Monisha G. Scott

The Human Antimicrobial Peptide LL-37 Is a Multifunctional Modulator of Innate Immune Responses

The role of LL-37, a human cationic antimicrobial peptide, in the immune system is not yet clearly understood. It is a widely expressed peptide that can be up-regulated during an immune response. In this report, we demonstrate that LL-37 is a potent antisepsis agent with the ability to inhibit macrophage stimulation by bacterial components such as LPS, lipoteichoic acid, and noncapped lipoarabinomannan. We also demonstrate that LL-37 protects mice against lethal endotoxemia. In addition to preventing macrophage activation by bacterial components, we hypothesized the LL-37 may also have direct effects on macrophage function. We therefore used gene expression profiling to identify macrophage functions that might be modulated by LL-37. These studies revealed that LL-37 directly up-regulates 29 genes and down-regulated another 20 genes. Among the genes predicted to be up-regulated by LL-37 were those encoding chemokines and chemokine receptors. Consistent with this, LL-37 up-regulated the expression of chemokines in macrophages and the mouse lung (monocyte chemoattractant protein 1), human A549 epithelial cells (IL-8), and whole human blood (monocyte chemoattractant protein 1 and IL-8), without stimulating the proinflammatory cytokine, TNFα. LL-37 also up-regulated the chemokine receptors CXCR-4, CCR2, and IL-8RB. These findings indicate that LL-37 may contribute to the immune response by limiting the damage caused by bacterial products and by recruiting immune cells to the site of infection so that they can clear the infection.

Impact of LL-37 on anti-infective immunity.

Host defense peptides (often called cationic antimicrobial peptides) have pleiotropic immunomodulatory functions. The human host defense peptide LL‐37 is up‐regulated at sites of infection and has little or no antimicrobial activity in tissue‐culture media but under the same conditions, demonstrates immunomodulatory effects on epithelial cells, monocytes, and dendritic cells (DC). These effects include the induction of chemokine production in a mitogen‐activated protein kinase‐dependent manner in epithelial cell lines and monocytes and profound alterations of DC differentiation, resulting in the capacity to enhance a T helper cell type 1 response. Although the exact mechanisms of interaction between LL‐37 and these cell types have not been elucidated, there is evidence for specific (i.e., receptor‐mediated) and nonspecific interactions. The relative significance of the direct antimicrobial activities and immunomodulatory properties of LL‐37 and other cationic host defense peptides in host defense remains unresolved. To demonstrate that antimicrobial activity was not necessarily required for protection in vivo, model peptides were synthesized and tested for antimicrobial and immunomodulatory activities. A peptide with no antimicrobial activity was found to be protective in animal models of Staphylococcus aureus and Salmonella infection, implying that a host defense peptide can protect by exerting immunomodulatory properties.

An anti-infective peptide that selectively modulates the innate immune response

We show that an innate defense–regulator peptide (IDR-1) was protective in mouse models of infection with important Gram-positive and Gram-negative pathogens, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and Salmonella enterica serovar Typhimurium. When given from 48 h before to 6 h after infection, the peptide was effective by both local and systemic administration. Because protection by IDR-1 was prevented by in vivo depletion of monocytes and macrophages, but not neutrophils or B- and T-lymphocytes, we conclude that monocytes and macrophages are key effector cells. IDR-1 was not directly antimicrobial: gene and protein expression analysis in human and mouse monocytes and macrophages indicated that IDR-1, acting through mitogen-activated protein kinase and other signaling pathways, enhanced the levels of monocyte chemokines while reducing pro-inflammatory cytokine responses. To our knowledge, an innate defense regulator that counters infection by selective modulation of innate immunity without obvious toxicities has not been reported previously.

Immunomodulatory Activities of Small Host Defense Peptides

ABSTRACT Recent studies have demonstrated that in addition to their antimicrobial activity, cationic host defense peptides, like the human cathelicidin LL-37, perform many activities relating to innate immunity, including the induction or modulation of chemokine and cytokine production, alteration of gene expression in host cells, and inhibition of proinflammatory responses of host cells to bacterial components such as lipopolysaccharide (LPS) in vitro and in vivo. To investigate if these properties are shared by smaller peptides, two cathelicidin peptides derived from bovine neutrophils, the 13-mer indolicidin and Bac2A, a linear 12-amino-acid derivative of bactenecin, were compared to the 37-amino-acid peptide LL-37. Indolicidin, like LL-37, inhibited LPS-induced tumor necrosis factor alpha (TNF-α) secretion, even when added up to an hour after the addition of Escherichia coli O111:B4 LPS to the human macrophage/monocyte-like THP-1 cell line. In contrast, Bac2A demonstrated no significant antiendotoxin activity. At low concentrations, indolicidin and LL-37 acted synergistically to suppress LPS-induced production of TNF-α. Indolicidin was analogous to LL-37 in its ability to induce the production of the chemokine interleukin-8 (IL-8) in a human bronchial cell line, 16HBE14o−, but it was unable to induce production of IL-8 in THP-1 cells. In contrast, Bac2A was unable to induce IL-8 in either cell type. Conversely, Bac2A was chemotactic for THP-1 cells at concentrations between 10 and 100 μg/ml, while indolicidin and LL-37 were not chemotactic at these concentrations for THP-1 cells. This indicates that in addition to the potential for direct microbicidal activity, cationic host defense peptides may have diverse and complementary abilities to modulate the innate immune response.

Cutting Edge: Cationic Antimicrobial Peptides Block the Binding of Lipopolysaccharide (LPS) to LPS Binding Protein

We investigated the mechanism by which cationic antimicrobial peptides block the activation of macrophages by LPS. The initial step in LPS signaling is the transfer of LPS to CD14 by LPS binding protein (LBP). Because many cationic antimicrobial peptides bind LPS, we asked whether these peptides block the binding of LPS to LBP. Using an assay that measures the binding of LPS to immobilized LBP, we show for the first time that a variety of structurally diverse cationic antimicrobial peptides block the interaction of LPS with LBP. The relative ability of different cationic peptides to block the binding of LPS to LBP correlated with their ability to block LPS-induced TNF-α production by the RAW 264.7 macrophage cell line.

Salmonella typhimurium Infection and Lipopolysaccharide Stimulation Induce Similar Changes in Macrophage Gene Expression

Changes in macrophage phenotype induced during infection result from the recognition of bacterial products as well as the action of bacterial virulence factors. We used the unprecedented opportunity provided by gene arrays to simultaneously study the expression of hundreds of genes during Salmonella typhimurium infection of macrophages and to assess the contribution of the bacterial virulence factor, LPS, in initiating the host responses to Salmonella. We found that S. typhimurium infection caused significant changes in the expression of numerous genes encoding chemokines, cell surface receptors, signaling molecules, and transcriptional activators at 4 h postinfection of the RAW 264.7 murine macrophage cell line. Our results revealed changes in the expression of several genes that had not been previously implicated in the host responses to S. typhimurium infection, as well as changes in the expression of several genes previously shown to be regulated by S. typhimurium infection. An overlapping spectrum of genes was expressed in response to virulent S. typhimurium and purified S. typhimurium LPS, reinforcing the major role of this surface molecule in stimulating the early response of macrophages to bacterial infection. The macrophage gene expression profile was further altered by activation with IFN-γ, indicating that host cell responses depend on the activation state of the cell.

Interaction and Cellular Localization of the Human Host Defense Peptide LL-37 with Lung Epithelial Cells

ABSTRACT LL-37 is a human cationic host defense peptide that is an essential component of innate immunity. In addition to its modest antimicrobial activity, LL-37 affects the gene expression and behavior of effector cells involved in the innate immune response, although its mode of interaction with eukaryotic cells remains unclear. The interaction of LL-37 with epithelial cells was characterized in tissue culture by using biotinylated LL-37 and confocal microscopy. It was demonstrated that LL-37 was actively taken up into A549 epithelial cells and eventually localized to the perinuclear region. Specific inhibitors were used to demonstrate that the uptake process was not mediated by actin but required elements normally involved in endocytosis and that trafficking to the perinuclear region was dependent on microtubules. By using nonlinear regression analysis, it was revealed that A549 epithelial cells have two receptors for LL-37B, with high and low affinity for LL-37, respectively. These results indicate the mode of interaction of LL-37 with epithelial cells and further our understanding of its role in modulating the innate immune response.

An α-Helical Cationic Antimicrobial Peptide Selectively Modulates Macrophage Responses to Lipopolysaccharide and Directly Alters Macrophage Gene Expression

Certain cationic antimicrobial peptides block the binding of LPS to LPS-binding protein and reduce the ability of LPS to induce the production of inflammatory mediators by macrophages. To gain a more complete understanding of how LPS activates macrophages and how cationic peptides influence this process, we have used gene array technology to profile gene expression patterns in macrophages treated with LPS in the presence or the absence of the insect-derived cationic antimicrobial peptide CEMA (cecropin-melittin hybrid). We found that CEMA selectively blocked LPS-induced gene expression in the RAW 264.7 macrophage cell line. The ability of LPS to induce the expression of >40 genes was strongly inhibited by CEMA, while LPS-induced expression of another 16 genes was relatively unaffected. In addition, CEMA itself induced the expression of a distinct set of 35 genes, including genes involved in cell adhesion and apoptosis. Thus, CEMA, a synthetic α-helical peptide, selectively modulates the transcriptional response of macrophages to LPS and can alter gene expression in macrophages.

Design of Host Defence Peptides for Antimicrobial and Immunity Enhancing Activities

Host defense peptides are a vital component of the innate immune systems of humans, other mammals, amphibians, and arthropods. The related cationic antimicrobial peptides are also produced by many species of bacteria and function as part of the antimicrobial arsenal to help the producing organism reduce competition for resources from sensitive species. The antimicrobial activities of many of these peptides have been extensively characterized and the structural requirements for these activities are also becoming increasingly clear. In addition to their known antimicrobial role, many host defense peptides are also involved in a plethora of immune functions in the host. In this review, we examine the role of structure in determining antimicrobial activity of certain prototypical cationic peptides and ways that bacteria have evolved to usurp these activities. We also review recent literature on what structural components are related to these immunomodulatory effects. It must be stressed however that these studies, and the area of peptide research, are still in their infancy.

Effect of Mycobacterial Phospholipids on Interaction of Mycobacterium tuberculosis with Macrophages

ABSTRACT This study demonstrates that pretreatment of macrophages with phosphatidylinositol, of either soya bean or mycobacterial origin, results in a down-regulation of the binding and uptake ofMycobacterium tuberculosis by the phagocytes. We also describe the novel observation that cardiolipin induces an increase in the binding and uptake of M. tuberculosis by macrophages. Neither phospholipid interacts with macrophages via the 2F8 epitope of scavenger receptor A, and treatment of macrophages with either phospholipid results in a down-regulation of CR3 function and tumor necrosis factor alpha production by the phagocyte. We have also shown that the ability of macrophages to interact with mycobacteria is greatly affected by an as yet unidentified product from the interaction of chloroform and polypropylene tubes.