Anti-infective peptide IDR-1002 augments monocyte chemotaxis towards CCR5 chemokines

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Highlights

  • IDR-1002 enhances monocyte chemotaxis towards CCL3 and CCL5.

  • Monocyte expression of CCR5 increases with IDR-1002 treatment.

  • CCL3-and CCL5-induced p38 MAPK activation is strengthened by IDR-1002.

  • CCR5 inhibition eliminates IDR-1002 enhancement of chemotaxis and signaling.

Abstract

Innate defense regulator (IDR) peptides are a class of immunomodulators which enhance and modulate host innate immune responses against microbial pathogens. While IDR-mediated protection against a range of bacterial pathogens is dependent on enhanced monocyte recruitment to the site of infection, the mechanisms through which they increase monocyte trafficking remain unclear. In this study, anti-infective peptide IDR-1002 was shown to enhance monocyte chemotaxis towards chemokines CCL3 and CCL5. This enhancement correlated with the selective upregulation of CCR5 surface expression by peptide-treated monocytes. It was found that IDR-1002 enhancement of monocyte chemotaxis was fully dependent on CCR5 function. Furthermore, IDR-1002 enhanced chemokine-induced monocyte p38 MAPK phosphorylation in a CCR5-dependent fashion. Overall, these results indicate that peptide IDR-1002 can selectively influence monocyte recruitment by host chemokines through the regulation of chemokine receptors.

Introduction

As the incidence of antibiotic-resistant pathogens continues to grow, so does the need for new therapeutic agents. Innate Defense Regulator (IDR)-peptides are a class of complex immunomodulators derived from endogenous host defense peptides [1] with the potent ability to combat bacterial pathogens through the complex regulation of the innate immune response. IDR-1, a peptide with no direct antimicrobial activity, confers prophylactic and therapeutic protection against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, and Salmonella enterica serovar Typhimurium in murine infection studies [2]. This protection correlates with an increase in monocyte/macrophage numbers, promotion of chemokine induction, and a suppression of inflammatory cytokine production at the site of infection. Similarly, IDR-1002, a derivative of bovine bactenecin, enhances host protection against S. aureus and Escherichia coli in murine infection models, again correlating with increased leukocyte recruitment and increased chemokine production at the infectious site [3]. Furthermore, IDR-1018, promotes wound healing and closure in porcine models of S. aureus-infected skin wounds [4]. These studies demonstrate the antimicrobial properties of IDR-peptides and highlight their potential as anti-infective therapeutics in situations of acute bacterial infection. While IDR-peptides show promise as novel antimicrobial agents, the mechanisms through which they enhance protection remain inadequately understood and must be further investigated.

The enhanced mobilization of monocytes during bacterial infection appears to be paramount in the anti-infective effects of IDR-peptides. This is further supported by the abolishment of peptide-mediated protection in animal models in which monocytes/macrophages were depleted [2], [3]. Thus to understand the anti-infective nature of IDR-peptides, we sought to elucidate the mechanisms through which they enhance monocyte recruitment. In a previous study, we determined that IDR-1002, a peptide with no direct chemotactic or chemokinetic effects, augments monocyte migration through a fibronectin network via the promotion of β1-integrin-mediated adhesion [5]. Observations in this study also implicated an adhesion-independent, chemokine-specific mechanism utilized by IDR-1002 to promote monocyte recruitment. Thus, we hypothesized that IDR-1002 could regulate monocyte responses towards chemokines, and in this manner promote monocyte mobilization.

In this study, IDR-1002 was found to enhance human monocyte chemotaxis towards CCL3 and CCL5, but not CCL2 or CCL7. This selective effect correlated with the increased surface expression of chemokine receptor (CCR)5, but not CCR1 or CCR2, by peptide-stimulated monocytes. Inhibition of CCR5 function eliminated the augmentation of monocyte chemotaxis by IDR-1002, demonstrating a chemokine-receptor-specific mechanism. Furthermore, IDR-1002 enhanced chemokine-induced phosphorylation of p38 MAP kinase in a CCR5-dependent manner. Overall, these results show an enhancement of human monocyte sensitivity to CCR5-chemokines through the selective upregulation of their receptor. These findings reveal a novel mechanism through which IDR-peptides enhance monocyte recruitment and thus enhance host protection against bacterial infection.

Section snippets

Reagents

Peptide IDR-1002 (VQRWLIVWRIRK-NH2) was synthesized by solid phase F-moc chemistry by CPC Scientific (Sunnyvale, CA). Chemokines CCL2, CCL3, CCL5, and CCL7 were obtained from R&D Systems (Minneapolis, MN).

Cell isolation and culture

PBMCs were isolated as previously reported [6]. Briefly, venous blood was collected from healthy volunteers using heparin-containing Vacutainer tubes (BD Bioscience, San Jose, CA) in accordance with UBC ethical guidelines. Blood was layered over Ficoll–Paque Plus (Amershan, Piscataway, NJ)

IDR-1002 enhances human monocyte chemotaxis towards CCL3 and CCL5

To investigate whether IDR-1002 regulated monocyte responses to endogenous chemoattractants, human monocyte chemotaxis assays were performed using a panel of human chemokines. The concentrations of chemokines used was based on their estimated maximal active dose range. Chemokines CCL2, CCL3, CCL5, and CCL7 were able to induce monocyte migration as expected (Fig. 1). Monocytes stimulated with IDR-1002 displayed no significant difference in migration towards CCL2 and CCL7. In contrast,

Discussion

It was demonstrated in this study that IDR-1002 enhanced human monocyte chemotaxis towards chemokines CCL3 and CCL5. The synergistic effect on monocyte migration between IDR-1002 and certain endogenous chemokines has significant implications in the role of IDR-peptides during an anti-infective immune response. The initial phase of microbial infection is in part characterized by the rapid production of endogenous immune-regulating mediators, including a diverse range of inflammatory cytokines.

Acknowledgements

This work was supported by the Canadian Institutes for Health Research (MOP-74493). R.E.W. Hancock is the recipient of a Canada Research Chair.

References (14)

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