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Frontiers | Emerging role of extracellular vesicles and exogenous stimuli in molecular mechanisms of peripheral nerve regeneration

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Therapeutic Potential of “Smart” Exosomes in Peripheral Nerve Regeneration

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Therapeutic Potential of “Smart” Exosomes in Peripheral Nerve Regeneration. PubMed, SCI, Scopus, ESCI, PMC indexed

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Ping Liu, 1, ² Jiang Peng, ² Gong-Hai Han, ³ Xiao Ding, ⁴ Shuai Wei, ⁴ Gang Gao, ⁵ Kun Huang, ⁶ Feng Chang, BS, 5,* and Yu Wang, PhD ²*

Abstract

Resident and inflammatory macrophages are essential effectors of the innate immune system. These cells provide innate immune defenses and regulate tissue and organ homeostasis. In addition to their roles in diseases such as cancer, obesity and osteoarthritis, they play vital roles in tissue repair and disease rehabilitation. Macrophages and other inflammatory cells are recruited to tissue injury sites where they promote changes in the microenvironment. Among the inflammatory cell types, only macrophages have both pro-inflammatory (M1) and anti-inflammatory (M2) actions, and M2 macrophages have four subtypes. The co-action of M1 and M2 subtypes can create a favorable microenvironment, releasing cytokines for damaged tissue repair. In this review, we discuss the activation of macrophages and their roles in severe peripheral nerve injury. We also describe the therapeutic potential of macrophages in nerve tissue engineering treatment and highlight approaches for enhancing M2 cell-mediated nerve repair and regeneration.

Keywords: nerve regeneration, macrophage, origin, polarization, function, nerve injury, nerve repair, tissue engineering, neural regeneration

Introduction

Macrophages have the powerful ability to phagocytize foreign bodies (Wynn et al., 2013). They are found in all tissues, where they act as sentinels that protect the tissue and ensure organ homeostasis. Tissue-specific macrophage types originate during development and include osteoclasts (bone), alveolar macrophages (lung), histiocytes (interstitial connective tissue), Kupffer cells (liver and gut), splenic macrophages (spleen), microglia (brain) and differentiated Schwann cells (peripheral nervous system) (Chen et al., 2015). Macrophages function as phagocytic antigen-presenting cells and have key roles in scavenging heterologous pathogens and transmitting danger signals; they are also involved in the formation of memory cells (Iijima and Iwasaki, 2014). However, they may also contribute to secondary infections (Gaya et al., 2015) and cancer metastasis (Keklikoglou and De Palma, 2014). Because of their roles in tissue repair, macrophages represent viable targets for disease treatment (Suzuki et al., 2014; Sadtler et al., 2016). A study by Suzuki et al. (2014) has indicated the potential therapeutic use of pulmonary macrophage transplantation for the treatment of hereditary pulmonary alveolar proteinosis. The application of macrophage-based therapies against peripheral nerve injury is attractive because the proximal and distal ends of the peripheral nerve participate in completely distinct events: proximal regeneration and distal denaturation (DeFrancesco-Lisowitz et al., 2015). Macrophages involved in distal degeneration promote the switch from the pro-inflammatory (M1) to the anti-inflammatory (M2) phenotype, which enhances proximal nerve regeneration (Mokarram et al., 2012). In the M2 macrophage subtypes, in addition to their common anti-inflammatory effects, they also have their respective functions. Briefly, M2a, M2b, M2c and M2d macrophages promote cell proliferation, cell maturation, resolution of inflammation and angiogenesis, respectively (Ferrante and Leibovich, 2012; Novak and Koh, 2013a, b; Chen et al., 2015; Gensel and Zhang, 2015). The M2 macrophage subtypes have great therapeutic potential given their documented anti-inflammatory effects and their respective functions in promoting tissue regeneration in engineered nerve tissue models (Mokarram et al., 2012).

In this review, we discuss the origin, polarization, type and function of macrophages, highlight their roles in peripheral nerve injury and describe their therapeutic value in neural tissue engineering.

We performed a literature search in PubMed from 2002 to December 2018 of studies published in English. The key words were macrophages [MeSH Terms], macrophage polarization and function, and peripheral nerve injuries [MeSH Terms]. The results were further screened by title/abstract, and non-Scientific Citation Index experiments and review articles. Furthermore, studies concerning central nervous system diseases and neoplasms were excluded.

Origin, Polarization, and Function of Macrophages

Tissue-resident macrophages and circulating macrophages (Geissmann et al., 2010; Davies and Taylor, 2015) originate from bone marrow hematopoietic stem cells (Wynn et al., 2013) (Figure 1 ). Before circulating macrophages enter the bloodstream, they must be stimulated by cytokines such as multi-colony stimulating factor (multi-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF) (Wu et al., 2017). Upon stimulation, these cells develop into monocytes, and then differentiate further into pre-macrophages. Finally, they become mature macrophages and are released into the circulation (Chen et al., 2015; Helft et al., 2015).

The macrophage polarization process transforms the macrophages' functions according to their current environment (Li et al., 2018). M1 macrophages are pro-inflammatory that secrete cytokines. M2 macrophages are anti-inflammatory macrophages that promote tissue repair. Because of this dual function, macrophages serve as potent immune effector cells. They play important roles in tissue homeostasis and disease rehabilitation, e.g., promoting the initiation and progression of tissue injury, as well as promoting wound healing and tissue remodeling. Macrophages show different functions at different disease stages; therefore their proper induction and elimination are necessary for efficient recovery (Duffield et al., 2005; Mantovani and Locati, 2013; Zhou et al., 2014). Heterogeneity and plasticity are hallmarks of macrophages (Gordon and Plűddemann, 2013; London et al., 2013) (Table 1 ). A variety of stimuli can induce different degrees of phenotypic polarization. In general, macrophage activation is classified as classic activation (M1) and selective activation (M2). M1 macrophages are induced by Toll-like receptor (TLR), ligands and interferon (IFN)-γ, and express CD32, chemokine (C-C motif) ligand 2 (CCL2), CD86, inducible nitric oxide synthase, HLA-DR, CD197, hypoxia inducible factor-1α (HIF-1α), IFN regulatory factor 1, myeloid differentiation primary response 88, TLR-2 and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Mueller and Schultze-Mosgau, 2011; Gensel and Zhang, 2015; Bardi et al., 2018). Polarized M1 macrophages mainly secrete pro-inflammatory factors, which aggravate inflammation and promote debris removal, sterilization and elimination of apoptotic cells (Gensel and Zhang, 2015). Compared with M1 macrophages, the activation pathway of M2 macrophages is complex. MS-275, a histone deacetylase inhibitor, induces the transformation of M1 to M2 type macrophages (Zhang and Schluesener, 2012). Additional studies have indicated that M2 macrophages can be induced by interleukin (IL)-4/IL-13 (M2a) to express insulin-like growth factor-1 (IGF-1), IL1RN and CD206. M2a macrophages promote anti-inflammatory effects such as cell proliferation and migration, production of growth factors and removal of apoptotic cells. M2b macrophages are induced by an immune complex and express CD86, TNFα, CD64, vascular endothelial growth factor (VEGF) and IGF-1. M2b macrophages promote cell maturation, tissue stabilization, angiogenesis and extracellular matrix synthesis. M2c macrophages are induced by the anti-inflammatory cytokines, IL-10 and transforming growth factor-β (TGF-β), expressing SLAM, Sphk-1, THBS1, HMOX-1, TGF-β, CD206 and CD163. They accelerate the resolution of inflammation, tissue repair and extracellular matrix synthesis, and produce growth factors. M2d macrophages are induced by the A2AR agonist and express high levels of VEGF, IL-10 and inducible nitric oxide synthase, and low levels of TNFα, IL-12 and arginase-1 (Gordon, 2003; Biswas and Mantovani, 2010; Fujiu et al., 2011; Ferrante et al., 2013; Sudduth et al., 2013; Chen et al., 2015). M2d macrophages derived from M1 macrophages have roles in angiogenesis and wound healing (Ferrante and Leibovich, 2012; Chen et al., 2015; Gensel and Zhang, 2015). With changes in the environment, M1 and M2 macrophages maintain a state of dynamic equilibrium. The dynamic switching of phenotype and function in macrophages is regulated by modulating signals (Jang et al., 2013; Mercalli et al., 2013). Recent studies have reported that M2 macrophages could also differentiate into M1 macrophages upon stimulation with lactoferrin-containing immune complexes, thus emphasizing their dynamic nature (Gao et al., 2018).

Table 1

Activation pathway

Stimulating factors

Surface markers/common features

Release factors

Functions

M1

Classic activation

TLR ligands, TNFα, INF-γ

CD32, CCL2, CD86, Nos2, HLA-DR, CD197, HIF-1α

TNFα, IL-1α, IL-1β, nitric oxide, metalloproteinases, reactive oxygen species

Pro-inflammatory, phagocytosis

M2

Recruitment of Macrophages to Injury Sites

The processes of macrophage recruitment and polarization are necessary for macrophage-mediated injury repair (Kucharova and Stallcup, 2018). Recruitment requires the help of chemokines or other signaling proteins. For example, AMPKa1 serves as a recruitment signal in the regeneration of skeletal muscle. In the absence of AMPKa1, macrophages do not acquire the M2 phenotype and have impaired phagocytosis (Lawrence and Fong, 2010). The CC chemokine, CCL2, and its receptor, CCR2, are responsible for monocyte trafficking in response to bone fractures (Xing et al., 2010). Mice deficient in CCR suffer from osteosclerosis (Binder et al., 2009). As a consequence, macrophage recruitment to the site of injury is necessary. After a peripheral nerve injury, monocyte chemoattractant protein-1 (MCP-1, also called CCL2), leukemia inhibitory factor, IL-1α, IL-1β and pancreatitis-associated protein III serve as the major monocyte/macrophage recruitment signals (Shamash et al., 2002; Tofaris et al., 2002; Namikawa et al., 2006; Van Steenwinckel et al., 2015). Macrophage infiltration is first observed at the site of injury 2-3 days post-injury and peaks at 7 days post-injury (Perry et al., 1987; Taskinen and Röyttä, 1997; Bendszus and Stoll, 2003; Mueller et al., 2003; Namikawa et al., 2006). To boost recruitment, infiltrated macrophages may secrete CCL2, TNFα, IL-1α and IL-1β (Shamash et al., 2002; Kiguchi et al., 2013). In nerve injury models, macrophage recruitment occurs even in the absence of the key recruitment signals, MCP-1 and IL-1β, as injured nerves also produce recruitment signals (Shamash et al., 2002). In Schwann cells, macrophage recruitment is regulated by the miR-327/CCL2 axis. When stimulated by the outside microenvironment, the increase in miR-327 expression inhibits macrophage recruitment, while CCL2 promotes macrophage recruitment (Zhao et al., 2017). In CCR2-deficient mice, macrophage infiltration to injured nerve sites is significantly decreased (Siebert et al., 2000; DeFrancesco-Lisowitz et al., 2015), which indicates that CCL2 is a primary pro-recruitment molecule. In summary, cytokine signaling is necessary for proper macrophage recruitment and subsequent injury repair.

After recruitment, the number of macrophages in the injured site increases. The macrophage subtypes do not increase simultaneously. Previous studies have indicated that macrophages, in most tissues, transit from M1 to M2 macrophages over time (Arnold et al., 2007; Dal-Secco et al., 2015). In addition to the above-mentioned polarization stimulation factors derived from injury, the M2a subtype can secret cytokines such as IL-10 and TGF-β, polarizing macrophages toward the M2c subtype directly (David and Kroner, 2011). When recruited and polarized, macrophages secrete cytokines to prepare for tissue repair.

Roles of Macrophages in Peripheral Nerve Injury

Nerve injury includes central nerve injury and peripheral nerve injury, both of which present challenges with respect to clinical treatment and functional recovery (Burtt et al., 2017), particularly those associated with the spinal cord. By contrast, skin and muscle tissues respond well to tissue repair mechanisms and show good recovery. The mechanisms governing these tissue-specific differences are not well understood, but it is clear that macrophages are key mediators of repair in all tissue types. Spinal cord injury can induce a chronic wound state that undergoes expansion and maintains demyelination, leading to impaired recovery and progressive tissue degeneration (Chiu et al., 2018). A spinal cord injury consists of three phases: inflammation, proliferation and remodeling (Gensel and Zhang, 2015). The central nervous system is unique in that complete regeneration after injury is rare, and relative to other injury forms, the repair process is complex and poorly understood. By contrast, peripheral nerves have strong regenerative capacity and macrophages play a core role in their repair. Injuries will induce distal segment of the nerve undergoing a series of cellular and molecular events that result in breakdown of the distal nerve segment, which is called Wallerian degeneration (Rotshenker, 2011), thereby promoting the regeneration of peripheral nerve. For these reasons, we will focus primarily on macrophage-mediated repair in the peripheral nerve injury setting. Unlike the central nervous system, where damaged neurons are usually unable to regenerate, axons in the peripheral nervous system can regenerate after injury (Figure 2 ).

After nerve injuries, especially severe and long-distance nerve injuries, local hypoxia and tissue necrosis secondary to inflammation are major obstacles for nerve repair and regeneration, which require a good microenvironment that is clean of necrotic tissue fragments, to promote angiogenesis, and the proliferation and migration of glial cells (Schwann cells). However, macrophages have the capacity to promote nerve repair (Cattin et al., 2015).

Macrophages respond to hypoxia by promoting angiogenesis

Damage to peripheral nerves promotes the recruitment and infiltration of many inflammatory cell types. Among the inflammatory cell types, only macrophages can sense (Cattin et al., 2015) and respond to hypoxic conditions through activation of the transcription factor, HIF-1α, which ultimately stimulates angiogenesis via VEGF activation (Pugh and Ratcliffe, 2003; Krock et al., 2011). In this case, macrophages in both the distal and proximal stumps are in hypoxic conditions (pO ₂ < 10 mmHg) (Young and Moller, 2010). At 2 days post-injury and before vascularization, a large number of hypoxic cells are present at the site of injury. This triggers HIF-1α synthesis in macrophages, followed by the expression of VEGF-A, which stimulates endothelial cell proliferation and migration. By day 3 post-injury, the proportion of hypoxic cells is substantially decreased, which indicates vascularization of the injured site (Cattin et al., 2015). Macrophages promote angiogenesis (Fantin et al., 2010) and maintain the health of endothelial cells by secreting VEGF-A (Lee et al., 2007). Macrophage-induced angiogenesis, which provides nutrition for the repair of the nerve tissue, is a critical step in injury repair. In addition, as macrophages are polarized into different subtypes, they can release cytokines to accelerate angiogenesis. For example, M2d macrophages secrete IL-10 and VEGF, which facilitate angiogenesis and the repair of blood vessels (Ferrante and Leibovich, 2012). Furthermore, angiogenesis provides a route for the migration of Schwann cells to the injury site.

Macrophages contribute to the migration and proliferation of Schwann cells

Macrophages promote inflammation through the production and release of pro-inflammatory cytokines. In addition, they serve as important antigen-presenting cells, stimulating an immune response to repair damage and accelerate recovery (Mueller et al., 2003). In addition to these roles, macrophages activate the proliferation and division of Schwann cells, which are vital glial cells in the peripheral nerve tissue (Armstrong et al., 2003). A previous study has indicated that after injury, Wallerian denaturation in the distal stump nerve occurs, and Schwann cells begin to dedifferentiate into Schwann cell precursors (Chen et al., 2015). After dedifferentiation, progenitor Schwann cells have a stronger phagocytosis and secretory capacity. These features aid in debris removal from the injury site and increase the release of nerve growth factor (NGF), brain-derived growth factor and ciliary nerve growth factor, which are key growth factors involved in nerve repair, or regeneration and proliferation of Schwann cells (Gong et al., 2014; Hoyng et al., 2014). Wallerian denaturation is also associated with axonal degeneration, compromise of the blood-nerve barrier, myelin breakdown and macrophage infiltration (Stoll and Muller, 1999). Therefore, it effectively promotes macrophage infiltration, which subsequently promotes migration and proliferation of Schwann cells. Additional studies have reported that M2a macrophages boost cell proliferation and cell migration by releasing IL-10 or TGF-β (Ferrante and Leibovich, 2012; Chen et al., 2015; Gensel and Zhang, 2015). In the hypoxic nerve injury microenvironment, macrophages sense low oxygen levels and promote angiogenesis, which allows endothelial cells to guide the direction of Schwann cell migration. Schwann cells use the polarized vasculature as a support to accelerate nerve repair and regeneration (Cattin et al., 2015), and synthesize the basement membrane for axon regeneration (Gong et al., 2014). Therefore, the macrophage-mediated migration and proliferation of Schwann cells has important implications in peripheral nervous system injuries.

Macrophages promote anti-inflammation and display enhanced scavenger function

In response to injury, macrophages are polarized into different subtypes to participate in damage repair. After activation, M1 macrophages show enhanced phagocytic ability along with increased scavenging of cellular debris and bacteria via upregulation of the scavenger receptor, CD36 (Sindrilaru and Scharffetter-Kochanek, 2013). M2a, M2b and M2c macrophages release anti-inflammatory cytokines, and increase cell proliferation and migration, tissue remodeling ( via release of Arginase and Ym1) and secretion of growth factors (Novak and Koh, 2013a, b). Therefore, when macrophages retain the M1 state, injured nerve tissues remain in an inflammatory and chronic wound state (Wang et al., 2007; Hu et al., 2011; Sindrilaru et al., 2011; Rigamonti et al., 2014). In addition, it has been reported that macrophages modulate the expression of galectin-1 (Gaudet et al., 2015), which has been implicated in the regulation of phagocytosis, inflammation/gliosis and axon growth after spinal cord injury. Moreover, galectin-1 has been shown to promote neural stem cell proliferation (Sakaguchi et al., 2006; Yamane et al., 2010).

Macrophages Application in Neural Tissue Engineering

Self-repair is not an option when faced with severe and long-distance nerve injuries. Fortunately, significant technological progress has been made in the field of neural tissue engineering. Typical engineered nerve tissue includes a nerve scaffold, seed cells and nerve factors (Gu et al., 2014). The nerve scaffold provides support and protection, and the seed cells and related nerve factors accelerate nerve regeneration. Tissue engineering has become particularly applicable for the treatment of severe peripheral nerve damage. Although great progress has been made in nerve tissue engineering for long-distance nerve defects, improvements to optimize nerve regeneration and function restoration in target organs are still needed. Engineering involves the fabrication of a biocompatible artificial nerve scaffold that mimics the natural extracellular matrix (Gu et al., 2014), and the addition of seed cells and related factors to the nerve scaffold to promote nerve regeneration. Often, stem cells are selected as seed cells for nerve tissue engineering. Stem cells have the potential to differentiate into Schwann cells, secrete neurotrophic factors, and assist in nerve regeneration and myelin formation (Ren et al., 2012), but they cannot regulate inflammation or clean up necrotic debris (Mokarram et al., 2012). Therefore, efforts to incorporate macrophages into the engineered nerve tissue are of great importance.

Artificial nerve conduits can be used to bridge the ends of damaged peripheral nerves. Macrophages are recruited to the lesion under the action of chemokines (Shamash et al., 2002; Tofaris et al., 2002; Namikawa et al., 2006; Van Steenwinckel et al., 2015). Recruitment to the injury site leads to activation of both the M1 and M2 subtypes. The coordination of M1 and M2 regulates the initial events of local inflammation (Mokarram et al., 2012). The distal segment of the nerve undergoes a series of cellular and molecular events that result in Wallerian degeneration (Rotshenker, 2011). During this process, cytokine production contributes to the recruitment of inflammatory cells such as M1 cells (Chen et al., 2007). M1 cells release additional pro-inflammatory cytokines such as TNFα, IL-1α and IL-1β, nitric oxide, reactive oxygen species and metalloproteinases (Murray and Wynn, 2011), which promote further tissue destruction. To control this process, activated M2 macrophages release the anti-inflammatory cytokines, IL-4/IL-13 and IL-10, which mediate tissue restoration and suppression of pro-inflammatory responses (Gordon, 2003). Therefore, engineered nerve tissues would benefit from the incorporation of macrophages, given their key roles in the regulation of inflammation (Mokarram et al., 2012). The ratio of pro-healing (M2) to pro-inflammatory (M1) macrophages directly correlates with the number of regenerated axons (Mokarram et al., 2012). Thus, methods that increase the ratio of M2/M1 subtypes and promote nerve regeneration have become increasingly popular. There are two ways to increase the M2/M1 ratio in nerve injury or artificial nerve conduits. The first method incorporates the chemotaxis and polarization of macrophages by cytokines, and the second method involves direct injection of M2 into the nerve conduit. For the former approach, physical methods have been used to adsorb macrophage chemotactic factors onto nerve conduits. However, the physically adsorbed components are free to diffuse from the scaffold surface into the surrounding tissue shortly after implantation. Therefore, the chance of recruiting and polarizing macrophages to the M2 state is small (Wang et al., 2012). To address this challenge, a combination of conjugated and adsorbed IL-10 was found to be an effective approach for inducing macrophage M2 polarization in the vicinity of scaffolds (Potas et al., 2015). IL-10 desorbed from the scaffold is free to promote the polarization of macrophages beyond the scaffold surface. Macrophage stimulation by exogenous IL-10 subsequently triggers the production of endogenous IL-10 (Mantovani et al., 2002), which is significantly increased in M2 macrophages. It is commonly thought that M2 macrophages respond after M1 macrophages have fulfilled their roles. However, the presence of M2 macrophages at early time points post-injury indicates that M2 cell activation is promoted by extrinsic factors. M2 macrophages not only overcome axonal growth inhibition, but also promote regeneration (Kigerl et al., 2009). Further studies have shown that engineered scaffolds treated with IL-4 or IFN-γ directly polarized macrophages toward the M2a subtype, and indirectly toward the M2c subtype. M2a macrophages secrete cytokines such as IL-10 and TGF-β (David and Kroner, 2011), and function in cell proliferation and migration, removal of apoptotic cells, cell maturation, tissue stabilization, angiogenesis, resolution of inflammation and tissue regeneration (Ferrante and Leibovich, 2012; Chen et al., 2015; Gensel and Zhang, 2015). These results indicate that adsorption of cytokines on the scaffolds increases the ratio of M2 macrophages, leading to significantly enhanced nerve regeneration. The explanation for this observation is associated with the vital functions of macrophages in wound healing. Specifically, the optimal balance of macrophage types promotes healing by regulation of the injury microenvironment, detection of hypoxia, induction of angiogenesis (Cattin et al., 2015; Moore et al., 2018) and preparation of the surrounding parenchyma for regeneration.

The second method for enhancing the local M2 macrophage population involves direct injection of macrophages into the injured nerve or nerve conduit. This method was validated by a recent study that incorporated a model of ischemia developed by unilateral femoral artery excision. Macrophages (M0, M1, M2) and phosphate-buffered saline were injected into the ischemic muscle 24 h after injury. Four days after treatment, M1 macrophage therapy restored perfusion and the number of endothelial cells, improved muscle morphology and fibrosis, and promoted functional recovery (Lu et al., 2011). The transplantation of M1 macrophages promoted a faster transition from the M1-like to M2-like phenotype (Gensel and Zhang, 2015). Although this application cannot be used to promote nerve tissue repair or regeneration, the results indicate that during damage repair no single macrophage subtype can effectively promote the repair of damaged tissue.

Conclusion and Perspective

Macrophages are key mediators of inflammation. Macrophage recruitment to the site of injury initiates a cascade of events that contribute to proper nerve tissue repair. Macrophages not only play an important role in the repair of damaged nerves, but also represent a therapeutic target for treatment of peripheral nerve injury. Owing to their pro- and anti-inflammatory nature, macrophages are able to protect tissues and promote repair. During tissue repair, M1 macrophages secrete pro-inflammatory cytokines, thereby enhancing inflammatory reactions and tissue necrosis. By contrast, M2 macrophages induce an anti-inflammatory response, accelerate tissue repair and represent a target for therapeutic treatment. Effective tissue repair requires coordination of M1 and M2 activities. M1 macrophages have powerful phagocytic effects and promote a suitable microenvironment for M2 macrophages to repair the injured tissue. Interventions that involve injection of M1 macrophages into the sites of injury appear to have the best therapeutic effects. The injected M1 cells accelerated both the removal of necrotic tissue and transformation of the M1 to M2 phenotype. While promising, these outcomes have only been demonstrated in a model of ischemic muscle injury, and have not been observed in the context of peripheral nerve injury. In some immune system diseases, M2 polarization is suppressed and the number of M1 macrophages is increased, leading to increased inflammation and the improper repair of the injured tissues.

Because the underlying mechanisms governing this process are poorly understood, further studies in this area are required for the successful development of novel therapeutic strategies. For example, a clear understanding of the specific therapeutic targets of macrophages is critical for the development of effective treatment strategies that involve macrophage activation. There is also uncertainty regarding the best methods for ascertaining what types of stimuli effectively promote macrophage M2 polarization. To be considered valuable for clinical applications, it is important to elucidate the regulatory mechanisms that control the transformation of macrophages to M2, and promote nerve repair and regeneration.

These findings shed light on improving peripheral nerve regeneration after scaffold modification by adsorption of cytokines such as IL-4, IFN-γ or IL-10, to promote macrophage M2 polarization. However, to promote nerve regeneration in neural tissue engineering, we need a good microenvironment, as well as good seed cells and nerve regeneration-related growth factors. Adsorption of M1 and M2 macrophage chemokines on scaffolds, or addition of polarized M1, and M1 macrophages as seed cells, may provide novel therapeutic targets for severe and long-distance nerve injuries. However, the amount and proportion of the two kinds of chemokines adsorbed on the scaffolds, and of polarized M1 and M2 macrophages added to the scaffolds need further study. The macrophage subtypes that respond to hypoxia, and promote the migration and proliferation of Schwann cells, and the mechanism remain to be further studied.

Additional files: Open peer review reports 1-3.

OPEN PEER REVIEW REPORT 1

OPEN PEER REVIEW REPORT 2

OPEN PEER REVIEW REPORT 3

Footnotes

Conflicts of interest: The authors declare no competing interests.

Financial support: This work was supported by the National Natural Science Foundation of China, No. 31771052 (to YW); the National Key Research & Development Program of China, No. 2017YFA0104701, 2017YFA0104702 and 2016YFC1101601; the National Basic Research Program of China (973 Program), No. 2014CB542201 (to JP); the Natural Science Foundation of Beijing, No. 7172202 (to YW); and the PLA Youth Training Project for Medical Science, No. 16QNP144 (to YW). None of the funding bodies plays any role in the study other than to provide funding.

Copyright license agreement: The Copyright License Agreement has been signed by all authors before publication.

Plagiarism check: Checked twice by iThenticate.

Peer review: Externally peer reviewed.

Open peer reviewers: Ozgur Boyraz, Gulhane Military Medical Academy, Turkey; Sheng Yi, Nantong University, China; Mark A. Yorek, University of Iowa, USA.

Funding: This work was supported by the National Natural Science Foundation of China, No. 31771052 (to YW); the National Key Research & Development Program of China, No. 2017YFA0104701, 2017YFA0104702 and 2016YFC1101601; the National Basic Research Program of China (973 Program), No. 2014CB542201 (to JP); the Natural Science Foundation of Beijing, No. 7172202 (to YW); and the PLA Youth Training Project for Medical Science, No. 16QNP144 (to YW).

P-Reviewers: Boyraz Q, Sheng Y, Yorek MA; C-Editor: Zhao M; S-Editors: Yu J, Li CH; L-Editors: Bell M, Wang L, Song LP; T-Editor: Liu XL

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Articles from Neural Regeneration Research are provided here courtesy of Wolters Kluwer -- Medknow Publications

# data[].attributes.attachment_link.title
Role of macrophages in peripheral nerve injury and repair

# data[].attributes.attachment_link.description
Resident and inflammatory macrophages are essential effectors of the innate immune system. These cells provide innate immune defenses and regulate tissue and organ homeostasis. In addition to their roles in diseases such as cancer, obesity and osteoarthritis, ...

# data[].attributes.attachment_link.title
Extracellular vesicle-encapsulated microRNA-23a from dorsal root ganglia neurons binds to A20 and promotes inflammatory macrophage polarization following peripheral nerve injury

# data[].attributes.attachment_link.description
Extracellular vesicles (EVs) are capable of transferring microRNAs (miRNAs or miRs) between two different types of cells and also serve as vehicles for delivery of therapeutic molecules. After peripheral nerve injury, abnormal expression patterns of miRNAs…

# data[].attributes.attachment_link.title
Exosomes and their microRNA cargo: new players in peripheral nerve
regeneration

# data[].attributes.attachment_link.description
Peripheral nerve injury is a major clinical problem and often results in
a poor functional recovery. Despite obvious clinical need, treatment strategies
have been largely suboptimal. In the nervous system, exosomes, which are
nano-sized extracellular…

# data[].attributes.attachment_link.title
Macrophage polarization in nerve injury: do Schwann cells play a role?

# data[].attributes.attachment_link.description
In response to peripheral nerve injury, the inflammatory response is almost entirely comprised of infiltrating macrophages. Macrophages are a highly plastic, heterogenic immune cell, playing an indispensable role in peripheral nerve injury, clearing debris…

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Abstract

Macrophages are present in all mammalian tissues and coexist with various cell types in order to respond to different environmental cues. However, the role of these cells has been underestimated in the context of peripheral nerve damage. More importantly, macrophages display divergent characteristics, associated with their origin, and in response to the modulatory effects of their microenvironment. Interestingly, the advent of new techniques such as fate mapping and single-cell transcriptomics and their synergistic use has helped characterize in detail the origin and fate of tissue-resident macrophages in the peripheral nervous system (PNS). Furthermore, these techniques have allowed a better understanding of their functions from simple homeostatic supervisors to chief regulators in peripheral neuropathies. In this review, we summarize the latest knowledge about macrophage ontogeny, function and tissue identity, with a particular focus on PNS-associated cells, as well as their interaction with reactive oxygen species under physiological and pathological conditions. We then revisit the process of Wallerian degeneration, describing the events accompanying axon degeneration, Schwann cell activation and most importantly, macrophage recruitment to the site of injury. Finally, we review these processes in light of internal and external insults to peripheral nerves leading to peripheral neuropathies, the involvement of macrophages and the potential benefit of the targeting of specific macrophages for the alleviation of functional defects in the PNS.

Graphical Abstract

Keywords: Nerve-resident macrophages, Macrophage polarization, Peripheral neuropathy, Wallerian degeneration, Neuroinflammation, Oxidative stress

Background

Most tissues in the body possess tissue-resident macrophage populations that are critical regulators of tissue homeostasis and host protection, in a diverse and changing environment. These cells are recognized as tissue phagocytes that derive from blood monocytes while their heterogeneity has also gained increased recognition. Despite some remaining controversies, the embryonic origin of key tissue-resident macrophage populations is now largely understood. The day-to-day maintenance and homeostasis of the resident macrophage pool is thus suggested to take place via two mechanisms: (i) proliferation of initially resident macrophages and (ii) monocyte infiltration and engraftment [ 1]. The question of which of these is favoured under physiological or pathological conditions in still unclear. Macrophages respond to environmental signals by reprogramming their metabolism and polarizing their phenotype/function in order to regulate their microenvironment in an active feedback loop. These cells express a plethora of cytokines, produce nerve growth factor (NGF) and ROS, and also regulate the composition of the extracellular matrix (ECM) [ 2- 6]. In the case of injury such as trauma or infection, it is well-known that various non-neuronal cells act at the site of injury, mainly in the form of Schwann cells (SC), and immune cells such as macrophages and fibroblasts [ 7, 8]. Macrophages, in particular, play an orchestrating role in peripheral nervous system (PNS) tissue injury and degeneration and later in the resolution of inflammation and tissue repair. Importantly, emerging evidence suggests that the phenotype of macrophages (pro-inflammatory versus anti-inflammatory) affects the outcome of regeneration [ 9]. "Healthy" neuroinflammation contributes to healing and remyelination through the production of several neurotrophic factors by all types of immune cells, the reduction of immune over-activity by growth factors produced by immune cells, the phagocytic clearance of inhibitory myelin debris and toxic substances, and the removal of chondroitin sulphate proteoglycans that impede remyelination and axonal regeneration [ 10]. In contrast, unresolved neuroinflammation can be detrimental to nerve structure and consequently, nerve function. In models of PNS axon regeneration in rodents, the local environment is permissive to regeneration for up to 4-8 weeks after injury, and becomes less trophic or atrophic afterwards, with changes in the ECM [ 6]. In this regard, there is a distinction between the terms PNS "axon regeneration" and PNS "nerve repair". Indeed, a small amount of evidence has shown that axons in the PNS may be able to regenerate and function properly after injury. However, nerve regeneration and clinical functionality after injury is probably not as optimal, hence in the best case, what occurs is rather nerve repair.

In this review, we summarize recent findings regarding nerve-resident macrophages, the current understanding of the mechanisms that underlie self-maintenance and imprinting of nerve-resident macrophages, as well as those controlling the access of monocytes to the macrophage niche. We revisit the process of Wallerian degeneration (WD), summarizing the critical steps and thereby the accompanying events of monocyte/macrophage recruitment into injured nerves, while taking into account the importance of the nerve-blood barrier in this process. Finally, we discuss the main PNS pathologies, specifically addressing the role of neuroinflammation and oxidative stress in their evolution and progression. Although many issues remain to be adequately resolved, new techniques are increasing our understanding of the role of macrophages in immunity and immunopathology, and additionally providing detailed insights on macrophage commitment to tissue niches and their behaviour in the case of internal or external insults. This, it is hoped, will pave the way for targeting of specific macrophages for alleviating nerve injuries associated with peripheral neuropathies.

Tissue macrophages: ontogeny and function

What we know about their origin

In 1926, Alexis Carrel and Albert H. Ebeling concluded after a series of experiments that: "a macrophage is merely a monocyte in a more active metabolic condition" [ 11]. Since then, the prevailing view has been that tissue macrophages are continuously repopulated by blood-circulating monocytes derived from progenitors present in the bone marrow. In 1972, a group of specialists from around the world proposed the mononuclear phagocyte system (MPS) as a classification of both monocytes and macrophages and their precursors. These authors described a family of cells based on their origin, morphology, function and kinetics [ 12]. Indeed, monocytes and macrophages rely on the same growth factors and transcriptional regulators (such as Purine-rich Box-1 (PU.1)), and share the expression of several surface markers (notably colony stimulating factor-1 (CSF1) receptor) [ 13]. However, in the last decade, this view has been revised after the emergence of a series of breakthrough publications. Impressive evidence, based on macrophage ontology, showed that some tissue-resident macrophages are first seeded during embryonic haematopoiesis without monocyte intermediates, and that these persist throughout the individual's life. As such, they are able to locally self-maintain independent from blood monocyte input [ 14- 18]. Given that haematopoiesis is well-conserved between Drosophila and vertebrates, studies in Drosophila larva showed that primitive haematopoiesis in the yolk sac produces erythroid cells and macrophage progenitors as the sole "white blood cells". These primitive macrophages give rise to many types of tissue-resident macrophages [ 19]. More recently, several fate mapping ontogeny studies have been performed in embryonic and adult mice [ 20- 22]. Foetal macrophage ontogeny showed that three successive waves of haematopoiesis (from embryonic day 7.5 (E7.5) till E10.5) occur during development (reviewed in [ 15]). The first wave is termed primitive haematopoiesis, taking place in the extra-embryonic yolk sac and giving rise to maturing macrophages. The second wave is intraembryonic and arises from erythro-myeloid progenitors (EMP) that migrate to the foetal liver and give rise to maturing monocyte/macrophages. The third wave arises from haemogenic endothelium and leads to the generation of immature haematopoietic stem cells (HSCs) that colonize the foetal liver as well as the foetal bone marrow. Eventually, mature HSCs in the bone marrow commit to their role in adult haematopoiesis. Importantly, many adult tissue-resident macrophages have been traced to embryonic origin, independently of provision from the bone marrow [ 15, 20].

The contribution of monocyte recruitment and resident macrophage proliferation to expansion of tissue macrophages is still controversial. To date, available evidence suggests that these two mechanisms are unlikely to be independent in the course of local macrophage expansion (for review, see [ 23]). In general, it is accepted that, at steady state, (1) embryonic macrophages, (2) adult monocyte-derived macrophages, and (3) daughter cells of adult monocyte-derived macrophages function all function together. In addition, in inflamed tissue, (4) recruited adult monocyte-derived cells are added to the already-diverse population [ 15]. However, for a given tissue, the contribution of distinct macrophage lineages/subtypes in these processes is currently unclear. To address this question, sophisticated approaches (such as fate mapping) are being developed and used to separate resident from infiltrating macrophages [ 23]. In their review, Ginhoux and Guilliams [ 15] classify adult tissues into those which exclusively contain yolk-sac-derived macrophages (brain, lung, liver, and epidermis), those with fast steady-state monocyte recruitment (dermis and gut), and tissues with slow steady-state input (pancreas and heart). Steady, low-grade metabolic, oxidative, or mechanical stress (as in physiological aging) can deplete these tissue-macrophage pools. Therefore, these conditions could drive homeostatic monocyte provision in order to replenish tissue-macrophage pools in a pattern similar to inflammation.

Monocytes, macrophages, and reactive species

ROS and RNS secretion is important in monocyte/macrophage surveillance in tissue homeostasis and first-line defence. Under physiological conditions, around 90% of endogenous ROS are produced in mitochondria by oxidative phosphorylation [ 52], and a minor amount comes from plasma membrane proteins, lipid metabolism, and the activity of cytosolic enzymes [ 53]. Normally, the body is equipped with a counter-acting defence system (such as glutathione and cytochrome p450) to "balance" continuous physiological ROS production. Nevertheless, the balance between ROS generation and the antioxidant system is slightly in favour of ROS, and a continuous low level of oxidative damage persists [ 54]. In turn, activated circulating monocytes and M1 macrophages increase ROS and RNS production after exposure to various signals, including pathogen-derived molecular patterns (PAMPs, such as lipopolysaccharide), damage-associated molecular patterns (DAMPs, such as high-mobility box 1 protein (HMGB-1), nucleotides, and DNA), cytokines (e.g. tumour necrosis factor-α (TNFα), interferon-γ), metabolic stress (e.g. hyperglycaemia, advanced glycation end-products, oxidized lipoproteins), endoplasmic reticulum stress, unfolded/misfolded protein accumulation, and some nanoparticles [ 29].

While both monocytes and macrophages are also subjected to oxidative damage caused by their own secretion of reactive species, macrophages are more robust than monocytes [ 55]. In order to survive and continue to function in such a hostile oxidative and inflammatory environment, macrophages are equipped with a network of protective mechanisms (notably the Nrf2 pathway and its downstream effectors, and the FOXO pathway) [ 29]. This difference may be linked to the modulatory effect of macrophages in which they kill excess monocytes during an inflammatory response. However, it is noteworthy that the mechanism of ROS sensing by macrophages (and other cells) is not fully clear. Despite this advantage in being protected from oxidative damage, macrophages are still not fully resistant to ROS-induced death. When oxidative stress is maintained for a long period of time, macrophages accumulate massive amounts of oxidized proteins and lipids, leading to metabolic dysfunction, phenotypic alterations and cell death. This is particularly relevant to human diseases such as inflammatory, autoimmune, cancerous, vascular, respiratory, and neurodegenerative diseases in which ROS are produced excessively [ 53]. Moreover, over time, innate repair mechanisms tend to be slowed and then overwhelmed, and organisms progressively lose physiological integrity due to the accumulation of cellular damage, contributing to aging and age-related diseases such as neurodegeneration.

Nerve-associated macrophages: where we are now

It is now four decades since sciatic nerve macrophages were first identified [ 56]. However, and in compelling contrast to central nervous system (CNS) microglia, PNS-resident macrophages are among the least studied subpopulations. Little is known about their cellular origin, capacity for self-maintenance, and their gene signature. Most research efforts are devoted to study CNS pathological mechanisms, where macrophages play pivotal roles. Recent observations in many CNS neurodegenerative diseases that PNS is a relevant disease target-especially in Multiple Sclerosis [ 57] could reshape research towards investigating systemic involvement in neurological diseases.

Where are these macrophages located? A peripheral nerve is made up of several bundles of axons, where individual axons are enveloped by myelinating or non-myelinating SC. Axons are embedded in an endoneural compartment made up of collagenous connective tissue and fibroblasts [ 58]. Axon fascicles are enclosed in the collagenous epineurium matrix that also contains small arteries and veins, as well as fibroblasts and macrophages [ 59- 61]. Studies also show that macrophages exist in the endoneurium, between myelin sheaths and in close contact with axons [ 56, 59, 62]. Normally, these macrophages continually survey the environment for threats and help to clear cellular debris.

Single-cell RNA sequencing, first used in 2009 [ 63], allows the remarkably fine characterization of specific cell subsets in a heterogeneous tissue. Only recently, this technique was used to characterize nerve-associated macrophages in mouse skin [ 35]. These cells are distinguished by their high expression of CX3CR1 marker (CX3CR1 hi), in comparison to other dermal macrophages. CX3CR1 hi nerve-associated macrophages closely interact with sensory nerves, both with respect to their transcriptome and their axon-scanning behaviour. Using fate mapping techniques, these cells were found to arise either from CX3CR1 hi prenatal progenitors, which are the dominant source in homeostasis, or from CX3CR1 low pro-inflammatory monocyte-derived macrophages [ 64] in the case of tissue injury; thus two distinct origins of these nerve macrophages can be differentiated. In an ear-punch murine model, these cells expand and contribute to myelin degradation, nerve regeneration, and wound healing [ 35]. The role of CX3CR1 in these highly committed peripheral nerve macrophages is not yet fully clear. Evidence from murine models indicates that CX3CR1 ligand CX3CL1 (also known as fractalkine FKN) triggers ROS production in CX3CR1 macrophages, as well as monocyte infiltration in sciatic nerves [ 65]. In addition, the chemokine FKN was shown to mediate neural/microglial interaction in neuropathic pain in rats [ 66].

A recent study thoroughly characterized sciatic nerve macrophages in mice [ 34]. Sciatic nerve macrophages presented a tissue-specific gene signature distinguishing them from other tissue macrophages (in the optic nerve, liver, lung, spleen, and peritoneum) and CNS microglia. The sciatic nerve macrophage profile was enriched by a unique gene set including Adam19, Cbr2, Cd209d, Foxred2, Fxyd2, Mgl2, Mmp9, Il1rl1, Kmo and Tslp. Single-cell profiling identified two spatially separated sciatic nerve macrophage types: Relmα⁺Mgl1⁺ cells in the epineurium and Relmα −Mgl1 − cells in the endoneurium (Fig. 1 ). By fate mapping, the authors showed that sciatic nerve macrophages do not derive from the early embryonic precursors colonizing the CNS, but originate primarily from late embryonic precursors and are replaced by bone marrow-derived macrophages over time. Finally, in contrast to the CNS, upon injury, the PNS macrophage pool is replenished by monocyte-derived macrophages. Indeed, investigating nerve-associated macrophages is a very topical area of research with significant challenges. Recently published studies, although few, have contributed greatly to the understanding of the striking functional plasticity of macrophages and of their potential role in peripheral neuropathies. Studying peripheral nerve immune responses in conditional knockout transgenic animals has provided further strong insights to aid the understanding of physiological/pathological crosstalk between the systemic immune system and peripheral nerve compartment.

Peripheral nerve disease: all roads lead to inflammation

Wallerian degeneration (WD): implications of neuronal immune response and macrophages

This process, initially named for Augustus Volney Waller who experimented on and described distal axon and myelin disintegration following nerve transection in 1850 [ 67] is now recognized to occur in other non-injury neuropathies, such as Alzheimer's disease [ 68]. Immediately after injury, and before transcriptional changes take place in the injured neuron, a retrograde injury signal is sent towards the nucleus [ 69]. The length of human axons presents a special challenge in comparison to animal axons, as injury signals must travel a long distance to reach the nucleus, located in the brain or spinal cord. Subsequently, the neuron upregulates the expression of growth-associated genes [ 9]. Meanwhile, the distal fibre ending undergoes "dying-back" degradation and clearance. The unusual extended survival of the slow Wallerian degeneration (Wld S) mutant mouse axons without their cell bodies [ 70] has fundamentally changed our view of axon degeneration. Subsequent studies have indicated that the Wld S gene product is an extraordinary fusion product of the ubiquitin ligase Ube4b gene and Nmnat1 (nicotinamide mononucleotide adenylyltransferase 1) [ 71]. This has resulted in a deeper understanding of WD at the molecular level. Specifically, maintaining axonal NAD⁺ levels by Nmnat2 enzyme activity is essential for axon protection [ 72]. Since that time, Sarm1 ((sterile α/Armadillo/TIR homology domain) was identified as a pro-degenerative axonal factor preserved throughout evolution [ 73]. A new piece has also been added to the puzzle with the discovery of the NAD⁺-depleting activity of SARM1 [ 74] acting downstream from Nmnat2 [ 75]. Figure 2 outlines the main players that control the WD pathway.

The recent discoveries of a link between Nmnat2 mutation and human diseases, in addition to the determination of protein structures involved in WD, is indeed promising in terms of deciphering underlying pathological mechanisms as well as for aiding drug development [ 68]. On the other hand, it has been well-established that the absence of Sarm1 in Drosophila [ 73], mouse [ 76] and Zebrafish [ 77] prevents the degradation of damaged axons. In addition, reducing or blocking the expression of Sarm1 ameliorates SC-resistance to chemotherapeutic agents, even after axon injury. However, macrophage recruitment to the injury site, and thus resolution of focal damage, was not affected [ 77]. Indeed, our knowledge to date is far from complete, and it is accepted that this process appears to be driven by a complex combination of cell populations to perform a wide spectrum of tasks at the site of injury. For instance, WD of the axon is accompanied by the trans-differentiation of SCs into a proliferative and repair phenotype, and the activation of a sterile immune response, specifically macrophages that are recruited by mechanisms that are both dependent and independent of blood monocyte infiltration [ 8, 14].

What attracts macrophages to the site of injury? After nerve insult, macrophages are among the first (2-3 days post-injury) and the most abundant cells to infiltrate the injury site, where monocytes/macrophages are recruited by factors produced by repair SCs, and then they further produce chemoattractants, such as CCL2, TNF-α, IL-1α and IL-1β, for greater macrophage infiltration [ 78]. CCL2 is the major chemokine for monocytes and macrophages. It binds with high affinity to the absence of receptor CCR2 which is expressed essentially by monocytes and macrophages [ 79] as well as by sensory neurons after injury [ 80]. Wang and colleagues [ 81] examined the immune capacity of neurons, particularly the role of SARM1 in Ccl2 induction. They reported that SARM1 acts via c-Jun kinase (JNK) and phospho-Jun to trigger the expression of CCL2 as well as CSF-1, CCL7, and CCL12 in DRG neurons. This neuronal immune response starts as early as 8 h post-injury, that is, even before the axon starts to degenerate. Moreover, blockade of the SARM1-JNK pathway abolishes the recruitment of immune cells as well as axon degeneration. However, genetic deletion of c-Jun has no effect on axon degeneration [ 82], and c-Jun deficiency effectively suppresses the neuronal immune response [ 81]. Thus, c-Jun directly targets the genes of immune factors expressed by the injury-afflicted neurons, only under pathological conditions.

On the other hand, it is worth mentioning that macrophages also express SARM1, which initially regulates the recruitment of transcription factors and RNA polymerase II to the Ccl5 promoter. It has been previously shown that Sarm1 −/− bone marrow-derived macrophages exhibits reduced Ccl5 expression compared with WT cells, for both TLR-dependent and -independent stimulation of cells [ 83]. However, more recently, Doran and colleagues [ 84] used CRISPR/Cas9-mediated SARM1 knockout and epitope-tagged mice to show that SARM1 is indeed expressed in macrophages but does not regulate nuclear transcription.

Peripheral neuropathies

The role of CNS microglia in neuropathic pain was first established in 2003 after observing the striking increase in microglial expression of purinoreceptor P2X ₄R after spinal cord injury in rats [ 101]. Later, more evidence appeared supporting immune cells not being passive observers in the nervous system, but rather active influencers in the initiation and/or progression of neuropathies. Specifically, macrophage activation contributes to experimental neuropathic pain states by releasing potent pro-inflammatory mediators, including TNFα, IL-1β, MCP-1, NGF, nitric oxide (NO) and prostanoids [ 102]. Moreover, peripheral nerve injury induces an increase in CSF1 in the DRG and spinal cord which is associated with induction of neuropathic pain [ 103].

The sciatic nerve ( Nervus ischiadicus) crush is one of the most common models of peripheral nerve injury in rodents. It is a valuable model to study peripheral axon regeneration, as well as providing insights into the failure of CNS axon regeneration [ 104]. More importantly, this model has served as a valuable tool to study the involvement of macrophages in the inflammatory and regenerative responses following nerve trauma. After sciatic nerve axonotmesis in mice, pro-inflammatory mediators such as IL-1β, Cox2, and TNF-alpha are rapidly produced (within 5-10 h) [ 5], as well as chemokines such as MIP-1α and monocyte chemoattractant protein 1 (MCP-1) (peaking at 24 h) [ 4]. Furthermore, after acute peripheral nerve injury, the expression of pro-inflammatory cytokines was seen to return to basal levels two days post-injury. Interestingly, Ydens and colleagues [ 105], showed that M2-like macrophages were triggered, showing a strong up-regulation of tissue repair markers (arginase-1, Ym1, and Trem2), thereby limiting the attack of the pro-inflammatory surge, resulting in a neuroprotective environment. The main cells acting at the site of injury were found to be resident endoneurial macrophages (9.5-fold above baseline). Their response was rapid: 2-3 days post-crush, while haematogenous macrophages started to appear at day 4 [ 106]. To evaluate the effect of injury on the recently characterized sciatic nerve macrophages, Relmα⁺Mgl1⁺ and Relmα −Mgl1 − (see above, and Fig. 1 ), a sciatic nerve crush mouse model was established. In the distal part of the sciatic nerve, Relmα⁺Mgl1⁺ cells did not respond, i.e. did not change their gene expression profile; whereas Relmα −Mgl1 − cells rapidly responded [ 34]. Interestingly, this could be expected as Relmα −Mgl1 − endoneurial cells were found to be in closer contact with the injured axon and transdifferentiating repair SCs. These cells then phagocytose debris at the injury site, and contribute to the disintegration of distal axons. While MHC class II expression is correlated with classical M1 phenotype, high or low MHC class II expression did not correlate with altered macrophage activation state at different time points after sciatic nerve crush. In addition, in this study, Relmα, Mgl2, Ym1 and arginase that are considered prototypical M2 markers, were neither co-expressed nor correlated with higher or lower MHC class II expression levels in this study [ 34]. This further highlights the severe limitations of applying the in vitro M1/M2- concept to in vivo macrophages.

Neurodegenerative diseases have a variety of causes but share some similar features: impaired mitochondrial function, increased oxidative stress and ROS production, inflammatory reactions, formation of protein aggregates and proteo-toxicity, and excito-toxicity. The counter-acting effects of axonal survival versus axonal degeneration factors determines whether an axon will be maintained or undergo self-destruction. In disease context, these events constitute a vicious cycle leading to neuron death [ 107]. In the following section, we discuss disease states in which inflammatory activity plays a direct causative role in peripheral neuropathies (PN), e.g. Guillain-Barré syndrome (GBS), as well as conditions where inflammation mediates PN secondary to underlying metabolic (diabetic polyneuropathy (DPN)) or genetic (Charcot-Marie-Tooth (CMT)) diseases or due to administration of anti-neoplastic drugs (chemotherapy-induced PN (CIPN)) (Fig. 3 ). As we will see, neuroinflammation is almost always accompanied by features of oxidative stress, which maintains or exacerbates the pathology. The interplay between macrophages and other cells in either development and continuation, or the correction of the disease milieu, will also be discussed, based on the available published data.

Diabetic polyneuropathy (DPN)

It is well-established that hyperglycaemia activates inflammatory and oxidative stress pathways, thus creating the diabetic milieu. These two axes interact at several cell-signalling levels leading, in the long-term, to microvascular complications and peripheral nerve dysfunction, known as diabetic polyneuropathy (DPN) [ 123- 125]. Uncontrolled persistent hyperglycaemia activates several metabolic pathways including polyol, protein kinase C (PKC), advanced glycation end-products (AGEs), and hexosamine pathways that alter the cellular metabolic state. AGEs stimulate microglia and macrophages to produce cytokines such as IL-1, IL-6, IL-17, TNFα, C reactive protein and chemokines such as CCL-2 and CXC [ 98]. In addition, hyperglycaemia-sensitive cells, such as endothelial cells, respond by increasing mitochondrial ROS and RNS production and vascular adhesion molecule formation. Furthermore, persistent hyperglycaemia modifies the glycosylation of myelin proteins, and thus their antigenicity, causing further infiltration and activation of monocytes, macrophages, and neutrophils, as well as ROS production. In contrast to the well-regulated ROS production in the anti-microbial response, metabolically generated ROS secretion in diabetic leukocytes is more impulsive and dysregulated. Macrophage infiltration further damages the myelin sheath and increases axon excitability, thus leading to oedema, inflammation, and cell necrosis. [ 98, 124, 125]. Under these conditions, Nrf-2 pathway has been shown to be suppressed leading to a downregulation in expression of antioxidant genes [ 124]. Therefore, persistent high glucose levels lead to a vicious circle of microvasculitis at nerve sites, neuroinflammation, and oxidative stress that damages peripheral nerves causing a broad range of sensori-motor symptoms.

Charcot-Marie-Tooth disease

Charcot-Marie-Tooth (CMT) disease is a heterogeneous group of diseases constituting the most common hereditary pathology of the PNS. Genetic mutations are manifested as lesions in myelin (CMT1) or axons (CMT2), and symptoms are variable and include distal muscle weakness and sensory perturbations [ 140]. Neuroinflammation has been described in both demyelinating and axonal CMT. CMT1-related myelin gene mutations lead to SC cytotoxicity, impaired myelination and consequently to nerve pathology. Interestingly, studies in rodent models of CMT1X [ 141], CMT1A [ 142] and CMT1B [ 143] showed that mutant SC express MCP-1/CCL2 through the MEK-ERK signalling pathway that guides pathogenic macrophage infiltration. In addition, CSF-1 knockout rescued the phenotype of CMT1X mice by stabilizing macrophage invasion and therefore preserving SC homeostasis despite the mutation [ 144]. More surprisingly, important cell-cell contacts were observed between the CSF-1-expressing fibroblasts and endoneurial macrophages in the diseased nerve, posing more questions about the complex cellular and molecular interactions in peripheral nerves [ 145]. On the other hand, mutations in the mitochondrial GDAP1 (ganglioside-induced differentiation-associated protein 1) gene cause axonal CMT and are associated with increased production of ROS and inflammatory mediators [ 146]. Furthermore, oxidative stress has also been described in CMT1A patients [ 147] and a rat model [ 92] and is thus suspected to play a role in the pathological process. However, the link between oxidative stress and macrophages is as yet not established in CMT disease.

Macrophages in therapy: potential and pitfalls

We have presented here the different phenotypes of macrophages depending on their microenvironment. These phenotypes have differential effects on the cellular biology and disease progression. The divergence in such phenotypes provides a therapeutic potential of monocyte/macrophage manipulation for the treatment of several pathologies including cancer, metabolic, autoimmune and neuroinflammatory diseases, as well as for regenerative medicine. This approach is already the subject of extensive study. For our purposes, macrophages are particularly relevant in therapeutics as these cells have (i) inherent plasticity that is well-suited to therapeutic manipulation; (ii) phagocytic abilities allowing them to be efficiently targeted with nanoformulated compounds, and (iii) critical roles in tissue repair. Among the possible therapeutic strategies, cell therapy by ex vivo-activated macrophages, or delivery of molecules and biomaterials to modulate the accumulation and phenotype of endogenous macrophages have been developed [ 148]. Attempts to inhibit the pro-inflammatory M1-like phenotype or promote an anti-inflammatory M2-like phenotype, by modulating the activity of inflammatory cytokines and transcription factors, have also been investigated. In diabetic mice, anti-IL-1β antibodies were shown to downregulate the M1-like phenotype and promote the M2-like phenotype, thus improving wound healing [ 149]. Similarly, topical application of peroxisome proliferator-activated receptor (PPAR)-γ agonists, known to promote an M2-like phenotype [ 89], was shown to improve wound healing in diabetic mice [ 150]. In a similar fashion, systemic M-CSF administration has been reported to promote remyelination following spinal cord injury in mice [ 151]. Likewise, transplantation of dental pulp stem cells suppressed inflammation in rat sciatic nerves by promoting macrophage polarization towards an anti-inflammatory phenotype and ameliorated diabetic polyneuropathy [ 152].

In a clinical context, data from the PNS are still lacking. Nevertheless, some clinical data have been collected from trials in different organ systems. For example, niacin administration in patients with Parkinson's disease was accompanied by improved quality of life through the attenuation of inflammation by shifting macrophage polarization from an M1 to M2 profile [ 153]. Moreover, the delivery of ex vivo-activated autologous M2 macrophages was shown to be beneficial in patients with heart failure [ 154] or stroke [ 155]. Multiple experimental and clinical studies have shown a protective role of MIF in GBS [ 113], although clinical trials are still unavailable. Several clinical trials have used parameters related to the macrophages, such as cytokine secretion, as primary or secondary clinical outcomes or biomarkers (for example NCT03321955).

It should be noted, however, that monocytes/macrophages may be less responsive to stimuli in the elderly and in patients with immune disorders, suggesting that macrophage-recruiting strategies may be less effective in these subjects. While targeting/using macrophages for therapy in diabetes, heart and CNS diseases shows great potential, macrophage manipulation in the context of the PNS should be seriously considered and its investigation encouraged for the development of next generation therapeutics. However this will also requires more efforts in the screening for biological effects using a combination of in vitro and in vivo assays.

Conclusions

It has now been fully accepted that monocytes are not direct precursors for many tissue-resident macrophages. Indeed, monocyte provision during homeostasis contributes partially to the tissue-resident macrophage pool, part of which is seeded prenatally [ 18]. However, the tissue-specific local environment is now known to be the most powerful controller of macrophage phenotype, regardless of their origin. Several models have been suggested to explain macrophage differentiation, including their day-to-day function, maintenance, population density and interaction with the surrounding microenvironment (or niche) [ 1]. These debates reflect the level of complexity and heterogeneity of these immune cells in tissue. For example, within a single tissue, macrophage heterogeneity is potentially underestimated due, in part, to the challenges involved in investigating small cell subsets, and the possible contamination of samples by other cells. New sophisticated techniques of single-cell sequencing and fate mapping, are now helping to overcome these limitations and are providing a significant contribution to the field in the so-called "single-cell era" [ 156]. Although limited, thus far, in comparison to their more extensive use in the study of CNS cells, applying these approaches to the PNS will certainly provide valuable information about the spatial, temporal, and functional distribution of tissue macrophages. These characteristics are expected to be even more complex in the pathological context. As data are lacking regarding this point, macrophage recruitment and origin in pathological states is an interesting substance for future investigation. Moreover, the current view that macrophages interact with primary sensory neurons in the peripheral tissues and the DRG to regulate not only inflammatory responses but also pain signals requires further study. One should keep in mind the difficulties in preparing libraries and analysing the large amount of data generated by single-cell sequencing [ 157]. Collaborative approaches across the globe such as the Human Cell Atlas [ https://www.humancellatlas.org] are creating an extensive catalogue to describe every single human cell type. Another challenge in studying macrophages associated with the PNS is the wide distribution of peripheral nerves within various tissue compartments; hence nerve-associated macrophages are exposed to distinct environmental signals. Indeed, epigenetic imprinting cannot be captured by transcriptomic or proteomic techniques, necessitating the synergistic use of other techniques to assess chromatin state. Recent compelling studies, in germ-free mice and/or adult mice treated with antibiotics, have emphasized the role of the microbiome in the time-dependent control of CNS microglia maturation and function [ 158- 160]. Hence, what might be the corresponding role of host microbiota in regulating PNS macrophage phenotype? This question requires future investigation.

On the other hand, the regeneration potential of the mammalian nervous system has been the subject of extensive research. While the acute inflammatory phase is destructive to peripheral nerves, proper WD, through immune cell clearance of myelin and axonal debris, is crucial for the repair process. Following insults to peripheral nerves, monocytes are recruited to the site of injury and resident macrophages are activated. These processes collectively termed neuroinflammation can be ultimately beneficial or detrimental to nerve function. Despite the regenerative capacity of PNS axons, clinical experience attests to pathological tissue remodelling and to disappointing functional recovery. The complex relationship between SCs, the axon, macrophages and endoneurial fibroblasts is the driver of these outcomes. In comparison to the CNS, nerve regeneration in the PNS may be incorrectly thought to be complete with little intervention [ 9]. However, successful PNS regeneration depends on the age of the patient and the delay before intervention, as well as on local factors, essentially the type, site, and severity of the injury, the recruitment of non-neuronal cells (fibroblasts and inflammatory cells), and the local microenvironment [ 7, 9]. Learning how to harness the benefits of neuroinflammation by polarizing macrophages into their regulatory/anti-inflammatory phenotype or enhancing the elimination of debris is currently the subject of extensive research. Moreover, we should stress that the interaction of the four local cell types mentioned above seems to be crucial in both physiological and pathological situations, making these interactions a good, yet challenging, target for potential therapeutics. In the future, directing research towards the modulation of macrophage phenotype may identify ways to help alleviate and reverse peripheral nerve insults through targeting both neuroinflammatory and oxidative stress pathways.

Acknowledgements

We are indebted to Dr. Ian Darby (Melbourne, Australia) for professional editing of this manuscript.

Abbreviations

BNB

Blood-nerve barrier

CIPN

Chemotherapy-induced peripheral neuropathy

CMT

Charcot-Marie-Tooth disease

CNS

Central nervous system

CSF1

Colony stimulating factor-1

DPN

Diabetic polyneuropathy

DRG

Dorsal root ganglia

ECM

Extracellular matrix

GBS

Guillain-Barré syndrome

HSC

Hematopoietic stem cells

IL

Interleukin

MCP-1

Monocyte chemoattractant protein 1

NGF

Nerve growth factor

NO

Nitric oxide

PN

Peripheral neuropathies

PNS

Peripheral nervous system

ROS

Reactive oxygen species

SC

Schwann cell

TNFα

Tumour necrosis factor-α

VEGF

Vascular endothelial growth factor

WD

Wallerian degeneration

Author contributions

ZM, MEM and AD conceptualized the manuscript outline. ZM performed the literature search, wrote the manuscript, and designed the figures. MEM, AR, FB and AD provided comments and proof-reading. All authors read and approved the final manuscript.

Funding

ZM is supported by a doctoral fellowship from the European Union (European Regional Development Fund). MEM is supported by a postdoctoral fellowship from the Association Française contre les Myopathies (AFM)-Téléthon. AR is supported by a postdoctoral fellowship from the European Union (cmRNABone- Horizon 2020, identifier code: H2020-SC1-BHC-2018-2020).

Availability of data and materials

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Footnotes

Publisher's Note
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Articles from Journal of Neuroinflammation are provided here courtesy of BMC

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The macrophage: a key player in the pathophysiology of peripheral neuropathies

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Macrophages are present in all mammalian tissues and coexist with various cell types in order to respond to different environmental cues. However, the role of these cells has been underestimated in the context of peripheral nerve damage. More importantly, ...

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Macrophage Activation in the Dorsal Root Ganglion in Rats Developing Autotomy after Peripheral Nerve Injury

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Autotomy, self-mutilation of a denervated limb, is common in animals after peripheral nerve injury (PNI) and is a reliable proxy for neuropathic pain in humans. Understanding the occurrence and treatment of autotomy remains challenging. The objective…