Dysregulation of monocyte/macrophage phenotype in wounds of diabetic mice
Highlights
► Diabetic wounds exhibit impaired healing and a persistent inflammatory response. ► Macrophages were isolated from excisional wounds of diabetic and non-diabetic mice. ► Macrophages in non-diabetic mice exhibit phenotypes associated with healing. ► Macrophages in diabetic mice exhibit a persistent pro-inflammatory phenotype. ► The persistent pro-inflammatory phenotype in diabetic mice may impede healing.
Introduction
Chronic wounds associated with diabetes, venous insufficiency and/or pressure represent a significant health problem with millions of patients afflicted and the associated treatment costing billions of dollars per year [1]. For diabetic patients, the lifetime incidence of a foot ulcer is up to 25% and up to 70% of these wounds remain unhealed after 20 weeks of standard treatment [2], [3], [4]. A common characteristic of these poorly healing wounds is a persistent inflammatory response, with prolonged accumulation of monocytes/macrophages (Mo/Mp) and elevated levels of pro-inflammatory cytokines [5], [6], [7], [8]. However, the role of Mo/Mp in the impaired healing of chronic wounds remains to be elucidated.
During normal wound healing, the classic functions of Mo/Mp are killing pathogens and clearing the wound of damaged tissue. Mo/Mp also directly promote healing, contributing to angiogenesis, collagen deposition and wound closure [9], [10], [11], [12]. In a recent study, we demonstrated that Mp depletion during wound healing in mice resulted in decreased wound levels of vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β1 [12], suggesting that Mp are an important source of these growth factors during normal wound healing. However, during impaired healing of diabetic mice, wounds exhibit prolonged accumulation of Mo/Mp and pro-inflammatory cytokines and proteases, reduced levels of various growth factors, delayed closure, and reduced angiogenesis and matrix deposition, all of which mimic chronic wounds in humans [13], [14], [15]. One explanation for these findings is that the diabetic environment may perturb the function of Mo/Mp, which in turn could contribute to impaired healing. Indeed, Mp isolated from polyvinyl alcohol sponges implanted subcutaneously in db/db mice exhibit impaired phagocytosis of apoptotic cells [16].
Different microenvironments have the potential for inducing a wide range of Mo/Mp phenotypes or functions [17], [18], [19], [20]. Bacterial products and pro-inflammatory cytokines are known to induce the classical pro-inflammatory, pathogen killing Mo/Mp phenotype associated with production of high levels of inflammatory cytokines (e.g. interleukin (IL)-12, tumor necrosis factor (TNF)-α and IL-1β) and reactive oxygen and nitrogen species. Anti-inflammatory cytokines, glucocorticoids, and modulators of glucose and lipid metabolism induce a broad spectrum of “alternative” Mo/Mp phenotypes including those that exhibit non- or anti-inflammatory and pro-tissue repair functions, typically characterized by expression of mannose receptor (CD206), scavenger receptors (e.g. CD36) and growth factors such as TGF-β1 and insulin-like growth factor (IGF)-1. Although the term “alternative” activation was first ascribed to Mo/Mp stimulated with interleukin (IL)-4, the term has become a generic name used for any form of non-classical activation.
The vast majority of our knowledge about Mo/Mp phenotypes has been obtained from in vitro experiments in which phenotypes have been studied using stimulation with a single cytokine or other effector, or perhaps two of these effectors. In vivo, there is potential for a multitude of effectors to act on Mo/Mp and the phenotype expressed is likely due to the net effect of all of these signals. Mo/Mp along with other inflammatory cells, keratinocytes, fibroblasts and endothelial cells likely contribute to, and are likely influenced by, this complex molecular environment. Although Mo/Mp phenotypes have been described for cells isolated from sponges implanted subcutaneously in mice [16], [21], the phenotype of Mo/Mp isolated directly from skin wounds has been described only recently [22], but not for diabetic wounds. Thus, little is known about the role and regulation of different Mo/Mp phenotypes in skin wound healing, either during normal healing or in diabetic wounds. Our hypothesis is that, during normal skin healing in non-diabetic mice, wound Mo/Mp exhibit a pro-inflammatory phenotype early following injury and then express a pro-healing phenotype as healing progresses. We also hypothesize that this phenotypic transition is impaired in diabetic db/db mice, which may contribute to deficient healing.
Section snippets
Animals
Diabetic db/db mice and non-diabetic db/+ controls on a C57Bl/6 background were obtained from Jackson Laboratories (Bar Harbor, ME). Experiments were performed on 12–16 week-old mice. All experimental procedures were approved by the Animal Care Committee at the University of Illinois at Chicago.
Excisional wounding
Mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (5 mg/kg) and their dorsum shaved and cleaned with betadine and then alcohol swab. Four 8 mm excisional wounds
Wound Mo/Mp differentiation
Although Mo/Mp play important roles in skin wound healing [9], [10], [11], [12], the phenotype(s) and regulation of skin wound Mo/Mp have not been elucidated. Thus, we isolated CD11b+/Ly6G− cells from excisional wounds to assess cells of the Mo/Mp lineage. We first measured expression of the Mo marker Ly6C and the Mp marker F4/80 – Ly6C expression decreases and F4/80 expression increases during Mo/Mp maturation [24], [25]. In CD11b+/Ly6G− wound cells isolated from db/+ mice, Ly6C mRNA
Discussion
Although Mp are required for normal wound healing, dysregulation of Mp function could contribute to impaired wound healing with diabetes [5], [6], [7], [8], [13], [14], [15]. The major finding of this study is that wound Mo/Mp of diabetic mice exhibit an impaired transition from pro-inflammatory to pro-healing phenotypes which may contribute to the pro-inflammatory wound environment and poor healing response. Wound Mo/Mp isolated from non-diabetic db/+ mice exhibited a pro-inflammatory
Acknowledgments
The authors thank Dr. Luisa DiPietro, University of Illinois at Chicago, for critical comments on a previous draft of this manuscript.
References (40)
- et al.
Differences in cellular infiltrate and extracellular matrix of chronic diabetic and venous ulcers versus acute wounds
J Invest Dermatol
(1998) - et al.
A transgenic mouse model of inducible macrophage depletion: effects of diphtheria toxin-driven lysozyme M-specific cell lineage ablation on wound inflammatory, angiogenic, and contractive processes
Am J Pathol
(2009) - et al.
Selective and specific macrophage ablation is detrimental to wound healing in mice
Am J Pathol
(2009) - et al.
The chemokine system in diverse forms of macrophage activation and polarization
Trends Immunol
(2004) - et al.
Large and sustained induction of chemokines during impaired wound healing in the genetically diabetic mouse: prolonged persistence of neutrophils and macrophages during the late phase of repair
J Invest Dermatol
(2000) - et al.
Blood monocytes consist of two principal subsets with distinct migratory properties
Immunity
(2003) - et al.
Systemic anti-TNFalpha treatment restores diabetes-impaired skin repair in ob/ob mice by inactivation of macrophages
J Invest Dermatol
(2007) - et al.
Human skin wounds: a major and snowballing threat to public health and the economy
Wound Repair Regen
(2009) - et al.
A review of the literature on topical therapies for diabetic foot ulcers. Part 2: advanced treatments
J Wound Care
(2009) - et al.
Topical therapies for diabetic foot ulcers: standard treatments
J Wound Care
(2008)