Abstract
Although inflammation is indispensable for the repair process in Wallerian degeneration (WD), the role of neutrophils in the WD repair process remains unclear. After peripheral nerve injury, neutrophils accumulate at the epineurium but not the parenchyma in the WD region because of the blood–nerve barrier. An increase or decrease in the number of neutrophils delayed or promoted macrophage infiltration from the epineurium into the parenchyma and the repair process in WD. Abundant neutrophil extracellular traps (NETs) were formed around neutrophils, and its inhibition dramatically increased macrophage infiltration into the parenchyma. Furthermore, inhibition of either MIF or its receptor, CXCR4, in neutrophils decreased NET formation, resulting in enhanced macrophage infiltration into the parenchyma. Moreover, inhibiting MIF for just 2 h after peripheral nerve injury promoted the repair process. These findings indicate that neutrophils delay the repair process in WD from outside the parenchyma by inhibiting macrophage infiltration via NET formation and that neutrophils, NETs, MIF, and CXCR4 are therapeutic targets for peripheral nerve regeneration.
Introduction
Although the peripheral nervous system (PNS), but not the central nervous system (CNS), can regenerate, clinical outcomes after peripheral nerve injury (PNI) are sometimes unsatisfactory, depending on the type and degree of injury. In particular, severe PNI, proximal injury, and reconstruction cases tend to exhibit poor recovery (Ciaramitaro et al, 2010; Grinsell & Keating, 2014). Achieving axon regeneration is not sufficient to induce meaningful functional recovery; rapid reinnervation is also required (Navarro, 2016). Despite advances in the research about peripheral nerve regeneration (Allodi et al, 2012; Gomez-Sanchez et al, 2017; Ketz et al, 2017; Hussain et al, 2020; Li et al, 2020), clinical treatments that are effective over microscopic sutures and reconstruction using autogenous nerve grafting have yet to be developed over the last half-century.
After PNI, the region requiring regeneration is divided into two parts, the injury site and Wallerian degeneration (WD) site. The former is initiated by a mechanical injury and characterized by swelling, hemorrhage, and necrosis of local cells (Mietto et al, 2015), whereas the latter is initiated by separation from the neuronal cell body, induces self-destructive pathways, and results in fragmentation of axons and the myelinated sheath (Wang et al, 2012). Although regeneration of injury sites is indispensable for peripheral nerve regeneration, regeneration of WD regions is equally or more important than that of injury site (Rotshenker, 2011). This is because WD can occur all the way to denervated peripheral organs, and axons must therefore regenerate over the entire WD region. In rodents, WD in the PNS begins within 24–48 h after injury (Beirowski et al, 2005), and axons become fragmented by the influx of calcium from extracellular and intracellular reservoirs and by the activation of proteases, followed by fragmentation of the myelin sheath through Schwann cells. The fragmented axons and myelin are subsequently phagocytosed and cleared by activated Schwann cells and macrophages. The Schwann cells then form a Büngner band—a substrate for regenerating axons—and remyelinate newly formed axons. This series of repair processes requires an inflammatory response, which is activated 3–7 d after PNI (Chen et al, 2021). Inhibition of the inflammatory response via knockout of toll-like receptor (TLR)2 and TLR4 or interleukin (IL)-6 results in reduced macrophage accumulation and a delay in myelin debris clearance and axon regeneration (Zhang et al, 2000; Boivin et al, 2007). In contrast, enhancement of the inflammatory response via administration of TNF-α or IL-6 promotes myelin debris clearance and axon regeneration (Gaudet et al, 2011; Fregnan et al, 2012; Mietto et al, 2015). These findings indicate that changes in the inflammation status of WD regions regulate the repair process, including axon regeneration. However, findings reported to date primarily concern macrophages; the role of neutrophils in the WD repair process remains to be fully elucidated.
Neutrophils exhibit a variety of functions related to tissue repair and damage, depending on tissue type and pathology. For instance, neutrophils are the first cells to infiltrate pathological regions via chemotaxis, where they mediate tissue homeostasis and repair processes through the removal of cellular debris and inducing the activation and differentiation of macrophages (Kolaczkowska & Kubes, 2013; Rosales, 2018; Kanashiro et al, 2020). In contrast, neutrophils can exacerbate tissue damage via the release of various mediators, such as reactive oxygen species (ROS) and matrix metalloproteases (MMPs) (Wang, 2018). For example, after traumatic brain injury, neutrophils infiltrate the parenchyma and release ROS and MMPs and activate microglia, leading to an expansion of secondary damage (Qin et al, 2005; Liu et al, 2018c). Indeed, suppression of neutrophil infiltration through CXCR2 knockout or the administration of neutralizing antibody was shown to promote neuronal survival after traumatic brain injury (Harris et al, 2005; Wang, 2018; Liu et al, 2018c). After injection of zymosan into the eye, by contrast, accumulated neutrophils were shown to promote axon regeneration of the optic nerve via the secretion of humoral factors (Sas et al, 2020). After PNI, neutrophil infiltration precedes the accumulation of macrophages at the injury site (Perkins & Tracey, 2000; Zuo et al, 2003), and IL-1R1/TNFR1 knockout or administration of Ly6G antibody inhibits macrophage accumulation and reduces neuropathic pain after injury (Nadeau et al, 2011). However, details regarding the spatiotemporal distribution and role of neutrophils in the WD repair process remain not fully clarified. Therefore, the purpose of the current study was to clarify the spatiotemporal distribution and role of neutrophils in the WD repair process.
The current study demonstrates that neutrophils accumulate only at the epineurium but not at the parenchyma in the WD region because of their inability to penetrate the blood–nerve barrier (BNB). Neutrophils delay the repair process by inhibiting the infiltration of macrophages from the epineurium to the parenchyma through the formation of neutrophil extracellular traps (NETs) via the macrophage migration inhibitory factor (MIF)-CXCR4 axis. These findings indicate that neutrophils delay the repair process in WD from outside of the parenchyma by inhibiting macrophage infiltration via NET formation and that neutrophils, NETs, MIF, and CXCR4 at the epineurium are therapeutic targets for peripheral nerve regeneration.
Results
Neutrophils accumulate at the epineurium but not the parenchyma in the WD regions despite chemokine expression
To clarify the spatiotemporal distribution of neutrophils after PNI, rat sciatic nerves were crushed and then perfused from 6 h to 1 wk after injury for subsequent histological evaluation. At the injury site, neutrophils identified based on typical nuclear morphology by hematoxylin and eosin (HE) staining accumulated at the parenchyma and epineurium (Perkins & Tracey, 2000; Zuo et al, 2003) (Fig 1A). In the WD area, neutrophils accumulated only at the epineurium but not at the parenchyma (Fig 1A). The neutrophils started to accumulate 6 h after injury and peaked 12 h after injury, with almost all neutrophils disappearing by 1 d after injury (Fig 1B). In the region of non-WD area, within the region 15 mm proximal to the injury site, neutrophils accumulated at the epineurium 12 h after injury (Fig 1C). Furthermore, immunolabeling analysis confirmed the presence of cells expressing the neutrophil markers, neutrophil elastase (NE) and Ly6G, only in the region in which type 1 collagen was deposited but not at the parenchyma, where type 1 collagen was absent (Fig 1D). The spatiotemporal distribution of macrophages was also investigated. Macrophages identified based on CD68 immunoreactivity were present only at the epineurium in the region of WD 6, 12 h, and 1 d after injury and started to accumulate at the parenchyma 3 d injury (Figs 1E and S1A). Then, the number of macrophages in the region of WD increased as a function of time (Fig S1A and B). These findings elucidate that neutrophils and macrophages accumulate only at the epineurium in the region of WD within 1 d after injury; later neutrophils disappear, and macrophages start to accumulate at the parenchyma.
Because chemokines are necessary to induce neutrophil and macrophage accumulation (Capucetti et al, 2020), the lack of their accumulation at the parenchyma of the WD region might have been because of low chemokine expression. Therefore, we examined the expression of CXCL1, 2, and 3 (Nadeau et al, 2011; Rajarathnam et al, 2019), which are major chemokines for neutrophil accumulation, and the macrophage accumulation cytokine CCL2 (Matsusuhima et al, 1989; Gschwandtner et al, 2019) by in situ hybridization analysis of sections 6 h after injury. A sciatic nerve locally injected with zymosan was used as a positive control. Expression of CXCL1, 2, 3, and CCL2 mRNA was detected in the zymosan-injected site (Fig S2) but not in the intact nerve (Fig 2A–D). Importantly, expression of CXCL1, 2, 3, and CCL2 was observed not only in and around the injury site (Fig 2E–H) but also at the parenchyma and the epineurium of the WD region (Fig 2I–L). These results demonstrate that neutrophils and macrophages accumulate only at the epineurium at the very early stage of WD despite chemokine expression.
The BNB inhibits infiltration of neutrophils into the parenchyma in the WD regions
The BNB of peripheral nerves controls the influx and efflux of cells and substances in the PNS to maintain homeostasis. Disruption of the BNB in pathological conditions allows inflammatory cells and cytotoxic substances to enter the parenchyma in the PNS, thereby exacerbating disease conditions (Liu et al, 2018a). As neutrophils cannot penetrate the blood–brain barrier (BBB) (Soares et al, 1995; Cho et al, 2015) and the epineurium does not have a BNB (Liu et al, 2018a), we hypothesized that neutrophils failed to accumulate at the parenchyma in the WD region because the BNB function was intact during neutrophil accumulation. To determine when the BNB starts to break down in WD, we evaluated leakage of IgG using immunolabeling analysis. The extravasation of high–molecular-weight substances such as IgG is a typical phenomenon of BBB breakdown (Kim et al, 2011; Ekmark-Lewén et al, 2013). In intact nerves, IgG immunoreactivity was observed only at the epineurium (Fig 3A), whereas IgG immunoreactivity was detected in and around the injury site continuously from 6 h to 7 d after injury (Fig 3A) (Kim et al, 2011; Hsieh et al, 2017). In the WD region, IgG immunoreactivity was observed only at the epineurium until 3 d after injury (Fig 3A); however, at 7 d after injury, IgG immunoreactivity was detected at the parenchyma as well (Fig 3A). These results indicate that BNB function is maintained in the WD region until 3 d after injury and that neutrophils accumulate only in areas in which the BNB has broken down.
Next, to determine whether the BNB inhibits neutrophil infiltration into the parenchyma in the WD region, we pharmacologically disrupted the BNB function. Mannitol and VEGF were injected continuously into the common iliac artery of adult rats for 30 min at 11 h after crush injury of the sciatic nerve (Zhang et al, 2000; Sun et al, 2012), followed by perfusion at 12 h after injury (Fig 3B). Control rats exhibited no IgG leakage, whereas rats that received mannitol and VEGF exhibited IgG leakage at the parenchyma throughout the region of WD (Fig 3C). Importantly, the number of neutrophils and macrophages accumulating at the parenchyma was significantly higher in forced BNB breakdown rats compared with control rats (Fig 3D–G), indicating that the BNB blocks the infiltration of neutrophils into the parenchyma in the WD region.
Reduction of neutrophil accumulation promotes the repair process in WD
To elucidate the role of neutrophils in the WD repair process, we reduced the number of neutrophils accumulating at the epineurium and examined macrophage accumulation, myelin debris clearance, and axon regeneration. To deplete neutrophils, an anti-PMN antibody was administered intraperitoneally 3 h before injury (Fig 4A) (Regal et al, 2015). Examination of the neutrophil count in the blood revealed that the number of neutrophils peaked 12 h after PNI and returned to normal 2 d after injury. In contrast, the administration of anti-PMN antibodies—but not control antibodies—completely abolished this increase in the number of neutrophils in the blood (Fig 4B). The accumulation of neutrophils at the epineurium in the WD region was apparently impaired by administration of the anti-PMN antibody (Fig 4C), decreasing 19–35% compared with the control (Fig 4C and D). Interestingly, administration of the anti-PMN antibody significantly increased the number of macrophages accumulating at the parenchyma at 12 h (Fig 4E and F) and 7 d after injury compared with the control (Fig S3E and F). Of note, the total number of macrophages at the parenchyma and epineurium did not significantly change (Fig S3A and B), and the expression of CCL2 immunoreactivity at the parenchyma also did not significantly change (Fig S3C and D), suggesting that the increase of macrophage accumulation at the parenchyma was because of enhance of the migration from the epineurium rather than the enhanced proliferation of existing macrophages at the parenchyma. Furthermore, by administration of the anti-PMN antibody, the amount of myelin debris was significantly reduced (Fig 4G and H), and the extent of axon regeneration was significantly increased (Fig 4I and J). These findings indicate that the reduction of the number of neutrophils accumulating at the epineurium within 1 d after injury promotes the WD repair process.
Increased neutrophil accumulation delays WD repair
Next, we explored the effect of the increase in neutrophil accumulation in the injured nerve on the WD repair processes. C57BL/6 mice received crush injuries in the sciatic nerves, followed by intravenous injection of syngeneic neutrophils (2 × 106/100 μl) derived from GFP mice 11 h later (Fig 5A). As a control, PBS (100 μl) was intravenously administered in the same manner as neutrophil infusion. The number of neutrophils in the blood 12 h after injury, which was 1 h after administration, increased 57.4% compared with the control (Fig 5B). In the WD region 12 h after injury, GFP-expressing neutrophils accumulated only at the epineurium (Fig 5C), and the number of neutrophils significantly increased 156.9–716.3% compared with the control (Fig 5D), supporting the prior finding that neutrophils accumulate at the epineurium but not the parenchyma.
At 7 d after injury, neutrophil infusion significantly decreased the number of macrophages accumulating at the parenchyma (Fig 5E and F), increased the amount of myelin debris (Fig 5G and H), and reduced the number of regenerating axons (Fig 5I and J). These findings indicate that the increase in the number of neutrophils accumulating at the epineurium significantly inhibits the WD repair process. The data of this and the preceding experiment indicate that neutrophils accumulate outside the parenchyma and inhibit the infiltration of macrophages into the parenchyma, thereby resulting in inhibition of the WD repair process.
NETs at the epineurium inhibit infiltration of macrophages into the parenchyma in WD
Because neutrophils release NETs to protect tissues from infection (Brinkmann et al, 2004) and to affect tissue repair (Zhu et al, 2021), we determined whether neutrophils release NETs at the epineurium in the WD region. NET formation was determined by triple immunolabeling against citrullinated histone 3 (CitH3), MPO, and DAPI (Gavillet et al, 2015; Magán-Fernández et al, 2019). Intact nerve exhibited no NET formation as expected (Fig 6A), whereas NET formation started to show up at the epineurium of the WD region from 6 h after injury and peaked at 12 h and disappeared at 3 d after injury (Fig 6A and B). NETs also surrounded macrophages at the epineurium at 12 h after injury (Fig 6C).
Next, we determined whether NET formation affects macrophage infiltration into the parenchyma. The sciatic nerve was circumferentially wrapped with a collagen sheet containing Cl-amidine (an inhibitor of peptidylarginine deiminase [PAD], an enzyme necessary for NET formation), deoxyribonuclease (DNase) I (a NET-degrading enzyme), or PBS as a control 10 h after injury, followed by perfusion 2 h later (Fig 7A). Both Cl-amidine and DNase I did not cause BNB breakdown (Fig 7B) but significantly reduced NET formation at the epineurium (Figs 7C and D and S4A), resulting in a significant increase in macrophage accumulation at the parenchyma (Fig 7E and F). As in the preceding experiment to reduce the number of accumulating neutrophils, the total number of macrophages at the parenchyma and epineurium and the expression of CCL2 at the parenchyma did not significantly change by the inhibition of NET formation (Fig S4B–E). Interestingly, the inhibition of NET formation significantly increased the number of neutrophils at the epineurium (Fig 7G and H), probably because the death of neutrophils induced by NETs and the cleavage of dead neutrophils by macrophages were attenuated (Farrera & Fadeel, 2013; Mutua & Gershwin, 2021). These results indicate that NET formation at the epineurium inhibits the infiltration of macrophages into the parenchyma in the WD region.
The MIF-CXCR4-NETs axis in neutrophils plays a central role in inhibiting macrophage infiltration
As the proinflammatory cytokine MIF reportedly induces NET formation (Rodrigues et al, 2020; Schindler et al, 2021), we investigated the expression of MIF in the WD region. Intact nerve exhibited no MIF immunoreactivity (Fig 8A), whereas it was detected in neutrophils accumulating at the epineurium in the WD region 12 h after injury (Fig 8A). In addition, the administration of an anti-PMN antibody to reduce accumulating neutrophils (Fig 4A–D) also significantly reduced the MIF immunoreactivity at the epineurium at 12 h after injury (Fig S5A and B). We therefore examined the effect of MIF on NET formation and macrophage infiltration. As in the preceding experiment, the sciatic nerve was circumferentially wrapped with a collagen sheet containing iso-1 (a MIF inhibitor) or dimethyl sulfoxide as a control 10 h after injury, followed by perfusion 2 h later (Fig 8B). Iso-1 did not impair the function of the BNB (Fig 8C), but it significantly inhibited NET formation (Fig 8D and E) and significantly increased the number of macrophages accumulating at the parenchyma (Fig 8F and G). In addition, as the experiment of the NET inhibition, MIF inhibition significantly increased the number of neutrophils at the epineurium (Fig 8H and I).
Next, we investigated the expression of CXCR4, a receptor for MIF, on neutrophils at the epineurium. No CXCR4 immunoreactivity was detected in the intact nerve but was observed in the neutrophils at the epineurium of the region of WD 12 h after injury (Fig 9A). Subsequently, as in the preceding experiment, a collagen sheet containing AMD 3100, a CXCR4 inhibitor, was applied onto the nerves 10 h after injury, followed by perfusion 2 h later (Fig 9B). AMD 3100 did not disrupt BNB function (Fig 9C) but significantly suppressed NET formation (Fig 9D and E) and substantially increased macrophage accumulation at the parenchyma in the WD region (Fig 9F and G). Furthermore, as the experiment of the NET inhibition and MIF inhibition, CXCR4 inhibition also significantly increased the number of neutrophils at the epineurium (Fig 9H and I). These results indicate that the MIF-CXCR4-NETs axis of neutrophils plays a central role in inhibiting the infiltration of macrophages from the epineurium into the parenchyma in the WD region.
Finally, we determined whether the MIF-CXCR4-NETs axis was involved in the WD repair process. In the same manner as described above, the WD region was wrapped with a collagen sheet containing iso-1 at 10 h after injury, followed by removal of the sheet with local irrigation 2 h later and perfusion at 7 d after injury (Fig 10A). Subjects treated with iso-1 exhibited a significant increase in the number of macrophages (Fig 10B and C) and a significant decrease in the amount of myelin debris at the parenchyma (Fig 10D and E), with significantly greater axon regeneration (Fig 10F and G). These results indicate that inhibition of MIF outside the parenchyma for just 2 h can significantly accelerate the WD repair process by promoting macrophage accumulation.
Discussion
The current study reveals that neutrophils accumulate only at the epineurium in the region of the WD and that neutrophils delay the WD repair process by inhibiting macrophage infiltration from the epineurium into the parenchyma via the release of NETs (Fig 11). Notably, neutrophil accumulation and disappearance at the outside of the WD region is completed within 1 d after injury. Despite this short-term and off-target accumulation, neutrophils substantially delay the WD repair process, including macrophage accumulation, myelin clearance, and axon regeneration. This is quite interesting because the initial short delay of the WD repair process results in a more prolonged impairment of repair. In addition, the observation that NETs inhibit macrophage infiltration enhances the understanding of how neutrophils regulate other immunological cells. Furthermore, the current study found that the MIF-CXCR4-NETs axis of neutrophils plays a central role in inhibiting macrophage infiltration into WD regions, identifying a new molecular target to accelerate WD repair. Inhibition of MIF for just 2 h significantly promoted the WD repair process, suggesting MIF inhibition as a potentially potent therapeutic approach for WD. These findings identified a novel function of neutrophils, elucidated details of the mechanism of WD repair, and identified therapeutic targets for PNI.
Even though chemokines were expressed at both the parenchyma and epineurium of the WD region, neutrophils accumulated only at the epineurium in regulating the WD repair process. This is quite interesting in three respects. First, this phenomenon seems specific to WD, in which the BNB is present and functions normally. Previous studies have rarely reported that neutrophils accumulate outside the pathological lesion but not inside the lesion. Second, neutrophils regulate the repair process at the outside of the lesion. Typically, neutrophils directly regulate inflammatory responses via the secretion of humoral factors, phagocytosis, and NET formation (Wang, 2018). Third, the epineurium is involved in the repair process in the PNS. The epineurium consists of blood vessels, mast cells, fibroblasts, and large amounts of collagen (Stratton et al, 2018), and few reports have indicated it regulates the repair process in the PNS. The current study found that immediately after PNI, the epineurium expressed chemokines, attracted inflammatory cells, and regulated the repair process via functions of accumulated neutrophils, indicating that the epineurium is more involved in the repair process in the PNS than previously thought. These phenomena might also occur with WD in the CNS, although further study is necessary because the rate of WD occurring in the CNS is much slower than that in the PNS (Vargas & Barres, 2007), and the inflammatory response in the CNS is not as robust as that in the PNS (Peruzzotti-Jametti et al, 2014).
Both the BBB and BNB are barrier systems that maintain the homeostasis of the nervous system at the border with the peripheral circulation. In the current study, the BNB blocked the penetration of neutrophils but not activated macrophages. This is similar to the BBB (Soares et al, 1995; McMahon et al, 2002; Corraliza, 2014; Cho et al, 2015) and seems reasonable as the BNB and BBB share a similar structure besides the lack of a glial boundary membrane composed of astrocytes (Nakatsuji, 2017). However, during WD, the BNB breaks down in PNS, but the BBB does not in CNS (Vargas & Barres, 2007). This difference might explain in part why WD occurs more rapidly in the PNS than the CNS (George & Griffin, 1994), although further study is needed.
In the current study, an (Daley et al, 2008; Chan et al, 2015) increase or decrease in the number of accumulating neutrophils, respectively, decreased or increased myelin clearance in WD. This is contrary to a previous study reporting that a decrease in accumulating neutrophils reduced myelin clearance in WD repair (Lindborg et al, 2017). Several factors could explain this discrepancy. To deplete neutrophils, the current study administered a PMN antibody once 3 h before injury, whereas the previous study administered a Ly6G antibody three times, including 2 d after injury, when neutrophils had already disappeared. Although the Ly6G antibody recognizes neutrophils specifically, a couple of studies reported that the systemic administration of this antibody reduced macrophages as well (Daley et al, 2008; Chan et al, 2015), raising a possibility that prolonged administration decreased the number of macrophages. In addition, the current study used rats, whereas the previous study used mice. Lastly, to quantify neutrophils and macrophages, the current study used histology, whereas the previous study used flowcytometry.
In the current study, promotion of axon regeneration was achieved by inhibiting the number of accumulating neutrophils in the region of WD. This is in conflict with a previous study that reported a decrease in the number of accumulating neutrophils did not affect the extent of axon regeneration after PNI (Nadeau et al, 2011). Several fundamental differences explain this contradictory result. First, the experimental models differed. The current study used a crush injury model, whereas the previous study used a reconstruction model with pre-degenerated sciatic nerve segments, which is not a model for WD. A second difference is that the previous study employed multiple administrations of Ly6G antibody until 4 d after injury. As explained above, prolonged systemic administration of Ly6G might reduce the number of macrophages (Daley et al, 2008; Chan et al, 2015).
Because it was first reported that activated neutrophils release NETs to degrade virulence factors and kill bacteria (Brinkmann et al, 2004), accumulating evidence has demonstrated that NETs not only protect tissues against infection (Jenne et al, 2013) but also play a role in tissue damage (Liu et al, 2016; Vaibhav et al, 2020). However, the role of NETs in PNS disorders remains unclear. Furthermore, whether NETs affect macrophage migration remains to be fully determined despite reports indicating that NETs activate macrophages (Nakazawa et al, 2016) and that macrophages phagocytose neutrophils in NETosis (Farrera & Fadeel, 2013). The current study demonstrated that NETs formed at the epineurium of the WD region and that inhibition of NET formation increased the accumulation of macrophages at the parenchyma. The quantification of the number of macrophages in the epineurium and the parenchyma revealed that NET inhibition did not increase the total number of macrophages in the region of WD (Fig S4C), suggesting that NET inhibition did not promote proliferation of macrophages. Furthermore, the macrophage density at the parenchyma increased (Fig 7F), and the expression of CCL2, the macrophage recruiting chemokine, at the parenchyma did not change by the NET inhibition (Fig S4D and E). Collectively, these findings indicate that NETs prevent macrophages to infiltrate from the epineurium to the parenchyma. Regarding the underlying mechanism, there are three possibilities. First, the effect of NETs as an attractant of macrophages is sufficient to keep macrophages locally at the epineurium, as shown in the previous studies that the neutrophil granular protein from NETs recruits circulating monocytes to the site of inflammation (Soehnlein et al, 2009; Almyroudis et al, 2013). Second, because NETs induce death of neutrophils (Remijsen et al, 2011), NET inhibition decreases dying neutrophils to be phagocyted by macrophages, resulting in more macrophage recruitment to the parenchyma. Third, NETs directly reduce migration ability of macrophages, as the previous report demonstrating that NETs inhibit monocyte migration in vitro (Hofbauer et al, 2020).
Neutrophils release NETs upon stimulation by various factors, such as ROS, myeloperoxidase, NE, PAD-4, and cytokines (Papayannopoulos et al, 2010; Metzler et al, 2011; Kirchner et al, 2012; Mutua & Gershwin, 2021). MIF, a pro-inflammatory cytokine, also stimulates neutrophils to release NETs via its receptor, CXCR4 (Rodrigues et al, 2020). In the current study, neutrophils accumulating at the epineurium in WD expressed MIF and CXCR4, and inhibition of either of these two molecules substantially suppressed NET formation, enhanced macrophage accumulation at the parenchyma, and promoted the WD repair process, indicating that the MIF-CXCR4 axis mediates NET formation and the WD repair process. Although there are contradictory reports indicating that MIF is required for repair after spinal cord injury and PNI (Nishio et al, 2002, 2009; Zhao et al, 2020), the current finding applies only when MIF is inhibited in the early stages after PNI. Collectively, these data suggest that MIF inhibition both inhibits and promotes the repair process after PNI, depending on its timing, duration, and location.
The current findings show that inhibiting the accumulation of neutrophils in the WD within 1 d after PNI or 2-h blockade of NET formation via inhibition of the MIF-CXCR4 axis promotes axonal regeneration. As depletion of neutrophils is likely to increase the probability of infection, and trauma is usually associated with an increased risk of infection, approaches aimed at reducing the number of circulating neutrophils do not seem practical for clinical application. Rather, a more practical approach would be inhibition of either NET formation or the MIF-CXCR4 axis at the epineurium for 2 h, as this would have a lower risk of infection and side effects and would be feasible when exposing an injured nerve during surgery after PNI. Therefore, neutrophils, NETs, and the MIF-CXCR4 axis are potentially potent targets for post-PNI therapy.
Materials and Methods
Animals
Adult male wild-type (WT) LEWIS rats (8–12 wk old; Charles River Laboratories Japan, Inc.), adult male WT C57BL/6 mice (8–10 wk old; Charles River Laboratories Japan. Inc.), and adult C57BL/6-Tg (CAG-EGFP) mice (Sankyo Laboratories Japan, Inc) were used in the current study. All animals were maintained at 20°C with a 12-h light/12-h dark cycle and free access to water and food. The study protocol was approved by the Local Ethical Committee of Hokkaido University (15-0097). Animals were anesthetized with both an intraperitoneal injection of a mixture of ketamine (Ketalar, Daiichi Sankyo Propharma Corporation) and medetomidine (Domitor, Orion Corporation) and in combination with inhalation of isoflurane (Escain, Pfizer).
Crush injury
The skin incision was made from the buttock to distal thigh to expose the sciatic nerve. Crush injuries were made by applying micro-mosquito forceps closed 1 min for rats and 30 s for mice as described previously (Endo et al, 2019). To mark an injury site, stay suture with an 8-0 nylon was placed at the epineurium just proximal to the injury site.
BNB breakdown
To disrupt BNB, 20% mannitol (30 ml/kg; Yoshindo) (Richner et al, 2019) with recombinant rat VEGF165 (15 μl/kg; PeproTech) (Shimizu et al, 2011) were infused into the blood stream through a common iliac artery. The infusion was initiated at 11 h after PNI and finished 30 min later, followed by perfused at 12 h after PNI. The disruption of BNB was determined by the leakage of IgG detected with immunolabeling (Poduslo et al, 1988).
Neutrophil depletion
Rats received intraperitoneal injections of 1 ml/kg rabbit anti-rat PMN antibody (#CLAD51140; Cedarlane) or 1 ml/kg rabbit normal serum (# CLSD403R; Cedarlane) as a negative control at 3 h before the injury. Subjects received daily intraperitoneal injections of antibiotics (5 mg/kg of enrofloxacin; Kyowa Medex Cox) to prevent infection. To quantify the number of neutrophils in blood stream, one drop of peripheral blood taken from the tail vein was smeared on a slide and stained with Wright Giemsa (Sigma-Aldrich).
Neutrophil infusion
Bone marrow cells were collected from femurs and tibias of adult GFP-expressing C57BL/6 mice by flushing bone marrow with PBS using a syringe with a 24-G needle. Collected cells were washed with PBS, followed by incubation with 10% RBC lysis buffer (#420302; BioLegend) at room temperature to eliminate erythrocytes. Neutrophils were isolated by a neutrophil isolation kit (# 130-097-658; MACs Miltenyi Biotec). The viability of isolated neutrophils was assessed by trypan blue (Thermo Fisher Scientific), and it was over 90%. Based on the previous studies (Nemzek et al, 2001; O’Connell et al, 2015), the total number of neutrophils circulating in the blood stream of an adult mouse was estimated as 2 × 106 cells. To double the number of circulating neutrophils, isolated neutrophils were suspended in PBS at 2 × 106 cells/100 μl and injected intravenously into the jugular vein of C57BL/6mice at 11 h after PNI. As a negative control, the same amount of PBS was intravenously administrated in the same manner.
Inhibition of NETs, CXCR4, and MIF
To inhibit NETs, CXCR4, and MIF, inhibitors were absorbed onto a collagen sheet (Pelnac). Cl-amidine of the PAD4 inhibitor (300 μg/50 μl/cm2, #10599; Funakoshi) and DNase I (200 U/50 μl/cm2, #314-08071; Nippongene) for NET inhibition (Kang et al, 2020), AMD 3100 (500 μg/50 μl/cm2, #10599; Abcam) for CXCR4 inhibition (Kawaguchi et al, 2009), and iso-1 (500 μg/50 μl/cm2, #ab142140; Abcam) for MIF inhibition (Liu et al, 2018b) were used. As a control, PBS (50 μl/cm2) was used except iso-1, which used 1% DMSO (50 μl/cm2) as a control. Ten hours after injury, the sciatic-nerve region 15–25 mm distal to injury sites was circumferentially wrapped with a 1-cm2 collagen sheet, followed by perfusion 2 h later. To investigate the effect of MIF inhibition on the repair process, a 2-cm2 collagen sheet containing isol-1(500 μg/50 μl/cm2, #ab142140; Abcam) was placed on the sciatic nerve to cover the region 5–25 mm distal to injury sites 10 h after injury. Two hours later, the sheet was removed, followed by repeated washing of sciatic nerves with saline. The control collagen sheet contained 1% DMSO. Subjects were perfused 1 wk after injury.
Histology
Subjects were perfused with 4% PFA (Nacalai tesque) in 0.1M PB (phosphate buffer). Sciatic nerves were dissected and incubated in 4% PFA overnight, followed by placement in 30% sucrose in 0.1 M PB for cryoprotection. Rat and mouse nerves were sagittally and axially sectioned at 10-μm intervals by a cryostat. Every four sections were mounted on the same slide. For HE staining, sections were incubated Mayer’s hematoxylin solution (Wako) and 1% eosin Y solution (Nacalai Tesque) and dehydrated by 100% ethanol (Fujifilm Wako Pure Chemical). For immuno-fluorescent labeling, sections were blocked for 1 h in 50 mM TBS with 0.25% Triton X-100 and 5% horse serum, followed by primary antibodies incubation overnight at 4°C. Primary antibodies were NE (1:200 rabbit from Abcam #ab68672) and Ly6G (1:200, rat from GeneTex #GTX40913) to detect neutrophils, CD68 (ED1; 1/500, mouse from Bio-Rad #MCA341R) to detect macrophages, biotin-conjugated donkey anti-rat IgG (1/500, rabbit from Jackson ImmunoResearch #712-065-150) to detect IgG, β3tublin (1/1,000, mouse from BioLegend #801202) or β3tublin (1/1,000, rabbit from Covance #PRB-435P) to detect axons, MBP (1/1,000, chicken from Aves lab #AB_2313550) to detect myelin, typeⅠcollagen (1/200, chicken from SouthernBiotech #1301-01), GFP (1/1,000, rabbit from Thermo Fisher Scientific #a6455), CCL2 (1/200, rabbit from Thermo Fisher Scientific #PA5-115555), citrullinated histone 3 (1/100, rabbit from Abcam #ab5103) and MPO (1/100, goat from R & D System #AF3667) to detect NETs (Gavillet et al, 2015; Magán-Fernández et al, 2019), CXCR4 (1/100, rabbit from Novus #NB100-56437), and MIF (1/200, rabbit from Thermo Fisher Scientific #PA527343). The following day, sections were incubated with Alexa fluorochrome–conjugated donkey secondary antibodies (1/1,000; Jackson ImmunoResearch) with DAPI (1/1,000; Sigma-Aldrich) for 1 h at room temperature. Sections were mounted on slides with Mowiol (Sigma-Aldrich).
In situ hybridization
Sections were treated with acetylation solution (2,2′,2″-nitrilotriethanol hydrochloride, 5N NaOH, and acetic anhydride) for 10 min to reduce nonspecific labeling and were then prehybridized for 30 min at room temperature in the hybridization buffer (50% formamide, 1M Tris–HCl, pH8.0, tRNA, 50× Denhardt’s, 5M NaCl, 0.5M EDTA, NLS, and dextran sulfate). The following digoxigenin (DIG)-labeled mRNA riboprobes were used for the hybridization: rat CXCL1 (nucleotides 289–835 bp;NM_030845.1), rat CXCL2 (nucleotides 268–762 bp; NM_053647.2), CXCL3 (nucleotides 283–806 bp; NM_001394590.1), and rat CCL2 (nucleotides 136–545; NM_031530). These probes were produced from an RT–PCR–derived cDNA library made from zymosan-induced lungs and heart and were subcloned into the pBluescript SK(+) plasmid vector. Hybridization was performed at 63.5°C overnight in a hybridization buffer supplemented with antisense and sense RNA probes (1/1,000). A sense probe was used as a negative control. The zymosan-induced sciatic nerve was evaluated as a positive control. After hybridization, sections were incubated in NTE buffer (5M NaCl, 1M Tris–HCl, pH8.0, 0.5M EDTA, and 20% tween) for 20 min, 20 mM iodoacetamide solution in NTE buffer for 20 min, NTE buffer for 10 min, and TNT buffer (5M NaCl, 1M Tris–HCl, pH7.4, and 20% tween) for 10 min. For detection of DIG, sections were blocked for 30 min with DIG mix solution, which was TNT buffer with 1% blocking reagent (Roche Diagnostics) and 4% normal sheep serum. Sections were then incubated with alkaline-phosphatase–conjugated sheep anti-DIG (1/500; Roche Diagnostics) for 1.5 h, followed by overnight incubation with 2% NBT/BCIP stock solution (Roche Diagnostics) diluted in buffer (1M Tris–HCl, 5M NaCl, AM MgCl2) at room temperature for chromogenic detection. Sections were dehydrated by 100% ethanol and xylene. All images were captured by an all-in-one microscope (BZ-X 710; Keyence).
Quantification
Quantification of neutrophils, macrophages, myelin debris, CCL2, and axons in rats was performed as described previously (Endo et al, 2019). In brief, three sections containing the middle part of the nerve on the same slide were used. Vertical 100-μm wide rectangular regions were set at 5-mm intervals from injury sites, and the total number of neutrophils and macrophages were divided by the total quantified area to calculate the cell density. Neutrophils and macrophages were identified by typical segmented or lobed morphology of the nucleus in HE-stained sections and CD 68 immunoreactivity surrounding DAPI, respectively. Neutrophils were quantified in all areas including epineurium or epineurium alone, and macrophages were quantified only at the parenchyma or both of parenchyma and epineurium. To quantify myelin debris and CCL2, the total area of the MBP or CCL2 immunoreactivity was divided by the total quantified area. For axon quantification, vertical lines were set at 5-mm intervals from injury sites. Axons were identified with β3tublin immunoreactivity, and the total number of axons crossing each line was divided by the length of each line. To calculate axon regeneration rate, each axon density was divided by the axon density at 5 mm proximal to the injury site. For quantification in mice, one transverse section of the sciatic nerve was used. The total number of neutrophils in the whole area was counted and divided by the whole area to calculate its density. For quantification of macrophages and myelin debris, a 100-μm wide rectangular region was set to cross the center of the transverse section, and the total number of CD68-positive cells and the total area of the MBP immunoreactivity at the parenchyma was quantified and divided by the quantified area to calculate its density. For quantification axons, a 50-μm wide rectangular region was set to cross the center of the transverse section, and the total number of axons was divided by the quantified area. For quantification of NET formation or MIF expression, a rectangle with sides of 50 and 100 μm was set in the middle of the epineurium, and the area of triple immunoreactivity of citrullinated histone 3, MPO, and DAPI or single immunoreactivity of MIF was divided by the area of the rectangle, which was 0.005 mm2.
Statistical analysis
All statistical analysis was performed using the JMP software (SAS). Normal distribution of data was assessed with the Shapiro–Wilk test. Multiple-group comparisons were performed by one-way ANOVA with the Tukey-Kramer test, and two-group a comparison was performed by an unpaired two-tailed t test. When P-value was smaller than 0.05, it was considered as statistically significant. Data in graphs were presented as the mean and SEM.
Acknowledgements
We thank M Endo for assistance with performing experiments. We are thankful to the Open Facility, Hokkaido University Sousei Hall for access to the cryostat. This work was supported by the Japan Agency for Medical Research and Development (JP21gm6210004), a Grant-in-Aid for Scientific Research (JP20K18016, JP20H03558), the Kobayashi Foundation, and the SEI Group CSR Foundation.
Author Contributions
Y Yamamoto: formal analysis, investigation, visualization, methodology, and writing—original draft, review, and editing.
K Kadoya: conceptualization, supervision, funding acquisition, project administration, and writing—original draft, review, and editing.
MA Terkawi: methodology.
T Endo: methodology.
K Konno: methodology.
M Watanabe: methodology.
S Ichihara: supervision.
A Hara: supervision.
K Kaneko: supervision.
N Iwasaki: supervision and project administration.
M Ishijima: supervision and project administration.
Conflict of Interest Statement
The authors declare that they have no conflict of interest.
- Received February 3, 2022.
- Revision received July 20, 2022.
- Accepted July 21, 2022.
- © 2022 Yamamoto et al.
This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).