Introduction
Chronic wounds are common in patients with vascular insufficiencies and underlying chronic conditions such as diabetes mellitus, as well as patients suffering from acute, extended trauma [
1]. Up to 15% of the more than 200 million diabetes patients worldwide develop a foot ulcer at some stage, leading to over one million amputations every year [
2]. The importance of chronic wounds in the pathway to lower limb amputation is paramount, as 84% of amputations are preceded by a diabetic foot ulcer [
3]. Chronic wounds and amputations in persons with diabetes often result in decreased physical, emotional and social function of patients, a reduced quality of life and major economic costs for both the patients, their families and society [
4,
5].
Sterile larvae (maggots) of the blowfly
Lucilia sericata are used for the treatment of different types of wounds including diabetic foot ulcers [
6‐
9]. The success rate of this therapy is around 68% for wounds unresponsive to conventional therapies, although some characteristics (chronic limb ischaemia, wound depth and age) may negatively influence the outcome [
8]. Besides the removal of necrotised tissue and infectious microorganisms, maggots may promote fibroblast migration and tissue remodelling [
10]. Moreover, maggot secretions potently inhibit the pro-inflammatory responses of human neutrophils without affecting their antimicrobial activities [
11]. Another prominent type of phagocyte in wounds is the monocyte. In response to chemotactic substances these cells migrate from the blood into the infected tissue to combat invading microorganisms. In addition, monocytes regulate the inflammatory process by secreting cytokines and growth factors, thereby recruiting more inflammatory cells as well as by antigen processing/presentation and lymphocyte activation.
In contrast to acute wound healing, chronic wounds are marked by a prolonged and dysregulated inflammatory phase. Inflammatory cells like neutrophils, monocytes and macrophages are not only present in excess numbers [
12‐
14], they also have enhanced production and release of pro-inflammatory cytokines, proteases and reactive oxygen species, leading to growth factor inactivation and tissue destruction [
15]. Therefore, inhibition of the pro-inflammatory responses of these cells could restrict their deleterious effects and thus contribute to healing processes. To obtain more insight into the mechanisms underlying the beneficial effects of medicinal maggots, this study focussed on the effects of maggot excretions and/or secretions on the pro-inflammatory activities of human monocytes.
Discussion
The main conclusion from the present study is that maggot secretions suppress the pro-inflammatory responses of monocytes without affecting their antimicrobial activities. This conclusion is based on the following observations.
First, secretions reduced production of the pro-inflammatory cytokines IL-12p40, TNF-α and MIF by lipopolysaccharides-stimulated monocytes, whereas production of anti-inflammatory cytokine IL-10 was enhanced. Addition of secretions to monocytes that had already been exposed to lipopolysaccharides resulted in similar effects on the cytokine profile, indicating that secretions can interfere with an ongoing inflammatory reaction (data not shown). The anti-inflammatory actions of secretions are not limited to modulation of the TLR-4 pathway, as secretions exerted similar effects on cells stimulated with a TLR-2 ligand. Furthermore, secretions inhibited the lipopolysaccharides-induced production of TNF-α and IL-12p40 by cells in whole blood (M. J. A. van der Plas and P. H. Nibbering, personal communication). However, production of the anti-inflammatory cytokine IL-10 by blood cells was not affected by secretions, suggesting that the secretions-induced increase in IL-10 production by purified monocytes may be counteracted by cellular/molecular components of whole blood. The suggestion that maggots produce IL-10 [
19] can be ruled out as it was withdrawn by the authors (K. Y. Mumcuoglu, Department of Parasitology, Hebrew University-Hadassah Medical School, Jerusalem, Israel, personal communication).
Second, secretions decreased the chemotactic response of monocytes to fMLP as well as to the chemotactic factors in supernatant fractions of (lipopolysaccharides-stimulated) monocyte cultures. These results are in agreement with our earlier finding that excretions/secretions reduced the migration of human neutrophils towards fMLP [
11]. The secretions-induced production of chemotactic factor MCP-1 and decreased production of migration inhibitor MIF by monocytes did not increase migration, indicating that secretions inhibited migration independently of the levels of these chemokines; participation of MIP-1β inhibition cannot be ruled out. The effect of secretions-induced increase of IL-8 and CD11b levels were not tested within our experimental setup. The increased expression of CD54 and CD11b on naive monocytes is unlikely to influence chemotaxis as monocytes are triggered when migrating into a wound.
Third, secretions did not affect the phagocytosis and intracellular killing of
S. aureus by freshly isolated monocytes and by monocytes cultured for 18 h. This is in agreement with our earlier findings that excretions/secretions had no effect on the phagocytosis and intracellular killing of
Candida albicans by neutrophils [
11]. Additionally, maggots aid in the removal of bacteria from wounds by ingesting bacteria together with liquefied necrotic tissue and subsequently killing them in their digestive tract [
20,
21]. An important implication of the above observations that secretions interfered in a similar fashion with activation of TLR-2 and TLR-4 pathways is that the reported differences in effects of maggots on survival of gram-positive and gram-negative bacteria [
22] are likely to be the result of antibacterial activity [
1] and not of differential modulation of immune cell responses.
Another major conclusion pertains to the mechanisms by which secretions exert their effects on monocytes. Our results showed that the effects of secretions on the cytokine/chemokine profiles of lipopolysaccharides- and lipoteichoic acid-stimulated monocytes were not caused by an altered sensitivity to these stimuli. In agreement with this, secretions also had no effect on expression of surface molecules involved in the recognition of bacterial products by (lipopolysaccharides-stimulated) monocytes, suggesting that secretions exert their effects either downstream of these receptors or on other, as yet unidentified (intracellular) binding partners and targets. Based on our earlier finding that excretions/secretions increased intracellular cAMP levels in neutrophils [
11], we assumed a similar mechanism by monocytes and, indeed, found that cAMP concentrations were dose- and time-dependently enhanced by secretions. Pre-treating monocytes with Rp-cAMPS, an inhibitor of cAMP-dependent protein kinase A-activation, attenuated the effects of secretions on lipopolysaccharides-stimulated cytokine production, indicating that maggots exert their effects on monocytes through a cAMP-dependent mechanism. Consistent with this, others have reported that activation of cAMP pathways is associated with reduced production of pro-inflammatory cytokines, including TNF-α, IL-12 and MIP-1β, without affecting IL-1β production and while enhancing production of IL-10 [
23,
24]. Furthermore, cAMP-elevation is connected to decreased migration [
25,
26], whereas phagocytosis by freshly isolated monocytes remains unaffected [
27]. However, elevation of cAMP was also associated with a moderate reduction in phagocytosis by incubated/stimulated monocytes and macrophages [
27,
28], a finding that seems to be in contrast with our data. This discrepancy can be explained by our observation that secretions enhanced the viability of monocytes, such that although the total phagocytosis of bacteria remained the same, the amount of phagocytosis per viable monocyte decreased. As secretions did not decrease expression of FcR, CR1 or CD11b (part of CR3), the reduction in phagocytosis per cell may be explained by interference of signalling pathways downstream of receptor activation [
28]. Interestingly, cAMP is known to inhibit apoptosis in several cell types [
29‐
31].
What could be the clinical relevance of the present findings? Although pro-inflammatory responses are essential for healing of acute wounds, they can be detrimental in chronic wounds where inflammation persists. Some histological data suggest that parts of chronic wounds seem to be stuck in different phases of healing with loss of the synchronicity that leads to rapid healing [
13]. Thus some part ready for epidermal resurfacing and fibroblast proliferation could be damaged by another part that is still in the inflammatory phase [
32]. Chronic leg ulcers are associated with elevated levels of pro-inflammatory cytokines, like TNF-α and MIF, compared with acute wounds [
33‐
35]. These cytokines enhance the production and release of a large variety of other pro-inflammatory cytokines [
36,
37] as well as the synthesis of several matrix metalloproteinases and serine proteases [
15,
37,
38]. When produced in excess these pro-inflammatory responses may cause deleterious extracellular matrix destruction [
39‐
41], as well as growth factor and protease inhibitor inactivation [
38,
42‐
44], and are responsible for the failure of wound healing. In addition, TNF-α activates phagocytes to produce reactive oxygen intermediates [
45,
46], which can be toxic to cells like endothelial cells, fibroblasts and leucocytes, and may further promote tissue proteolysis by potentiating the effects of several proteinases, while inactivating proteinase inhibitors [
47,
48]. Together, pro-inflammatory responses may be responsible for maintaining the chronic status of chronic wounds. Furthermore, TNF-α is also related to the formation of ulcers through its enhancement of plasminogen activator inhibitor-1 production [
49,
50], which can lead to impaired lysis of pericapillary fibrin cuffs [
51]. Importantly, although the mechanisms underlying the immunomodulatory effects of maggot secretions on monocytes are not fully elucidated, the findings from the present in vitro study show that maggot secretions potently inhibit the pro-inflammatory activities of monocytes. Secretions decrease migration of cells to the wound and reduce the amount of pro-inflammatory cytokines of the cells located in the wound, while their overall antibacterial activities are unaltered. Consequently, the release of other pro-inflammatory cytokines, reactive oxygen intermediates and proteases will diminish, bringing tissue destruction to a halt and possibly resulting in an environment beneficial for healing.
The exciting beneficial effect of maggots in treatment of diabetic foot ulcers and other chronic wounds, as found in clinical studies [
6‐
9], could be explained by the phenomena described in this study. Apart from the direct antibacterial features of maggots observed in other studies [
20,
21] and our earlier observations that excretions/secretions can inhibit the formation and breakdown of bacterial biofilms [
1], we found that maggots seem to preserve the important antibacterial function of human phagocytes, while protecting the fragile regenerating wound-bed against inflammation and tissue destruction by the same inflammatory cells.