They change according to their surrounding environment and needs, and perform their due functions. Therefore, selective targeting of macrophage subpopulations for pro-healing therapy may provide an attractive strategy in regenerative processes. However, the understanding of spatiotemporal cues of each subpopulation of macrophages during the repair process is still limited.
We must further explore the plasticity of macrophages and discover more functional subgroups to enable better regulation of the repair process to promote wound repair and regeneration. As wounds heal, the tight regulation of the macrophage phenotype switching from a M1-proinflammatory to a M2-anti-inflammatory pro-healing phenotype contribute to the smooth progress of the repair process 76 — However, under pathological condition, such as ageing, obesity, infection, and diabetes, M1 macrophages in wounds were restrained with an incomplete switch to M2 phenotype, resulting in the stall of the repair process at inflammatory phase 10 , 79 , 80 Figure 2.
The mechanisms behind the persistent inflammatory macrophage phenotype in chronic wounds have been gradually identified. Figure 2 Macrophages originate from bone marrow in acute and chronic wounds. High numbers of bone marrow-produced monocytes under elderly, obese and diabetes may lead to increased number of macrophage in chronic wound.
Transition from M1- to M2- phenotypes is impaired in chronic wounds. Multiple studies reported that macrophages are prone to dysregulation under pathophysiological conditions such as ageing, obesity, and diabetes, and their aberrant activities are present not only at the wound site, but also in bone marrow BM and blood circulation 2 , 81 — Alteration in the number and function of macrophage have been revealed in chronic wounds, which are closely related to hematopoietic disruption and particularly to myeloid skewing 67 , 84 — This is known as increased monopoiesis Figure 2.
In aging mice, the hematopoietic stem and progenitor cells HSPCs showed restricted diversity and a preferential myeloid-biased differentiation, which was reflected by enhanced accumulation of the myeloid progenitor in aged mice The comparative analysis of expression profiles of HSPCs suggested that the myeloid differentiation related genes in aged mice, such as Runx1, Hoxb6 , and Osmr are upregulated while lymphopoiesis genes are downregulated compared with those of young mice Dramatic changes in the HSPCs milieu or niche in aging mice were attributed to the aging process and myeloid differentiation bias.
Niche factors, such as proinflammatory cytokine RANTES, secreted from aging stromal cells and differentiated blood cells have been considered as a major factor in this HSPCs subtype shift 89 , Ergen et al. Obesity and aging share many common traits in term of immunity and metabolism 91 , Several studies showed that obesity modifies the composition of adipose tissue in BM and restrains the generation of osteoblastic cells from mesenchymal progenitors, thereby affecting BM homeostasis.
Moreover, adipocytes in BM are key limiting factors of the hematopoietic function in obesity through direct and indirect means 84 , Obesity induced oxidative stress is a key driver for the aberrant HSPCs activity skewed toward the granulocyte-macrophage progenitors via dysregulation of the expression of Gfi1 in HSPCs 84 , Additional studies indicate that an epigenetic-based mechanism is involved in the programming of macrophages biased toward a proinflammatory phenotype under obesity.
This epigenetic feature is passed down to the macrophages at the wound sites, making them predisposed toward a proinflammatory phenotype Preprogrammed HSPCs committed towards the myeloid lineage and increased proportions of BM myeloid progenitors have also been found in diabetic mice during homeostasis and following injury, which results in an enhanced myeloid output 95 — An elevated number of circulating monocytes was also revealed in diabetic patients 2.
These results suggest a mechanistic link between neutrophils and monocytes. Research conducted by Nagareddy et al. However, the opposite trend was observed in the study by Yan et al. The above findings demonstrate that the wound M1-dominant macrophage phenotype is set at the BM level. The microenvironment in the BM changes significantly under pathological conditions, such as aging, obesity, and diabetes, which predetermines the gene expression of HSPCs and dysregulates their differentiation potential and function.
Therefore, these findings reveal a novel therapeutic approach for chronic wounds. Presently, there are two theories regarding changes in the number of macrophages in chronic wounds.
One is that a large number of macrophages infiltrate in chronic wounds as the results of increased monopoiesis, accompanied by an increase mobilization to the bloodstream. The other is that the number of macrophages is significantly reduced during the inflammatory phase in diabetic mice, which is attributed in part to a weakened chemotaxis into the wound 65 , The inconsistency in wound macrophage numbers between studies could result from technical differences in the evaluation of wound macrophage and requires further research.
Several reports showed that this was not only related to the functional modifications in HSCs that are passed down to macrophage progeny, but that it was also associated with local effects mediated by the wound microenvironment 2 , 43 , The wound microenvironment has a predominant role on the behavior and functionality of healing cells.
Macrophages are highly plastic and can change their phenotype and functions in accordance with local microenvironmental signals According to the non-healing wound research, a large number of studies focused on diabetes-related chronic wounds. Both mouse and human diabetic wound conditioned media prefer to induce a proinflammatory macrophage phenotype of BM- derived macrophage in vitro , which suggests an imbalanced microenvironment 43 , Cell senescence is a normal physiological process in the repair process.
Conversely, it prevents fibrosis by driving the senescence of myofibroblasts The rapid and effective clearance of these cells is essential for optimal repair outcomes , In contrast, an excessive amount of senescent cells in the wound or a disturbance of senescent cell clearance may lead to impaired wound healing.
Similar to those found in wounds of elderly, a large amount of senescent cells accumulate in skin wounds of diabetic mice, and macrophages account for a large proportion of these cells Wilkinson et al.
SASP is an important approach for a small number of senescent cells in tissues to exert significant local biological effects, and it is implicated in the occurrence of numerous chronic diseases — For example, proinflammatory SASP is thought to be involved in the development of insulin resistance and type II diabetes mellitus in both mice and humans , And the SASP secreted by macrophages derived from wounds of diabetic mice was enriched in CXCR2 ligands, which induced fibrotic markers of fibroblasts and had the potential to promote the senescence in fibroblasts Furthermore, wounds in diabetic mice treated with the CXCR2 antagonist showed reduction in macrophage senescence and local inflammation and facilitated wound closure, suggesting a novel avenue of targeting the CXCR2 receptor for potential therapeutic developments Therefore, the senescence of macrophages not only impairs their polarization from a proinflammatory phenotype to one that supports reparative processes, but also affects the wound microenvironment and biological functions of other repair cells through paracrine effects.
This burden, in turn, augments pro-inflammatory activity and sustains the inflammatory phase The hyperglycemic microenvironment affects the polarization of macrophages in both direct and indirect ways. Huang et al. The M1 type marker, CCR7, was significantly upregulated in THP-1 under the high glucose condition as compared to the normal glucose condition, which suggested a biased M1 macrophage M2 macrophages are characterized as reductive macrophages, suggesting the redox regulation in macrophages physiology M2 phenotype activation stimulates increased arginase-1 activity and is accompanied by reduced ROS and NO generation.
Diabetic subjects have higher serum levels of TNF-a, MCP-1 that is associated with failure to heal in diabetic foot wounds Further, these proinflammatory can form a positive feedback loop to sustain a persistent proinflammatory wound macrophage phenotype.
For example, Mirza et al. BM—derived macrophage exhibited a proinflammatory wound macrophage phenotype when cultured with conditioned medium of chronic wounds, supporting the notion that proinflammatory mediators are involved in the persistent inflammatory phenotype of macrophages in wounds The increased accumulation of proinflammatory activity in wounds is derived from adipocytes due to their hypertrophy under diabetic condition 18 , Recently, numerous discoveries supported the concept that diabetes and metabolic syndrome are systemic inflammatory diseases This strong association with adipocyte hypertrophy leads to lipotoxicity and excessive production of chemokine and cytokines Moreover, the overexpansion of adipocyte triggers a stress state and eventually results in apoptosis to release inflammatory mediators and attract macrophages to the adipose tissue This causes further infiltration of inflammatory cells, intensifying the secretion of inflammatory mediators, creating a systemic low-grade inflammatory state that may participate in high inflammation states of chronic wounds Consequently, anti-inflammatory treatments may not only improve senescence and insulin resistance but also play an active role in accelerating wound repair.
Neuroregulatory factors are likewise altered in the wounds of diabetics. However, the definite molecular mechanism remains unclear. Chronic wounds are frequently accompanied by the invasion and infection of bacteria due to their unique microenvironment. Multiple species of bacteria have been isolated from chronic wounds, and several also affect the M1 to M2 polarization of macrophages For example, Pseudomonas aeruginosa P.
It prolongs the presence of M1 macrophages in chronic wounds in two ways. Therefore, P. In summary, the unique and complex wound microenvironment of chronic wounds inhibits the polarization of macrophages from the inflammatory to the repair phenotype. Therefore, the inflammation phase of healing cannot transition to the proliferative phase in chronic wounds, and the proliferation of connective, endothelial, and epithelial tissue cannot be further completed Consequently, targeted changes in the unbalanced microenvironment of chronic wounds may be of great significance to promote healing.
Simultaneously, the imbalanced microenvironment changes have multiple-aspects and levels, determining that the multi-target treatment may be more effective than the single-target treatment in local wound treatment. Sustained increases in the number of wound macrophages and the dysregulation of their phenotypes, caused both by intrinsic alterations in HSPCs and by a local sophisticated microenvironment, lead to impaired wound healing.
Recently, several therapeutic approaches aimed at restoration of macrophage function have garnered significant attention. Methods include systemic treatment via oral administration of drugs and local treatment through neutralizing antibodies, MSCs, and biomaterials are discussed.
As mentioned in the previous section, the majority of macrophages in wounds are derived mainly from HSPCs. These already predisposed progenitor cells are partially responsible for the dysregulated macrophage polarization and prolonged inflammation in chronic wounds, which suggests that a HSPCs based therapy may improve the function of macrophages from the source.
Docosahexaenoic acid DHA is an omega-3 fatty acid, which is prerequisite for cell growth, development, and metabolic functions in mammals. It is also the precursor of several molecules that regulate the resolution of inflammation. Further, Jia et al. The same study implies that oral DHA in diabetic patients is able to correct the impaired plasticity of HSPCs, thereby improving resolution of inflammation, stimulating the transition into the proliferation stage, and thus promoting wound repair Chronic low-grade systemic inflammation is a common pathophysiological property of aging, obesity and diabetes 82 , , It is associated with a higher content of pro-inflammatory macrophages — Prolonged and persistent systemic inflammation can be destructive to various tissues and impair wound healing Consequently, anti-inflammatory strategies focus on the inflammation resolution to stop or dampen the inflammatory response, which may ameliorate the systemic and local inflammation state.
Apart from treatment of the primary disease, such as diabetes, improving complications caused by it, such as chronic wounds, requires further study. Over-activated M1 macrophages contribute to the hyper-inflammation state of chronic wounds by secreting high levels of pro-inflammatory factors. Neutralizing antibodies aim against these factors that silence M1 macrophages are a promising strategy for enhancing wound healing.
Goren et al. Further, Arginase, as a specific phenotypic marker of M2 macrophages, is also widely expressed in other wound cells including keratinocytes, fibroblasts, and endothelial cells.
It has been found to play critical roles in inflammatory response and cellular functions through controlling the local arginine concentration and regulating nitric oxide production , Decreased arginase level has been found in tissue of chronic non-healing And local inhibition of arginase activity by genetic and pharmacological means significantly impedes wound repair with enhanced inflammatory infiltrate and delayed re-epithelialization Contrastly, Kavalukas et al. The inconsistency of these results may be related to the model used in the research, the means and degree of inhibiting arginase activity.
Therefore, treatment strategies based on arginase needs further research. Peace or inhibition of overactivated signaling pathways is another alternative approach.
The AGE-RAGE signaling pathway is significantly enhanced in diabetic wounds, inhibiting macrophage polarization and M2 phenotypic macrophage function Anti-RAGE antibody-applied wounds increased the number of neutrophils phagocytized by macrophages and promoted the phenotypic switch of macrophages from proinflammatory to pro-healing activities Oxygen radical scavengers have been reported to provide positive effects in the treatment of chronic wounds For example, topically applied mepenzolate bromide to the wound bed considerably corrected the excessive and prolonged ROS production, and further decreased the level of pro-inflammatory cytokines and increased the level of pro-healing cytokines, results in a promotion of macrophage M2 phenotype polarization, thus accelerating the wound closure rate Chronicity was reversed in non-healing wounds by treatment with these antioxidants They could elicit phenotypic changes from the M1 gene signature to M2 gene signature via indication of the nuclear factor erythroid 2-related factor 2 Nrf2 , a key member of antioxidant regulators The treatment strategies presented in this review are therefore presented principally to weaken and combat the adverse factors in chronic wounds, balance the inflammatory wound microenvironment, promote the polarization of macrophages, and finally facilitate the wound repair process into the proliferative stage.
CD has been proposed as a specific marker for macrophages with an anti-inflammatory phenotype. It has been shown that using a modified nanoparticle, polyethylenimine PEI grafted with a mannose receptor ligand Man-PEI to induce CD in human primary macrophages lead to changes in the secretion profile and induced anti-inflammatory responses Further, Ferreira et al.
They found that polarized M2 macrophages have the ability to promote a faster wound healing by interacting with keratinocytes and fibroblasts This precise targeting method can avoids limitations and side effects due to the heterogeneity of chemical-based therapy on different cells and may become an important strategy to reverse chronic wounds.
Chronic wounds are characterized by the polarization barrier of macrophages leading to a higher proportion of M1 phenotypic macrophages and a lower proportion of M2 phenotypic macrophages.
A direct addition of exogenous M2 macrophages to the wound may promote wound repair. These activated macrophages with anti-inflammatory properties may be beneficial for wound repair. Presently, this method lacks sufficient supporting data, and further research is required.
Mesenchymal stem cells MSCs are high multi-potentiality residing in different tissues including BM, adipose tissue, the umbilical cord, and skin , Their application in acute and chronic wound models has been successful, resulting in resolution of wound inflammation, and enhancement of angiogenesis and acceleration of wound closure — Evidence indicates that MSCs exert powerful modulating effects on the immune system, in particular with regard to the immunoregulatory function on macrophages One of the mechanisms of MSC action on macrophages is via their recruitment.
For example, BM-MSCs conditioned medium significantly accelerates migration of macrophages in vitro Subcutaneous injection and topical application of BM-MSCs conditioned media in wounds increased proportions of macrophages and endothelial progenitor cells compared with control group, thus enhancing wound healing Another mechanism of MSC action on macrophages is by augmentation macrophages to engulf apoptotic neutrophils, which is attributed to upregulation of the intercellular adhesion molecule-1 ICAM-1 on macrophages or enhanced release of soluble extracellular superoxide dismutase SOD3 from MSCs — Another mechanism of MSC action on macrophage is via enhancement of M1-M2 polarization and increasing the frequency of M2 macrophages in wounds.
Several in vitro studies showed that macrophage facilitated differentiation into M2 phenotypes when co-cultured with MSCs or MSCs derived secretomes — Furthermore, MSCs were found to regulate the macrophage phenotype in vivo A panel of MSC secreted mediators were studied for their immunomodulatory mechanisms.
Other possible mechanisms must be further developed and validated. Currently, MSCs resolve the unrestrained and prolonged inflammation of chronic wounds in terms of numerous aspects, and are ideally suited for the treatment of chronic wounds Further, MSCs have a unique property, namely, the capacity to sense the microenvironment in which they are located, which determines that its immunoregulatory function of MSCs reveals high plasticity , Compared with the chemical-based or growth factor-based strategies, this marks an excellent property and allows for refinement of MSC-based cell therapies in the future , Biomaterials can provide suitable environments that enhance inherent biological activities and functions in repairing cells through appropriate biochemical cues e.
An increasing amount of evidence shows that they can also influence the flexible nature of macrophages in wounds. Immunomodulatory biomaterials, particularly the natural biomaterials, have shown regulation of the macrophage fate , The decellularized dermal scaffold DDS is a skin tissue that removes cellular components and retains the ECM structure, which was shown to play a therapeutic role in wound repair DDS in particular can regulate the transition of macrophages from the M1 pro-inflammatory phenotype to the M2 pro-repairing phenotype, thus promoting macrophage polarization , He et al.
Physical and chemical properties of biomaterials have a profound impact on the cellular behavior. For example, a high molecular weight of HA caused macrophages to take on anti-inflammatory features, whereas a low molecular weight of HA resulted in the activation of M1 macrophages — Biomaterials composed of collagen and highly sulfated HA derivatives promoted switching from the M1 to M2 phenotype of macrophages.
Keratin biomaterials have also been used in wound repair studies, and their ability to tune inflammation has been confirmed — In vitro studies have shown that primary macrophages inoculated into high molecular weight extracted keratin and keratin peptide coatings facilitated differentiation into M2c macrophages with anti-inflammatory behavior Therefore, immunomodulatory biomaterials with the goal of promoting macrophage polarization present an innovative repair strategy for chronic wounds.
Biological materials not only have the ability of immunological regulation, but also serve as carriers of cells and active molecules to enhance survival rates of transplanted cells and avoid the burst release of active molecules and rapid degradation. Therefore, combining the above-mentioned immunomodulatory biomaterials with active molecules that promote the polarization of macrophages to M2 phenotype may achieve a better pro-healing function than using materials or cells or active molecules alone.
An increasing body of evidence points to the benefits of such approaches in inert implants , Bioactive molecules or cells have been pursued to enhance wound repair, and they have been successfully administered through the different delivery systems, such as sponge scaffolds , , polymer , , hydrogel — , and nanofibers , , which prove to be effective in preclinical studies. Notably, increasing evidence reveals the immunomodulatory effects of biomaterials, which may become important approaches of treating chronic wounds, while the mechanism responsible for this response has rarely been explored.
In future research, a large number of studies are required to examine the potential mechanism of immunomodulatory materials, and offers guidance for the design of more desirable biological materials to improve tissue repair and regeneration. Notably, the therapeutic perspective and specific signal targets differ in the existing methods. We consider using existing multiple treatments in combination to achieve synergistic effects between each other, striving for the better therapeutic effects for the treatment of chronic wounds.
Step 3: This interaction is known to occur through different cell surface receptors — most notably, the Dectin 1 receptor. Activation of the dectin-1 receptor has been shown to induce proliferation and migration of keratinocytes, which enhance the growth of healthy granulation tissue and re-epithelialisation.
Step 4: This triggers cellular signalling cascades, which stimulate macrophages, growth factors and cell-signalling molecules required for healing. Step 5: The chain reaction of these microbiological processes induces proliferative, angiogenic and epithelialisation responses as required, restarting the healing process in stalled wounds. Macrophages secrete matrix metalloproteases MMPs , which are critical in autolytic debridement of necrotic tissue, as well as cytokines and growth factors — notably, fibroblast growth factor, epidermal growth factor, transforming growth factor-beta and interleukin-1 — that encourage granulation and epithelialisation [5].
Your email address will not be published. This site uses Akismet to reduce spam. Moreover, gene expression analysis of day 15 wounds showed increased expression of alternative activation markers such as Arginase-1 and YM-1 in M2 wounds, indicating persistence of M2 polarization phenotype even after 15 days of macrophage injection.
Although expression of cytokines between the different groups did not differ at this time point, M2 injected wounds presented a significant reduction in expression of MMP2 compared to controls.
MMPs have been implicated in wound repair by their ability to remodel extracellular matrix components and indiscriminate pharmacologic inhibition of MMPs significantly delays wound healing in wild type mice, indicating their substantial role in skin repair and re-epithelialization [29] , [30]. A reduction in MMPs levels might therefore contribute to the phenotype of delayed healing in M2a and M2c wounds in our experiment.
Reduced expression of MMP2 in M2a and M2c wounds could be a consequence of its reduced expression by M2a and M2c wound macrophages, as our in-vitro analysis of M2a and M2c cells has indicated. Our observation that murine M2 macrophages express reduced MMP2 levels is supported by studies in human macrophages where alternative activation was found to decrease mRNA levels and gelatinase activity of MMP2 [31]. Microscopic examination of wounds in the diabetic mice indicated that M2 macrophage injected wounds presented a phenotype of delayed re-epithelialization, with a younger and more immature connective tissue as reflected by collagen birefringence under polarized light, and with persistence of neutrophils in the wound bed.
Neutrophils negatively influence wound repair probably because this cell type is capable of destroying normal tissue. Their depletion was previously shown to accelerate wound healing in both wild-type and diabetic mice [32] , and delayed wound closure in diabetic mice has been attributed to prolonged persistence of neutrophils and macrophages in the late phases of repair [33].
Although it is still not clear if increased neutrophil numbers in M2 injected wounds in our experiments are due to late infiltration of these cells or to defective resolution of inflammation and neutrophil clearance, we can hypothesize that neutrophil persistence retards the healing process of these wounds.
Our studies are in line with a recent report by Dreymueller et al. Comparable to our findings, ESDM treated wounds presented reduced re-epithelialization and significantly higher cellularity compared to controls, indicating persistence of inflammation during the late phases of repair. It can be hypothesized that M2 macrophages might switch towards a more M1-like phenotype upon injection to the early wounds which exhibit a pro-inflammatory milieu. However, the discrepancy in phenotype between M2 and the control M0 injected wounds would argue against a reverse polarization of exogenously administered cells by the wound environment.
Conversely we can speculate that exogenously administered M2 cells, but not the non-polarized M0 controls, might induce the M1 macrophages present in the wounds at the initial phases of repair, to adopt a less inflammatory profile that ultimately negatively affects healing.
Injection of activated macrophages into wounds of patients was shown to promote wound healing [36] , while rapid recruitment and activation of macrophages into murine wounds accelerates tissue repair [37]. In line, ablation of macrophages during the early phases of repair [6] or loss of TNF [38] , was shown to retard re-epithelialization and wound closure of murine cutaneous wounds.
Therefore it can be hypothesized that some degree of initial pro-inflammatory response is not damaging, but on the contrary, necessary in order to activate the subsequent phases of healing, and is the unrestrained, prolonged or excessive activation of inflammation that is detrimental.
The administration of M2 macrophages during the early inflammatory phases in our experiments might have disrupted this delicate balance. The potential beneficial effect of M2 macrophage administration in later phases of healing needs to be evaluated.
Taken together, our study shows that, at least during murine cutaneous healing responses, manipulation of the wound environment by exogenous administration of M2-polarized macrophages does not represent an effective therapeutic strategy. Cells were analyzed in quadruplicate samples after 24 h of polarization.
Statistical significance was evaluated analyzing M2a and M2c expression levels compared to M0, by t-test. Representative images of picrosirius red collagen deposition stained wounds. Picrosirius red stained sections were analyzed under bright field light A-D or under polarized light E-H.
Intense red birefringence seen in saline and M0 wounds is a consequence of uniform packing of newly deposited collagen fiber bundles that are arranged in parallel arrays while minimal organization of collagen bundles results in little red birefringence intensity as seen in M2a and M2c wounds. Magnification x. We wish to thank Dr. Konings for qPCR primers.
Wrote the paper: NJ SX. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Macrophages play a crucial role in all stages of cutaneous wound healing responses and dysregulation of macrophage function can result in derailed wound repair. Introduction Cutaneous wound healing is a complex and highly dynamic process in which the interaction between resident cells of the skin, inflammatory leukocytes, extracellular matrix components and soluble mediators is essential.
Materials and Methods 2. Morphometric analysis Wounds were photographed every two days and wound area was quantified using Image J. Results 3. Exogenously administered macrophages localize in wounds for several days To determine the fate of exogenously injected macrophages into skin wounds, bone marrow derived macrophages were differentiated from ROSAEGFP transgenic mice [22] and 0.
Download: PPT. Figure 3. Figure 4. M2 macrophages retain the M2 polarization profile 15 days post-injury At 15 days post-injury, mice were sacrificed and wounds were isolated for analysis. Figure 5.
Figure 6. Figure 7. Discussion The role of macrophages during cutaneous wound healing responses has been the subject of intense research over the past decades. Supporting Information. Figure S1. Figure S2. Table S1. Acknowledgments We wish to thank Dr.
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