Endotoxin (Lipopolysaccharide) Neutralization by Innate Immunity Host-Defense Peptides

Abstract

Binding of lipopolysaccharide (LPS) to macrophages results in proinflammatory cytokine secretion. In extreme cases it leads to endotoxic shock. A few innate immunity antimicrobial peptides (AMPs) neutralize LPS activity. However, the underlying mechanism and properties of the peptides are not yet clear. Toward meeting this goal we investigated four AMPs and their fluorescently labeled analogs. These AMPs varied in composition, length, structure, and selectivity toward cells. The list included human LL-37 (37-mer), magainin (24-mer), a 15-mer amphipathic ␣-helix, and its D,L-amino acid structurally altered analog. The peptides were investigated for their ability to inhibit LPS-mediated cytokine release from RAW264.7 and bone marrow-derived primary macrophages, to bind LPS in solution, and when LPS is already bound to macrophages (fluorescence spectroscopy and confocal microscopy), to compete with LPS for its binding site on the CD14 receptor (flow cytometry) and affect LPS oligomerization. We conclude that a strong binding of a peptide to LPS aggregates accompanied by aggregate dissociation prevents LPS from binding to the carrier protein lipopolysaccharide-binding protein, or alternatively to its receptor, and hence inhibits cytokine secretion. Lipopolysaccharide (LPS),2 also termed endotoxin, is an integral structural component of the outer membrane of Gram-negative bacteria (1). LPS is released from the bacteria during cell division, cell death, or in particular, as a result of antibiotic treatment against bacterial infection (2, 3). Upon its release, LPS is recognized by mononuclear phagocytes (monocytes and macrophages), which are part of the innate immunity of the host, and activates them. This results in an increase in their phagocytic activity and significantly enhances the secretion of proinflammatory cytokines such as tumor necrosis factor-␣ (TNF-␣), interleukin-6 (IL-6), and others (4 -6). Although pro-inflammatory cytokine secretion is essential for the development of the local inflammatory response, an unbalanced and overproduction of such cytokines may lead to septic shock characterized by endothelial damage, loss of vascular tone, coagulopathy, and multiple system organ failure, often resulting in death (7,8). The activation mechanism of macrophages by LPS starts when LPS (through its toxic entity, lipid A) binds with LPSbinding protein (LBP), accelerating the binding of LPS to CD14, the primary receptor of LPS, which is expressed mainly on macrophages (9 -11). The LPS-CD14 complex initiates intracellular signaling by interacting with the transmembrane protein Toll-like receptor-4 (TLR-4), which activates the NF-B transcription factor, resulting in the production and secretion of pro-inflammatory cytokines (12-16).In an attempt to understand the mechanism of macrophage stimulation by LPS, two major approaches have been reported. The first one utilized LPS receptor antagonists including anti-CD14 antibodies, anti-LBP antibodies, and lipid A analogs, all of which bind to essential components particip...

Chicken cathelicidin-2-derived peptides with enhanced immunomodulatory and antibacterial activities against biological warfare agents

“…In line with our observations, Nan et al [12] showed that substitution of Trp by Lys residues in the Trp-rich peptide indolicidin resulted in decreased hydrophobicity and loss of LPS-neutralising activity. Previously it was shown that inhibition of the pro-inflammatory response is mainly established by direct binding of cationic HDPs to LPS [11,13]. In line with these observations, we found a correlation between LPS-neutralising activities and LPS binding.…”

Cathelicidin LL-37 Increases Lung Epithelial Cell Stiffness, Decreases Transepithelial Permeability, and Prevents Epithelial Invasion by Pseudomonas aeruginosa

“…1E). This finding is in agreement with several studies showing inactivation of the antibacterial function of LL-37 by LPS (35)(36)(37)(38)(39). It is interesting to note that the elastic modulus of A549 cells treated with a premixed solution of LPS and LL-37 is significantly lower than the elastic modulus of cells treated with either agonist separately.…”

“…This effect could indicate that the natural response of lung epithelium is to increase its stiffness in an attempt to prevent bacterial entry during infection, and the increased presence of LL-37 is able to preemptively induce and bolster this stiffening response. Alternately, LL-37 is able to bind to LPS, which could also result in a reduction in proinflammatory stimuli (39). However, this interaction might also result in a gradual functional sequestration of LL-37 during the course of the infection, depending on the relative amounts of LPS and LL-37.…”

Anti-inflammatory action of an antimicrobial model peptide that suppresses the TRIF-dependent signaling pathway via inhibition of toll-like receptor 4 endocytosis in lipopolysaccharide-stimulated macrophages

“…Flow cytometric analysis of CD14 indicated that the peptide treatment did not affect the expression of CD14 in the macrophage cells (Fig 6A). As CD14 functions as the primary receptor of LPS to activate TLR4 signaling [28,29], we examined whether WALK11.3 interfered with LPS binding to the macrophage cells. The known LPS binding inhibitor PMB significantly inhibited FITC-conjugated LPS binding to the cells.…”