Differential Internalization of Thrombin-Derived Host Defense Peptides into Monocytes and Macrophages.

Proteolytic cleavage of thrombin generates C-terminal host defense peptides exerting multiple immunomodulatory effects in response to bacterial stimuli. Previously, we reported that thrombin-derived C-terminal peptides (TCPs) are internalized in monocytes and macrophages in a time- and temperature-dependent manner. In this study, we investigated which endocytosis pathways are responsible for the internalization of TCPs. Using confocal microscopy and flow cytometry, we show that both clathrin-dependent and clathrin-independent pathways are involved in the internalization of the prototypic TCP GKY25 in RAW264.7 and human monocyte-derived M1 macrophages, whereas the uptake of GKY25 in monocytic THP-1 cells is mainly dynamin-dependent. Internalized GKY25 was transported to endosomes and finally lysosomes, where it remained detectable for up to 10 h. Comparison of GKY25 uptake with that of the natural occurring TCPs HVF18 and FYT21 indicates that the pathway of TCP endocytosis is not only cell type-dependent but also depends on the length and composition of the peptide as well as the presence of LPS and bacteria. Finally, using neutron reflectometry, we show that the observed differences between HVF18 and the other 2 TCPs may be explained partially by differences in membrane insertion. Taken together, we show that TCPs are differentially internalized into monocytes and macrophages.

Effect of PEGylation on Host Defense Peptide Complexation with Bacterial Lipopolysaccharide.

Conjugation with poly(ethylene glycol) ("PEGylation") is a widely used approach for improving the therapeutic propensities of peptide and protein drugs through prolonging bloodstream circulation, reducing toxicity and immunogenicity, and improving proteolytic stability. In the present study, we investigate how PEGylation affects the interaction of host defense peptides (HDPs) with bacterial lipopolysaccharide (LPS) as well as HDP suppression of LPS-induced cell activation. In particular, we investigate the effects of PEGylation site for KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), a peptide displaying potent anti-inflammatory effects, primarily provided by its N-terminal part. PEGylation was performed either in the N-terminus, the C-terminus, or in both termini, keeping the total number of ethylene groups (n = 48) constant. Ellipsometry showed KYE28 to exhibit pronounced affinity to both LPS and its hydrophobic lipid A moiety. The PEGylated peptide variants displayed lower, but comparable, affinity for both LPS and lipid A, irrespective of the PEGylation site. Furthermore, both KYE28 and its PEGylated variants triggered LPS aggregate disruption. To investigate the peptide structure in such LPS complexes, a battery of nuclear magnetic resonance (NMR) methods was employed. From this, it was found that KYE28 formed a well-folded structure after LPS binding, stabilized by hydrophobic domains involving aromatic amino acids as well as by electrostatic interactions. In contrast, the PEGylated peptide variants displayed a less well-defined secondary structure, suggesting weaker LPS interactions in line with the ellipsometry findings. Nevertheless, the N-terminal part of KYE28 retained helix formation after PEGylation, irrespective of the conjugation site. For THP1-Xblue-CD14 reporter cells, KYE28 displayed potent suppression of LPS activation at simultaneously low cell toxicity. Interestingly, the PEGylated KYE28 variants displayed similar or improved suppression of LPS-induced cell activation, implying the underlying key role of the largely retained helical structure close to the N-terminus, irrespective of PEGylation site. Taken together, the results show that PEGylation of HDPs can be done insensitively to the conjugation site without losing anti-inflammatory effects, even for peptides inducing such effects through one of its termini.

Bioinformatic Analysis of the Wound Peptidome Reveals Potential Biomarkers and Antimicrobial Peptides.

Wound infection is a common and serious medical condition with an unmet need for improved diagnostic tools. A peptidomic approach, aided by mass spectrometry and bioinformatics, could provide novel means of identifying new peptide biomarkers for wound healing and infection assessment. Wound fluid is suitable for peptidomic analysis since it is both intimately tied to the wound environment and is readily available. In this study we investigate the peptidomes of wound fluids derived from surgical drainages following mastectomy and from wound dressings following facial skin grafting. By applying sorting algorithms and open source third party software to peptidomic label free tandem mass spectrometry data we provide an unbiased general methodology for analyzing and differentiating between peptidomes. We show that the wound fluid peptidomes of patients are highly individualized. However, differences emerge when grouping the patients depending on wound type. Furthermore, the abundance of peptides originating from documented antimicrobial regions of hemoglobin in infected wounds may contribute to an antimicrobial wound environment, as determined by in silico analysis. We validate our findings by compiling literature on peptide biomarkers and peptides of physiological significance and cross checking the results against our dataset, demonstrating that well-documented peptides of immunological significance are abundant in infected wounds, and originate from certain distinct regions in proteins such as hemoglobin and fibrinogen. Ultimately, we have demonstrated the power using sorting algorithms and open source software to help yield insights and visualize peptidomic data.

Proteolytic signatures define unique thrombin-derived peptides present in human wound fluid in vivo.

The disease burden of failing skin repair and non-healing ulcers is extensive. There is an unmet need for new diagnostic approaches to better predict healing activity and wound infection. Uncontrolled and excessive protease activity, of endogenous or bacterial origin, has been described as a major contributor to wound healing impairments. Proteolytic peptide patterns could therefore correlate and "report" healing activity and infection. This work describes a proof of principle delineating a strategy by which peptides from a selected protein, human thrombin, are detected and attributed to proteolytic actions. With a particular focus on thrombin-derived C-terminal peptides (TCP), we show that distinct peptide patterns are generated in vitro by the human S1 peptidases human neutrophil elastase and cathepsin G, and the bacterial M4 peptidases Pseudomonas aeruginosa elastase and Staphylococcus aureus aureolysin, respectively. Corresponding peptide sequences were identified in wound fluids from acute and non-healing ulcers, and notably, one peptide, FYT21 (FYTHVFRLKKWIQKVIDQFGE), was only present in wound fluid from non-healing ulcers colonized by P. aeruginosa and S. aureus. Our result is a proof of principle pointing at the possibility of defining peptide biomarkers reporting distinct proteolytic activities, of potential implication for improved diagnosis of wound healing and infection.

Pseudomonas aeruginosa elastase cleaves a C-terminal peptide from human thrombin that inhibits host inflammatory responses.

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.

The Thrombin-Derived Host Defense Peptide GKY25 Inhibits Endotoxin-Induced Responses through Interactions with Lipopolysaccharide and Macrophages/Monocytes.

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.

Host defense peptides of thrombin modulate inflammation and coagulation in endotoxin-mediated shock and Pseudomonas aeruginosa sepsis.

Gram-negative sepsis is accompanied by a disproportionate innate immune response and excessive coagulation mainly induced by endotoxins released from bacteria. Due to rising antibiotic resistance and current lack of other effective treatments there is an urgent need for new therapies. We here present a new treatment concept for sepsis and endotoxin-mediated shock, based on host defense peptides from the C-terminal part of human thrombin, found to have a broad and inhibitory effect on multiple sepsis pathologies. Thus, the peptides abrogate pro-inflammatory cytokine responses to endotoxin in vitro and in vivo. Furthermore, they interfere with coagulation by modulating contact activation and tissue factor-mediated clotting in vitro, leading to normalization of coagulation responses in vivo, a previously unknown function of host defense peptides. In a mouse model of Pseudomonas aeruginosa sepsis, the peptide GKY25, while mediating a modest antimicrobial effect, significantly inhibited the pro-inflammatory response, decreased fibrin deposition and leakage in the lungs, as well as reduced mortality. Taken together, the capacity of such thrombin-derived peptides to simultaneously modulate bacterial levels, pro-inflammatory responses, and coagulation, renders them attractive therapeutic candidates for the treatment of invasive infections and sepsis.

Maggot secretions skew monocyte-macrophage differentiation away from a pro-inflammatory to a pro-angiogenic type.

BACKGROUND: Maggots of the blowfly Lucilia sericata are used for the treatment of chronic wounds. Earlier we reported maggot secretions to inhibit pro-inflammatory responses of human monocytes. The aim of this study was to investigate the effect of maggot secretions on the differentiation of monocytes into pro-inflammatory (MØ-1) and anti-inflammatory/pro-angiogenic macrophages (MØ-2) as these cells play a central role in wound healing. METHODOLOGY/PRINCIPAL FINDINGS: Freshly isolated monocytes were incubated with secretions and GM-CSF or M-CSF for 6 days and then stimulated with LPS or LTA for 18 h. The expression of cell surface molecules and the levels of cytokines, chemokines and growth factors in supernatants were measured. Our results showed secretions to affect monocyte-macrophage differentiation leading to MØ-1 with a partial MØ-2-like morphology but lacking CD163, which is characteristic for MØ-2. In response to LPS or LTA, secretions-differentiated MØ-1 produced less pro-inflammatory cytokines (TNF-alpha, IL-12p40 and MIF) than control cells. Similar results were observed for MØ-2 when stimulated with low concentrations of LPS. Furthermore, secretions dose-dependently led to MØ-1 and MØ-2 characterized by an altered chemokine production. Secretions led to MØ-2, but not MØ-1, producing enhanced levels of the growth factors bFGF and VEGF, as compared to control cells. The expression of cell-surface receptors involved in LPS/LTA was enhanced by secretions, that of CD86 and HLA-DR down-regulated, while receptors involved in phagocytosis remained largely unaffected. CONCLUSIONS: Maggot secretions skew the differentiation of monocytes into macrophages away from a pro-inflammatory to a pro-angiogenic type.

Maggot excretions/secretions inhibit multiple neutrophil pro-inflammatory responses.

There is renewed interest in the use of maggots (Lucilia sericata) to aid in healing of chronic wounds. In such wounds neutrophils precipitate tissue damage rather than contribute to healing. As the molecules responsible for the beneficial actions of maggots are contained in their excretions/secretions (ES), we assessed the effects of ES on functional activities of human neutrophils. ES dose-dependently inhibited elastase release and H(2)O(2) production by fMLP-activated neutrophils; maximal inhibition was seen with 5-50 microg of ES/ml. In contrast, ES did not affect phagocytosis and intracellular killing of Candida albicans by neutrophils. Furthermore, 0.5 microg of ES/ml already inhibited neutrophil migration towards fMLP. ES dose-dependently reduced the fMLP-stimulated expression of CD11b/CD18 by neutrophils, suggesting that ES modulate neutrophil adhesion to endothelial cells. ES did not affect the fMLP-induced rise in [Ca(2+)](i) in neutrophils, indicating that ES act down-stream of phospholipase C-mediated activation of protein kinase C. In agreement, ES inhibited PMA-activated neutrophil functional activities. ES induced a rise in intracellular cAMP concentration in neutrophils and pharmacological activators of cAMP-dependent mechanisms mimicked their inhibitory effects on neutrophils. The beneficial effects of maggots on chronic wounds may be explained in part by inhibition of multiple pro-inflammatory responses of activated neutrophils by ES.

Monomethylfumarate affects polarization of monocyte-derived dendritic cells resulting in down-regulated Th1 lymphocyte responses.

Psoriasis vulgaris, a type-1 cytokine-mediated chronic skin disease, can be treated successfully with fumaric acid esters (FAE). Beneficial effects of this medication coincided with decreased production of IFN-gamma. Since dendritic cells (DC) regulate the differentiation of T helper (Th) cells, this study focussed on effects of monomethylfumarate (MMF, bioactive metabolite of FAE) on polarization of monocyte-derived DC. MMF-incubated, lipo-polysaccharide-stimulated DC (MMF-DC) produced dramatically (p<0.05) reduced levels of IL-12p70 and IL-10 (8+/-4% and 20+/-4%, respectively) compared to control DC. MMF-DC were mature. MMF affected polarization of DC irrespective of polarization factor(s) and ligands for the various Toll-like receptors used. Coculture of MMF-DC with naive and primed allogenous Th cells resulted in lymphocytes producing less IFN-gamma, i.e. 59% and 54% of that by the respective Th cells cocultured with control DC. IL-4 production by primed, but not naive Th cells cocultured with MMF-DC was decreased as compared to cocultures with control DC. IL-10 production by naive and primed Th cells cocultured with MMF-DC and control DC did not differ. In addition, MMF inhibited LPS-induced NF-kappaB activation in DC. Together, beneficial effects of FAE in psoriasis involve modulation of DC polarization by MMF such that these cells down-regulate IFN-gamma production by Th cells.

Furin is a chemokine-modifying enzyme: in vitro and in vivo processing of CXCL10 generates a C-terminally truncated chemokine retaining full activity.

Chemokines comprise a class of structurally related proteins that are involved in many aspects of leukocyte migration under basal and inflammatory conditions. In addition to the large number of genes, limited processing of these proteins by a variety of enzymes enhances the complexity of the total spectrum of chemokine variants. We have recently shown that the native chemokine CXCL10 is processed at the C terminus, thereby shedding the last four amino acids. The present study was performed to elucidate the mechanism in vivo and in vitro and to study the biological activity of this novel isoform of CXCL10. Using a combination of protein purification and mass spectrometric techniques, we show that the production of C-terminally truncated CXCL10 by primary keratinocytes is inhibited in vivo by a specific inhibitor of pro-protein convertases (e.g. furin) but not by inhibition of matrix metalloproteinases. Moreover, CXCL10 is processed by furin in vitro, which is abrogated by a mutation in the furin recognition site. Using GTPgammaS binding, Ca(2+) mobilization, and chemotaxis assays, we demonstrate that the C-terminally truncated CXCL10 variant is a potent ligand for CXCR3. Moreover, the inverse agonist activity on the virally encoded receptor ORF74 and the direct antibacterial activity of CXCL10 are fully retained. Hence, we have identified furin as a novel chemokine-modifying enzyme in vitro and most probably also in vivo, generating a C-terminally truncated CXCL10, which fully retains its (inverse) agonistic properties.