TAM receptors mediate the Fpr2-driven pain resolution and fibrinolysis after nerve injury

Abstract

Nerve injury causes neuropathic pain and multilevel nerve barrier disruption. Nerve barriers consist of perineurial, endothelial and myelin barriers. So far, it is unclear whether resealing nerve barriers fosters pain resolution and recovery. To this end, we analysed the nerve barrier property portfolio, pain behaviour battery and lipidomics for precursors of specialized pro-resolving meditators (SPMs) and their receptors in chronic constriction injury of the rat sciatic nerve to identify targets for pain resolution by resealing the selected nerve barriers. Of the three nerve barriers-perineurium, capillaries and myelin-only capillary tightness specifically against larger molecules, such as fibrinogen, recuperated with pain resolution. Fibrinogen immunoreactivity was elevated in rats not only at the time of neuropathic pain but also in nerve biopsies from patients with (but not without) painful polyneuropathy, indicating that sealing of the vascular barrier might be a novel approach in pain treatment. Hydroxyeicosatetraenoic acid (15R-HETE), a precursor of aspirin-triggered lipoxin A4, was specifically upregulated at the beginning of pain resolution. Repeated local application of resolvin D1-laden nanoparticles or Fpr2 agonists sex-independently resulted in accelerated pain resolution and fibrinogen removal. Clearing macrophages (Cd206) were boosted and fibrinolytic pathways (Plat) were induced, while inflammation (Tnfα) and inflammasomes (Nlrp3) were unaffected by this treatment. Blocking TAM receptors (Tyro3, Axl and Mer) and tyrosine kinase receptors linking haemostasis and inflammation completely inhibited all the effects. In summary, nanoparticles can be used as transporters for fleeting lipids, such as SPMs, and therefore expand the array of possible therapeutic agents. Thus, the Fpr2-Cd206-TAM receptor axis may be a suitable target for strengthening the capillary barrier, removing endoneurial fibrinogen and boosting pain resolution in patients with chronic neuropathic pain.

Keywords: Blood–nerve barrier; Chronic constriction injury; Fibrinogen; Nanoparticles; Pain resolution; Resolvin D1; TAM receptors.

Fig. 1

Neuropathic pain resolves 6 weeks after nerve injury. a Schematic illustration of the experimental setup: Male and female Wistar rats underwent CCI or sham surgery and were examined using the displayed tests throughout 9 weeks. b Mechanical allodynia and c thermal hypersensitivity were assessed weekly (n = 6–8). d The print area and e stand time presented as the ratios of values from right to left hindpaws were recorded using the Catwalk gait analysis system (n = 6–7). f The covered distance and g the mean speed of CCI and sham animals were recorded with voluntary running wheels (n = 8). h The summary of all behavioural tests including von Frey and Hargreaves from both male and female rats. The heatmap shows the ratios between the arithmetic means of CCI and sham groups. Data in graphs are shown as mean ± SEM, *p < 0.05. Two-way repeated-measures ANOVA with Tukey’s multiple comparison. PWT paw withdrawal threshold, PWL paw withdrawal latency, CCI chronic constriction injury, VWR voluntary wheel running, ANOVA analysis of variance

Fig. 2

The perineurial barrier remains open up to 9 weeks, while the myelin barrier stays intact. a Depiction of two ex vivo immersion techniques for analysing the perineurial and myelin barriers after CCI or sham. b Representative images of sciatic nerve cross sections and c, d quantification of endoneurial fluorescence intensity of nerves immersed in Fluo (332 Da) and Evans blue albumin (EBA, 68 kDA) ex vivo. Dashed lines indicate the endoneurial regions. Scale bar: 300 µm. e Representative brightfield and fluorescence images of teased nerve fibres from the proximal part of the sciatic nerve after immersion in 70 kDa FITC-dextran. Scale bar: 100 µm. f Quantification of the fluorescence intensity measured in the internodal regions of the teased fibres. (n = 138–296 from 5 to 6 animals per group, pairwise Mann–Whitney U tests). The relative gene expression of g Cldn1, h Tjp1 and i Cldn19 in the ligation parts of ipsilateral and contralateral sciatic nerves after 1, 3 and 6 weeks after CCI (n = 5–6). All data are shown as mean ± SEM, *p < 0.05 compared to control at the indicated time points, two-way ANOVA with Šidák’s multiple comparisons unless indicated otherwise

Fig. 3

The capillary barrier reseals specifically for fibrinogen in parallel with neuropathic pain. a Schematic illustration of the capillary permeability test: Evans blue albumin (EBA, 68 kDa) was intravenously injected. Cross sections at distinct positions distal and proximal from the ligations were collected and analysed. b Representative images of sciatic nerve cross sections. The dashed lines indicate the endoneurial regions. Scale bar: 300 µm. c Depiction of the spatial and temporal EBA extravasation into the endoneurium through endoneurial vessels. Sham values were averaged and plotted on each position as reference. Dashed lines mark the positions that were used for the calculations for the areas under the curve in d Quantification analysis of the areas under the curves distal and proximal from 0 to 6 mm from the ligations were plotted (n = 3–6). *distal vs. sham; #distal vs. proximal p < 0.05 two-way ANOVA with Bonferroni’s multiple comparison test. e Representative images of fibrinogen immunoreactivity in the sciatic nerve 1 week after surgery. The dashed lines indicate the endoneurial region. f Quantification of the intensity of fibrinogen immunoreactivity within the endoneurial regions (n = 5–6). Scale bar: 300 µm. Relative gene expression of g Plat and h Cldn5 in sciatic nerves after CCI (n = 5–6). i Summary of all permeability tests. The heatmap displays the ratios between the arithmetic means of CCI and sham groups. All data are shown as mean ± SEM, *p < 0.05 two-way ANOVA with Šidák’s multiple comparisons

Fig. 4

More endoneurial fibrinogen in sural nerve biopsies from patients with painful polyneuropathies. Sural nerve biopsies from patients were immunofluorescently stained. Representative images of human sural nerves stained for a fibrinogen and c tissue plasminogen activator (tPA). The dashed lines indicate the endoneurial regions. Scale bar: 300 µm. b, d Quantifications of the signal intensity of fibrinogen and tPA. Patients were grouped by pain occurrence (n = 16, Student’s t tests with Welch’s correction)

Fig. 5

Unbiased lipidomic analysis selectively identified higher levels of the LXA4 precursor 15R-HETE at 3 weeks, initiating the resolution phase. a Arachidonic-, docosahexaenoic- and eicosapentaenoic acids-derived metabolites were measured by LC-MSMS in the sciatic nerve, and their cognate receptors were assessed using qPCR. b Relative levels of the 11 detected lipid metabolites are depicted. Only 15R-HETE (circled in red) had a significant upregulation in CCI specifically at 3 weeks compared with sham. c Relative mRNA expression of the respective SPM receptor after CCI; CL contralateral, IL ipsilateral. All data are shown as mean ± SEM, n = 5–6, two-way ANOVA with Šidák’s multiple comparisons. *p < 0.05; ANOVA: analysis of variance. d Expression levels as counts per million (CPM) of the SPM receptors from intact and injured human sural nerve tissues collected 2 weeks after a full transection injury (n = 6, Student’s t tests with Welch’s correction), as obtained from previously published bulk RNAseq data [72]

Fig. 6

Local application of the Fpr2 agonist BML-111 initiates pain resolution, macrophage polarization and fibrinogen clearance. a CCI-operated animals received a perineurial injection of BML-111 or vehicle (PBS). The animals were retested with Hargreaves and von Frey at 1, 2, 3 and 6 h. As soon as reflexive tests at 6 h were finished, the animals received a second injection from day 18 to 24 and were killed at day 25. Blue arrows indicate the daily injection/test programme. T: reflexive testing; syringe: injection. b Mechanical allodynia was measured after the first BML-111 injection on day 18 (n = 6, two-way repeated-measures ANOVA with Tukey’s multiple comparisons). Paw withdrawal thresholds for c mechanical and d thermal hypersensitivities of baseline measurements show an accumulative effect of perineurial BML-111 (n = 5–6, two-way repeated-measures ANOVA with Tukey’s multiple comparisons). e, f Representative images and quantification of fibrinogen immunostaining in the sciatic nerve after BML-111 injections. The dashed lines indicate the endoneurial region. Scale bar: 300 µm (n = 6, Student’s t tests with Welch’s correction). g-k Relative mRNA expression of Tnfα, Arg1, Cd206, Nlrp3 and Plat after BML-111 injections (n = 6, Student’s t tests with Welch’s correction). l Mechanical allodynia was assessed daily for 1 week after injections were discontinued (n = 6, two-way repeated-measures ANOVA with Tukey’s multiple comparisons). ANOVA analysis of variance. All data are shown as mean ± SEM, *p < 0.05

Fig. 7

Locally applied RvD1-laden nanoparticles (NPs) induce pain resolution, macrophage polarization and fibrinogen clearance after CCI. a Treatment plan for RvD1-NPs in CCI-operated male rats: The animals received a perineurial injection of RvD1-NPs or empty NPs. They were tested at baseline, 1, 2, 3 and 6 h from days 18 to 24, and killed on day 25. Blue arrows indicate daily injection/test programme. T reflexive testing; syringe: injection. b Estimated amount of released RvD1 from NPs by a dialysis experiment. The NP solution was diluted in PBS and incubated at 37 °C in dialysis tubes. The RvD1 concentration of the dialysates was subsequently determined by LC-MSMS (n = 1). c Mechanical allodynia was measured after the first RvD1-NP injection on day 18 (n = 6, two-way repeated-measures ANOVA with Tukey’s multiple comparisons). d Mechanical hypersensitivities measured before the injections during the injection period (n = 6, two-way repeated-measures ANOVA with Tukey’s multiple comparisons). e Representative images of fibrinogen immunostaining in sciatic nerve cross sections after RvD1-NP injections. The dashed lines indicate the endoneurial region. Scale bar: 300 µm. f Quantification of the intensity of fibrinogen immunoreactivity within the endoneurial regions (n = 6, Student’s t tests with Welch’s correction). g-k Relative mRNA expression of Tnfα, Arg1, Cd206, Nlrp3 and Plat after daily RvD1-NP injections (n = 6, Student’s t tests with Welch’s correction). l Fluxes of FITC-labelled fibrinogen through monolayers of primary HDMECs were measured in vitro. Extracellular FITC-fibrinogen fluorescence intensity was assessed to evaluate paracellular barrier integrity, while intracellular FITC-fibrinogen was assessed to evaluate transcellular passage, potentially via endocytosis (n = 7–9, one-way ANOVA with multiple comparisons). NP empty nanoparticles, RvD1-NP RvD1-laden nanoparticles, ANOVA analysis of variance, PBS phosphate-buffered saline, CCI chronic constriction injury, HDMECs human dermal microvascular endothelial cells. Scale bar: 300 µm. All data are shown as mean ± SEM, *p < 0.05

Fig. 8

Blocking TAM receptors inhibits Fpr2-mediated pain resolution and fibrinogen clearance. a Proposed model of pain relief through Fpr2 activation and involvement of the TAM receptor family of receptor tyrosine kinases (Tyro3, Axl and MerTK) in macrophages and Schwann cells leading to fibrinogen clearance: For details, see text. b, c Male Wistar rats received BML-111 injections combined with prior injections of the TAM receptor inhibitor RU-301 for 1 week. Paw withdrawal thresholds for b mechanical and c thermal hypersensitivities (n = 6, two-way repeated-measures ANOVA with Tukey’s multiple comparisons). d, e Representative images of fibrinogen immunostaining and quantification within the endoneurial regions in sciatic nerve cross sections after BML-111 with or without RU-301 injections. The dashed lines indicate the endoneurial region (n = 6, Student’s t tests with Welch’s correction). Scale bar: 300 µm. f, g Relative mRNA expression of Cd206 and Plat after 1 week of BML-111 administration with and without TAM blocking