Toll-like receptor signaling is critical for Wallerian degeneration and functional recovery after peripheral nerve injury

Abstract

Toll-like receptors (TLRs) bind specific components conserved among microorganisms as well as endogenous ligands produced by necrotic cells, injured axons, and the extracellular matrix. Here, we investigated whether TLRs are involved in regulating the immune response, Wallerian degeneration (WD), and nerve regeneration after sciatic nerve lesion. Early expression of interleukin-1beta and monocyte chemoattractant protein-1 was compromised in the sciatic nerve distal stump of mice deficient in TLR signaling. In addition, significantly fewer macrophages were recruited and/or activated in the sciatic nerve distal stump of TLR2-, TLR4-, and MyD88-deficient mice compared with wild-type littermates, whereas WD, axonal regeneration, and recovery of locomotor function were impaired. In contrast, animals that received a single microinjection of TLR2 and TLR4 ligands at the site of sciatic nerve lesion had faster clearance of the degenerating myelin and recovered earlier than saline-injected control rats. Finally, rats that had altered innate immune response through dexamethasone treatment exhibited three times more myelin debris in their sciatic nerve distal stump and a significant delay in recovery of locomotor function. Our results provide strong evidence that TLR signaling plays a critical role in orchestrating the innate immune response leading to efficient and rapid clearance of inhibitory myelin debris and nerve regeneration.

Figure 1.

Early expression of IL-1β is abolished in the sciatic nerve distal stump of mice deficient in TLR signaling. A, B, D, E, Representative dark-field photomicrographs showing IL-1β mRNA expression in C57BL/6J (A, D) and MyD88-ko (B, E) mice at 1.5 h (A, B) and 24 h (D, E) after sciatic nerve lesion. All photomicrographs were taken at the same level of the sciatic nerve distal stump. C, F, Quantification of ISH signal for IL-1β mRNA [in mean gray values (MGV)] in the sciatic nerve distal stump of TLR2-ko, MyD88-ko, and TLR4^d mice and their respective wild-type littermates at 1.5 h (C) and 24 h (F) after lesion (n = 4–6 per group per time point). G, qRT-PCR analyses confirmed that IL-1β expression is severely compromised in TLR2-, TLR4-, and MyD88-deficient mice at 24 h after sciatic nerve lesion (n = 3–5 per group). Data are expressed as the ratio to 18S ribosomal RNA (rRNA). H, Protein levels for IL-1β, as detected using cytokine arrays and expressed as I.O.D., are significantly reduced in TLR2-, TLR4-, and MyD88-deficient mice compared with their respective wild-type littermates at 24 h after sciatic nerve lesion (n = 3 per group). All data are expressed as mean ± SEM. Statistical significance bars were added to graphs to more directly infer whether any two means are statistically significant from one another. ***p < 0.001, **p < 0.01, and *p < 0.05 compared with the control group. NS, Not statistically significant. Scale bar, 125 μm.

Figure 2.

Early expression of MCP-1 is compromised in the sciatic nerve distal stump of mice deficient in TLR signaling. A, B, D, E, Representative dark-field photomicrographs showing expression for MCP-1 mRNA in C57BL/6J (A, D) and MyD88-ko (B, E) mice at 1.5 h (A, B) and 24 h (D, E) after sciatic nerve lesion. All photomicrographs were taken at the same level of the sciatic nerve distal stump. C, F, Quantification of ISH signal for MCP-1 mRNA [in mean gray values (MGV)] in the sciatic nerve distal stump of TLR2-ko, MyD88-ko, and TLR4^d mice and their respective wild-type littermates at 1.5 h (C) and 24 h (F) after lesion (n = 4–6 per group per time point). All data are expressed as mean ± SEM. ***p < 0.001, **p < 0.01, and *p < 0.05 compared with the control group. NS, Not statistically significant. Scale bar, 125 μm.

Figure 3.

Deficiency in TLR signaling compromises macrophage recruitment/activation and delays myelin debris clearance, axonal regeneration, and locomotor recovery after sciatic nerve lesion. A–D, Representative fluorescence photomicrographs taken from longitudinal sections of the sciatic nerve distal to the lesion showing CD68-immunopositive macrophages in C57BL/6J (A), TLR2-ko (B), C3H/HeOUJ (C), and TLR4^d (D) mice at 7 d. E, Quantification of the number of CD68⁺ macrophages in the sciatic nerve distal stump of TLR2-ko, TLR4^d, MyD88-ko, and wild-type mice at 7 d after lesion (n = 8 per group). F–I, Bright-field photomicrographs showing myelin stained with LFB in the sciatic nerve distal stump of C57BL/6J (F), TLR2-ko (G), C3H/HeOUJ (H), and TLR4^d (I) mice at 7 d. J, Quantification of LFB staining of myelin in the degenerating sciatic nerve distal stump of mice deficient in TLR signaling at 7 d (n = 8 per group). K, Quantification of the number of axons that had regenerated up to 4 mm distal to the site of lesion, as visualized by GAP-43 immunofluorescence, at 4 d after lesion. L, M, Recovery of locomotor functions from a sciatic nerve lesion, as determined by the SFI, in TLR2-ko, MyD88-ko, and TLR4^d mice compared with their respective control groups (WT) over a period of 49 d (n = 12 per group). ***p < 0.001, **p < 0.01, and *p < 0.05 compared with the control group. Scale bar, 50 μm.

Figure 4.

Activation of TLR signaling through intraneural injections of TLR2 and TLR4 ligands enhances the recruitment and myelin phagocytic activity of macrophages and accelerates myelin debris clearance and locomotor recovery after sciatic nerve lesion. A, Quantification of CD68 and iba1immunoreactivity (ir) in the degenerating sciatic nerve distal stump of rats that received a single microinjection of PBS (Ctl), zymosan (ZYM; a TLR2 ligand), or LPS (a TLR4 ligand) at the site of lesion (3 d time point; n = 8 per group). B, C, Representative fluorescence photomicrographs showing iba1-immunopositive macrophages in a PBS-injected (B) and a LPS-injected (C) rat. Arrows indicate the lesion epicenter. D, E, Confocal high-power photomicrographs showing examples of iba1⁺ macrophages (green) that have ingested myelin debris (red), as detected using the ORO staining. Arrows point to double-labeled cells. F, Quantification of the number of macrophages that have ingested myelin debris in the sciatic nerve distal stump of rats treated with PBS, ZYM, or LPS at 3 d after lesion per injection (n = 8 per group). G, Quantification of LFB staining of myelin in the degenerating sciatic nerve distal stump at 7 d (n = 6–7 per group). H, Recovery of locomotor functions from a sciatic nerve lesion in rats given microinjections of PBS or TLR ligands (n = 10 per group). ***p < 0.001 and *p < 0.05 compared with the Ctl group. Scale bars: B, C, 260 μm; D, 8.5 μm; E, 14 μm.

Figure 5.

Alteration of immune responses by glucocorticoid treatment delays myelin debris clearance and recovery of locomotor functions after sciatic nerve lesion. A, Quantification of the recruitment/activation of CD68⁺ macrophages in the sciatic nerve distal stump of rats treated with DEX, MP, or saline (Ctl) at 7 d after sciatic nerve lesion (n = 5–6 per group). B, Quantification of LFB staining of myelin in the degenerating sciatic nerve distal stump of rats treated with glucocorticoid agents or saline at 7 d (n = 5–6 per group). C, Bright-field photomicrographs showing myelin debris stained with LFB in the sciatic nerve of saline- and DEX-treated rats at 7 d after lesion. Arrows indicate the lesion site. D, Recovery of locomotor functions from a sciatic nerve lesion, as determined by the SFI, in rats treated with DEX, MP, or saline (n = 10–11 per group). ***p < 0.001, **p < 0.01, and *p < 0.05 compared with the Ctl group. Scale bar, 260 μm.