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/proangiogenic 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 monocytemacrophage 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-a, 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

Light microscopy analysis of the effect secretions on the differentiation of monocytes to macrophages.

<p>Monocytes were differentiated to MØ-1 in the presence of GM-CSF (A) or in the presence of GM-CSF and 35 µg of secretions/ml (B) and their morphology evaluated. Similarly, monocytes were differentiated to MØ-2 in the presence of M-CSF (C) and in the presence of M-CSF and secretions (D). Results indicated in days are from a representative experiment.</p

LPS-induced growth factor production by secretions differentiated macrophages.

<p>The results for control macrophages are expressed as the median value and range, and are set at 100%. The effect of secretions is expressed as a percentage relative to the production of growth factors by control cells. Results are means±SEM of at least six experiments. Values are significantly (*p<0.05 and **p<0.005) different from those for macrophages stimulated with LPS. ND: not detectable.</p

LPS-induced chemokine production by secretions differentiated macrophages.

<p>The results for control macrophages are expressed as the median value and range, and are set at 100%. The effect of secretions is expressed as a percentage relative to the chemokine production by control cells. Results are means±SEM of at least six experiments. Values are significantly (*p<0.05 and **p<0.005) different from those for macrophages stimulated with LPS. ND: not detectable.</p

Expression of surface receptors by secretions-differentiated macrophages.

<p>Results, expressed as the mean fluorescence intensity (MFI), are means±SEM of 6–11 experiments. Values are significantly (*p<0.05 and **p<0.005) different from those for control cells. ND: not detectable.</p

LPS-induced cytokine production by secretions differentiated macrophages.

<p>The results for the control macrophages are expressed as the median value and range, and are set at 100%. The effect of secretions is expressed as a percentage relative to the cytokine production by control cells. Results are means±SEM of at least six experiments. Values are significantly (*p<0.05 and **p<0.005) different from those for macrophages stimulated with LPS.</p

Cytokine production in response to LTA.

<p>We measured the production of IL-12p40 (A,B), TNF-α (C,D), IL-6 (E,F) and IL-10 (G,H) by control and secretions-differentiated MØ-1 and MØ-2 induced by a range of LTA. The results, expressed in ng/ml, are means±SEM of 12 experiments. Open bars: control macrophages; filled bars: secretions differentiated macrophages. *p<0.05 for differences from control macrophages.</p

Thrombin-Derived Host-Defense Peptides Modulate Monocyte/Macrophage Inflammatory Responses to Gram-Negative Bacteria

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.Published versio

Cytokine production in response to LPS.

<p>We measured the production of IL-12p40 (A,B) and IL-10 (C,D) by control and secretions-differentiated MØ-1 and MØ-2 in response to a range of LPS. The results, expressed in ng/ml, are means±SEM of 9–10 experiments. Open bars: control macrophages; filled bars: secretions differentiated macrophages. *p<0.05 for differences from control macrophages.</p