ABSTRACT
Surgical site infections (SSI) are a clinical and economic burden. Suture-associated SSI may develop when bacteria colonize the suture surface and form biofilms that are resistant to antibiotics. Thrombin-derived C-terminal peptide (TCP)-25 is a host defense peptide with a unique dual mode of action that can target both bacteria and the excessive inflammation induced by bacterial products. The peptide demonstrates therapeutic potential in preclinical in vivo wound infection models. In this study, the authors set out to explore whether TCP-25 can provide a new bioactive innate immune feature to hydrophilic polyglactin sutures (Vicryl). Using a combination of biochemical, biophysical, antibacterial, biofilm, and anti-inflammatory assays in vitro, in silico molecular modeling studies, along with experimental infection and inflammation models in mice, a proof-of-concept that TCP-25 can provide Vicryl sutures with a previously undisclosed host defense capacity, that enables targeting of bacteria, biofilms, and the accompanying inflammatory response, is shown.
Subject(s)
Bacterial Infections , Polyglactin 910 , Humans , Mice , Animals , Polyglactin 910/therapeutic use , Sutures , Inflammation/drug therapy , Surgical Wound Infection/drug therapy , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Bacterial Infections/drug therapy , PeptidesABSTRACT
There is a clinical need for conceptually new treatments that target the excessive activation of inflammatory pathways during systemic infection. Thrombin-derived C-terminal peptides (TCPs) are endogenous anti-infective immunomodulators interfering with CD14-mediated TLR-dependent immune responses. Here we describe the development of a peptide-based compound for systemic use, sHVF18, expressing the evolutionarily conserved innate structural fold of natural TCPs. Using a combination of structure- and in silico-based design, nuclear magnetic resonance spectroscopy, biophysics, mass spectrometry, cellular, and in vivo studies, we here elucidate the structure, CD14 interactions, protease stability, transcriptome profiling, and therapeutic efficacy of sHVF18. The designed peptide displays a conformationally stabilized, protease resistant active innate fold and targets the LPS-binding groove of CD14. In vivo, it shows therapeutic efficacy in experimental models of endotoxin shock in mice and pigs and increases survival in mouse models of systemic polymicrobial infection. The results provide a drug class based on Nature´s own anti-infective principles.
Subject(s)
Lipopolysaccharides , Toll-Like Receptors , Animals , Mice , Swine , Lipopolysaccharides/metabolism , Toll-Like Receptors/metabolism , Inflammation/pathology , Peptides/chemistry , Peptide Hydrolases , Lipopolysaccharide Receptors/metabolismABSTRACT
There is an urgent need for treatments that not only reduce bacterial infection that occurs during wounding but that also target the accompanying excessive inflammatory response. TCP-25, a thrombin-derived antibacterial peptide, scavenges toll-like receptor agonists such as endotoxins and lipoteichoic acid and prevents toll-like receptor-4 dimerization to reduce infection-related inflammation in vivo. Using a combination of biophysical, cellular, and microbiological assays followed by experimental studies in mouse and pig models, we show that TCP-25, when delivered from a polyurethane (PU) material, exerts anti-infective and anti-inflammatory effects in vitro and in vivo. Specifically, TCP-25 killed the common wound pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, in both in vitro and in vivo assays. Furthermore, after its release from the PU material, the peptide retained its capacity to induce its helical conformation upon endotoxin interaction, yielding reduced activation of NF-κB in THP-1 reporter cells, and diminished accumulation of inflammatory cells and subsequent release of IL-6 and TNF-α in subcutaneous implant models in vivo. Moreover, in a porcine partial thickness wound infection model, TCP-25 treated infection with S. aureus, and reduced the concomitant inflammatory response. Taken together, these findings demonstrate a combined antibacterial and anti-inflammatory effect of TCP-25 delivered from PU in vitro, and in mouse and porcine in vivo models of localized infection-inflammation. STATEMENT OF SIGNIFICANCE: Local wound infections may result in systemic complications and can be difficult to treat due to increasing antimicrobial resistance. Surgical site infections and biomaterial-related infections present a major challenge for hospitals. In recent years, various antimicrobial coatings have been developed for infection prevention and current concepts focus on various matrices with added anti-infective components, including various antibiotics and antiseptics. We have developed a dual action wound dressing concept where the host defense peptide TCP-25, when delivered from a PU material, targets both bacterial infection and the accompanying inflammation. TCP-25 PU showed efficacy in in vitro and experimental wound models in mouse and minipigs.
Subject(s)
Staphylococcal Infections , Wound Infection , Animals , Anti-Bacterial Agents , Inflammation/drug therapy , Mice , Peptides/pharmacology , Polyurethanes , Pseudomonas aeruginosa , Staphylococcal Infections/drug therapy , Staphylococcus aureus , Swine , Swine, Miniature , Wound Infection/drug therapyABSTRACT
There is a link between high lipopolysaccharide (LPS) levels in the blood and the metabolic syndrome, and metabolic syndrome predisposes patients to severe COVID-19. Here, we define an interaction between SARS-CoV-2 spike (S) protein and LPS, leading to aggravated inflammation in vitro and in vivo. Native gel electrophoresis demonstrated that SARS-CoV-2 S protein binds to LPS. Microscale thermophoresis yielded a KD of â¼47 nM for the interaction. Computational modeling and all-atom molecular dynamics simulations further substantiated the experimental results, identifying a main LPS-binding site in SARS-CoV-2 S protein. S protein, when combined with low levels of LPS, boosted nuclear factor-kappa B (NF-κB) activation in monocytic THP-1 cells and cytokine responses in human blood and peripheral blood mononuclear cells, respectively. The in vitro inflammatory response was further validated by employing NF-κB reporter mice and in vivo bioimaging. Dynamic light scattering, transmission electron microscopy, and LPS-FITC analyses demonstrated that S protein modulated the aggregation state of LPS, providing a molecular explanation for the observed boosting effect. Taken together, our results provide an interesting molecular link between excessive inflammation during infection with SARS-CoV-2 and comorbidities involving increased levels of bacterial endotoxins.
Subject(s)
COVID-19/complications , Inflammation/etiology , Lipopolysaccharides/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism , Animals , Binding Sites , COVID-19/immunology , COVID-19/virology , Cytokine Release Syndrome/etiology , Cytokine Release Syndrome/immunology , Disease Models, Animal , Gram-Negative Bacterial Infections/complications , Gram-Negative Bacterial Infections/immunology , Humans , In Vitro Techniques , Lipid A/chemistry , Lipid A/immunology , Lipid A/metabolism , Lipopolysaccharides/chemistry , Lipopolysaccharides/immunology , Mice , Mice, Inbred BALB C , Mice, Transgenic , Models, Immunological , Models, Molecular , Molecular Docking Simulation , Protein Binding , Protein Interaction Domains and Motifs , Respiratory Distress Syndrome/etiology , Risk Factors , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunologyABSTRACT
There is a clinical need for improved wound treatments that prevent both infection and excessive inflammation. TCP-25, a thrombin-derived peptide, is antibacterial and scavenges pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide, thereby preventing CD14 interaction and Toll-like receptor dimerization, leading to reduced downstream immune activation. Here, we describe the development of a hydrogel formulation that was functionalized with TCP-25 to target bacteria and associated PAMP-induced inflammation. In vitro studies determined the polymer prerequisites for such TCP-25-mediated dual action, favoring the use of noncharged hydrophilic hydrogels, which enabled peptide conformational changes and LPS binding. The TCP-25-functionalized hydrogels killed Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa bacteria in vitro, as well as in experimental mouse models of subcutaneous infection. The TCP-25 hydrogel also mediated reduction of LPS-induced local inflammatory responses, as demonstrated by analysis of local cytokine production and in vivo bioimaging using nuclear factor κB (NF-κB) reporter mice. In porcine partial thickness wound models, TCP-25 prevented infection with S. aureus and reduced concentrations of proinflammatory cytokines. Proteolytic fragmentation of TCP-25 in vitro yielded a series of bioactive TCP fragments that were identical or similar to those present in wounds in vivo. Together, the results demonstrate the therapeutic potential of TCP-25 hydrogel, a wound treatment based on the body's peptide defense, for prevention of both bacterial infection and the accompanying inflammation.
Subject(s)
Hydrogels/chemistry , Inflammation/complications , Inflammation/drug therapy , Peptides/therapeutic use , Wound Infection/complications , Wound Infection/drug therapy , Animals , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Disease Models, Animal , Drug Compounding , Endotoxins , Humans , Inflammation/microbiology , Leukocyte Elastase/metabolism , Mice , Microbial Sensitivity Tests , Peptides/chemistry , Peptides/pharmacokinetics , Protein Structure, Secondary , Proteolysis/drug effects , Pseudomonas aeruginosa/drug effects , Rheology , Staphylococcus aureus/drug effects , Swine , Thrombin/metabolism , Wound Infection/microbiologyABSTRACT
Background: A surgical site infection (SSI) is believed to be the result of an exaggerated inflammatory response. Objective: Examine the relationship between clinical status and inflammation biomarkers in full-thickness skin grafting wounds. Methods: Twenty patients planned for facial full-thickness skin grafting were enrolled. A week after surgery, all graft wounds were clinically assessed using a 3-step scale for inflammation (low, moderate, high). All wounds were swabbed for routine microbiological analysis and assessment of numbers of aerobic bacteria. Tie-over dressings from all patients were collected and used for wound fluid extraction and subsequent analysis of MMPs, cytokines, and NF-κB inducing activity. Results: Wounds with a high degree of inflammation contained increased total MMP activity (P ≤ 0.05) in their corresponding fluids. Likewise, the level of the cytokines IL-1ß, IL-8, IL-6, TNF-α was analyzed, and particularly IL-1ß was discriminatory for highly inflamed wounds (P ≤ 0.01). Moreover, bacterial loads were increased in highly inflamed wounds compared to wounds with a low degree of inflammation (P ≤ 0.01). NF-κB activation in the monocytic cell line THP-1 was significantly higher when these cells were stimulated by wound fluids with a high degree of inflammation (P ≤ 0.01). Growth of S. aureus in wounds did not vary between wounds with different degrees of inflammation (chi-square 3.8, P = 0.144). Conclusion: Biomarkers analyzed from tie-over dressings correlated to clinical wound healing in full-thickness skin grafting.
ABSTRACT
Host-defense peptides play a fundamental role in the innate immune system by modulating inflammatory responses. Previously, it was shown that the thrombin derived host-defense peptide GKY25 inhibits LPS-induced responses of monocytes and macrophages in vitro, ex vivo, and in vivo. In this study, the effect of GKY25 on the interaction of monocytes/macrophages with Gram-negative bacteria was explored. Electron microscopy analysis showed that fibrin slough from non-healing wounds, colonized with Staphylococcus aureus and Pseudomonas aeruginosa, contains C-terminal thrombin epitopes associated with these bacteria extracellularly and in phagosomes of leukocytes. Live imaging of RAW 264.7 cell cultures showed binding of GKY25 to Escherichia coli BioParticles extracellularly, and colocalization intracellularly. Although peptide binding did not alter the rate of phagocytosis, GKY25 reduced NF-κB/AP-1 activation and subsequent cytokine release in response to both heat-killed and live bacteria. Notably, preincubation of RAW 264.7 cells with peptide did increase BioParticle uptake in a dose-dependent manner. Taken together, the thrombin-derived host-defense peptide GKY25 binds to bacteria extracellularly and colocalizes with bacteria intracellularly, thereby reducing pro-inflammatory responses.
ABSTRACT
Host defense peptides have recently gained much interest as novel anti-infectives owing to their ability to kill bacteria and simultaneously modulate host cell responses. The cationic host defense peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), derived from the C terminus of human thrombin, inhibits proinflammatory responses in vitro and in vivo, but the mode of action is unclear. In this study, we show that GKY25, apart from binding bacterial LPS, also interacts directly with monocytes and macrophages in vitro, ex vivo, and in vivo. Moreover, GKY25 inhibits TLR4- and TLR2-induced NF-κB activation in response to several microbe-derived agonists. Furthermore, GKY25 reduces LPS-induced phosphorylation of MAPKs p38α and JNK1/2/3. FACS and electron microscopy analyses showed that GKY25 interferes with TLR4/myeloid differentiation protein-2 dimerization. The results demonstrate a previously undisclosed activity of the host defense peptide GKY25, based on combined LPS and cell interactions leading to inhibition of TLR4 dimerization and subsequent reduction of NF-κB activity and proinflammatory cytokine production in monocytes and macrophages.
Subject(s)
Antimicrobial Cationic Peptides/pharmacology , Lipopolysaccharides/immunology , Macrophages/drug effects , Monocytes/drug effects , Peptide Fragments/pharmacology , Thrombin/pharmacology , Amino Acid Sequence , Animals , Antimicrobial Cationic Peptides/immunology , Cell Line , Cytokines/biosynthesis , Enzyme Activation/drug effects , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Lymphocyte Antigen 96/metabolism , Macrophages/immunology , Male , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinase 14/metabolism , Molecular Sequence Data , Monocytes/immunology , NF-kappa B/metabolism , Peptide Fragments/immunology , Phosphorylation/drug effects , Protein Multimerization , Signal Transduction/immunology , Thrombin/immunology , Thrombin/metabolism , Toll-Like Receptor 2/immunology , Toll-Like Receptor 4/immunologyABSTRACT
In several studies plant lectins have shown promise as transgenic resistance factors against various insect pests. We have here shown that pea seed lectin is a potential candidate for use against pollen beetle, a serious pest of Brassica oilseeds. In feeding assays where pollen beetle larvae were fed oilseed rape anthers soaked in a 1% solution of pea lectin there was a reduction in survival of 84% compared to larvae on control treatment and the weight of surviving larvae was reduced by 79%. When a 10% solution of pea lectin was used all larvae were dead after 4 days of testing. To further evaluate the potential use of pea lectin, transgenic plants of oilseed rape (Brassica napus cv. Westar) were produced in which the pea lectin gene under control of the pollen-specific promoter Sta44-4 was introduced. In 11 out of 20 tested plants of the T0-generation there was a significant reduction in larval weight, which ranged up to 46% compared to the control. A small but significant reduction in larval survival rate was also observed. In the T2-generation significant weight reductions, with a maximum of 32%, were obtained in 10 out of 33 comparisons between transgenic plants and their controls. Pea lectin concentrations in anthers of transgenic T2-plants ranged up to 1.5% of total soluble protein. There was a negative correlation between lectin concentration and larval growth. Plants from test groups with significant differences in larval weights had a significantly higher mean pea lectin concentration, 0.64% compared to 0.15% for plants from test groups without effect on larval weight. These results support the conclusion that pea lectin is a promising resistance factor for use in Brassica oilseeds against pollen beetles.