly upregulated in the airways of COPD patients co-localizes with several antimicrobial proteins expressed in the airways. In vitro, OPN bound lactoferrin, secretory leukocyte peptidase inhibitor (SLPI), midkine, human beta defensin-3 (hBD-3), and thymic stromal lymphopoietin (TSLP) but showed low or no affinity for lysozyme and LL-37. Binding of OPN impaired the antibacterial activity against the important bacterial pathogens Streptococcus pneumoniae and Pseudomonas aeruginosa. Interestingly, OPN reduced lysozyme-induced killing of S. pneumoniae, a finding that could be explained by binding of OPN to the bacterial surface, thereby shielding the bacteria. A fragment of OPN generated by elastase of P. aeruginosa retained some inhibitory effect. Some antimicrobial proteins have additional functions. However, the muramidase-activity of lysozyme and the protease inhibitory function of SLPI were not affected by OPN. Taken together, OPN can contribute to the impairment of innate host defense by interfering with the function of antimicrobial proteins, thus increasing the vulnerability to acquire infections during COPD.