Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 35 (50), 16431-42

Role of IL-10 in Resolution of Inflammation and Functional Recovery After Peripheral Nerve Injury

Affiliations

Role of IL-10 in Resolution of Inflammation and Functional Recovery After Peripheral Nerve Injury

Bruno Siqueira Mietto et al. J Neurosci.

Abstract

A rapid proinflammatory response after peripheral nerve injury is required for clearance of tissue debris (Wallerian degeneration) and effective regeneration. Unlike the CNS, this response is rapidly terminated in peripheral nerves starting between 2 and 3 weeks after crush injury. We examined the expression and role of the anti-inflammatory cytokine IL-10 in the resolution of inflammation and regeneration after sciatic nerve crush injury in mice. IL-10 mRNA increased over the first 7 d after injury, whereas at the protein level, immunofluorescence labeling showed IL-10(+) cells increased almost 3-fold in the first 3 weeks, with macrophages being the major cell type expressing IL-10. The role of IL-10 in nerve injury was assessed using IL-10-null mice. Increased numbers of macrophages were found in the distal segment of IL-10-null mice at early (3 d) and late (14 and 21 d) time points, suggesting that IL-10 may play a role in controlling the early influx and the later efflux of macrophages out of the nerve. A chemokine/cytokine PCR array of the nerve 24 h after crush showed a 2- to 4-fold increase in the expression of 10 proinflammatory mediators in IL-10(-/-) mice. In addition, myelin phagocytosis in vitro by LPS stimulated bone-marrow-derived macrophages from IL-10-null mice failed to downregulate expression of proinflammatory chemokines/cytokines, suggesting that IL-10 is required for the myelin-phagocytosis-induced shift of macrophages from proinflammatory to anti-inflammatory/pro-repair phenotype. The failure to switch off inflammation in IL-10-null mice was accompanied by impaired axon regeneration and poor recovery of motor and sensory function.

Significance statement: An appropriately regulated inflammatory response after peripheral nerve injury is essential for axon regeneration and recovery. The aim of this study was to investigate the expression and role of the anti-inflammatory cytokine IL-10 in terminating inflammation after sciatic nerve crush injury and promoting regeneration. IL-10 is rapidly expressed by macrophages after crush injury. Its role was assessed using IL-10-null mice, which showed that IL-10 plays a role in controlling the early influx and the later efflux of macrophages out of the injured nerve, reduces the expression of proinflammatory chemokines and cytokines, and is required for myelin-phagocytosis-induced shift of macrophages from proinflammatory to anti-inflammatory. Furthermore, lack of IL-10 leads to impaired axon regeneration and poor recovery of motor and sensory function.

Keywords: IL-10; anti-inflammatory cytokine; inflammation; macrophage; peripheral nerve; regeneration.

Figures

Figure 1.
Figure 1.
Changes in expression of IL-10 in the distal segment of the sciatic nerve after crush injury. A, Graph showing the fold change in IL-10 mRNA levels at different days postinjury (dpi) compared with uninjured nerve, detected by qRT-PCR. Note the gradual increase in expression over the first 7 d. n = 3–4 samples of 2 pooled nerves per time point; mean ± SEM; ****p < 0.0001 from uninjured nerve. B, Graph showing changes in the number of IL-10+ cells detected by immunofluoresence labeling of the sciatic nerve at a distance of ∼3 mm distal to the injury. n = 3 per time point; mean ± SEM; ***p < 0.001, ****p < 0.0001 from uninjured nerve. C, Quantification of double-immunofluoresence labeling of the nerve with antibodies against IL-10 and CD11b. Note that the vast majority of the IL-10+ cells in the distal nerve segment are CD11b+ macrophages. Counts were done at a distance of ∼3 mm distal to the injury. n = 3 per time point; mean ± SEM; ***p < 0.001, ****p < 0.0001 compared with CD11b cells in each group. DF, Double-immunofluorescence labeling for IL-10 (i) and CD11b (ii), and merged images (iii) of the nerve at 3 (D), 14 (E), and 21 (F) dpi. Note the increase in IL-10 labeling over time after injury and the presence of IL-10 labeling in foamy phagocytic macrophages at the later time point (E, F). Arrows point to double-labeled cells. Scale bar, 100 μm.
Figure 2.
Figure 2.
Changes in macrophage numbers in injured nerve and response of BMDM to myelin phagocytosis. A, Graph showing changes in the number of CD11b+ macrophages in the uninjured and injured distal nerve segment at different times after crush injury in WT and IL-10-null (IL-10−/−) mice. Note the significant increase in macrophages in the nerves of IL-10-null mice compared with WT mice at 3, 14, and 21 d postinjury (dpi). n = 4–5 nerves per time point per group; mean ± SEM; *p < 0.05. BF, Graphs showing response of BMDM from WT and IL-10−/− mice to LPS stimulation with and without myelin phagocytosis. Note that myelin phagocytosis (myelin + LPS) by WT BMDM reduces the expression of TNF, IL-6, CCL3, and CCL4. n = 3; mean ± SD; *p < 0.05. In contrast, myelin phagocytosis by BMDM from IL-10−/− mice does not significantly reduce expression of these molecules, except for CCL4, compared with stimulation with LPS alone. Cytokine and chemokine levels were significantly higher in IL10−/− cells after myelin + LPS compared with WT cells. #p < 0.05. Note also the 3-fold increase in CCL2 expression in LPS-stimulated IL-10−/− cells compared with WT cells. #p < 0.05.
Figure 3.
Figure 3.
Changes in expression of macrophage markers in WT and IL-10−/− nerves at different times after crush injury. Shown are changes in proinflammatory M1 markers CD16/32 (A), CD86 (B), and anti-inflammatory M2 markers CD206 (C), CD163 (D), and Arg-1 (E). Note the increase in CD16/32 and CD86 (A, B) and decrease in CD206 and Arg-1 (CE) expression in IL-10-null nerves compared with WT nerves. Double labeling for CD206 and CD86 (F) and CD16/32 and CD163 (G) show that, in IL-10-null nerves, a greater number of CD206+ macrophages also express the proinflammatory M1 marker CD86 (F) and fewer CD16/32+ macrophages express the anti-inflammatory M2 marker CD163 (G) at various times after injury. n = 4–5 mice per time point per group; mean ± SEM; **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 4.
Figure 4.
Immunofluorescence labeling of M1 macrophage markers CD16/32 in WT (A) and IL-10−/− (C) mice and CD86 in WT (B) and IL-10−/− (D) mice at 3 (i), 7 (ii), 14 (iii), and 21 (iv) days postinjury (dpi). CD16/32 expression increases rapidly in both WT (A) and IL-10−/− (C) nerves after injury. Note, however, the increased labeling at 3 d in IL-10−/− compared with WT nerves. In contrast, CD86 is expressed by very few cells in both WT (B) and IL-10−/− (D) nerves, but is increased in IL-10−/− compared with WT nerves. Scale bar, 100 μm.
Figure 5.
Figure 5.
Immunofluorescence labeling of M2 macrophage markers CD206 in WT (A) and IL-10−/− (C) mice and CD163 in WT (B) and IL-10−/− (D) mice at 3 (i), 7 (ii), 14 (iii), and 21 (iv) days postinjury (dpi). In both genotypes, the number of cells expressing CD206 increased with dpi, however, the labeling is more intense at the earlier time point of 3 d (Ai). The expression of CD163 decreased with time after injury in both WT (BiBiv) and IL-10−/− (DiDiv) nerves. Note, however, that the number of CD206 and CD163-labeled cells are lower in the IL-10−/− compared with the WT nerves. Scale bar, 100 μm.
Figure 6.
Figure 6.
Double-immunofluorescence labeling of Arg-1 in WT (A) and IL-10−/− (C) mice at 3 (i) and 7 (ii) days postinjury (dpi). The sections are double labeled for the macrophage marker CD11b (B, D). Note that Arg-1 is expressed in some CD11b+ macrophages at 3 dpi in WT nerves (Ai) and this is decreased in IL-10−/− nerves at 3 dpi (Ci). No Arg-1 staining is seen at 7 dpi in either genotype (Aii, Cii); note the abundance of CD11b+ macrophages in these regions. Scale bar, 100 μm. E, Fold increase in the expression of 10 chemokines and cytokines that were statistically significantly increased in IL-10-null nerves compared with WT nerves at 24 h after crush injury detected with a PCR array screen. The horizontal bar represents level in injured WT nerves. n = 3–4; p < 0.05 for all values shown. Il6, Interleukin 6; Ltb, lymphotoxin β; Thpo, thrombopoietin.
Figure 7.
Figure 7.
Recovery of motor and sensory function and axon regeneration. A, Graph showing motor recovery using the SFI. Note that IL-10-null mice show significantly reduced recovery at 21 and 28 d postinjury (dpi). n = 5–6; mean ± SD; *p < 0.05. B, Graph showing sensory recovery assessed using the von Frey hair test. Note that the WT mice begin to show recovery from mechanical allodenia by 28 dpi, whereas the IL-10-null mice show no improvement. n = 5; mean ± SEM; *p < 0.05. C, D, Immunofluorescence labeling for GAP-43 to visualize regenerating axons in WT (C) and IL-10−/− (D) nerves at 3 dpi; asterisks indicate the proximal end of the nerve near the site of crush injury; arrows indicate labeled axons distally. Note the longer growth in the WT nerve (C) compared with the IL-10−/− nerve (D); quantification is given in the text. Scale bar, 500 μm.

Similar articles

See all similar articles

Cited by 25 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback