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. 2015 Feb 24;112(8):2581-6.
doi: 10.1073/pnas.1423221112. Epub 2015 Feb 9.

Neuroinflammation Triggered by β-glucan/dectin-1 Signaling Enables CNS Axon Regeneration

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Free PMC article

Neuroinflammation Triggered by β-glucan/dectin-1 Signaling Enables CNS Axon Regeneration

Katherine T Baldwin et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Innate immunity can facilitate nervous system regeneration, yet the underlying cellular and molecular mechanisms are not well understood. Here we show that intraocular injection of lipopolysaccharide (LPS), a bacterial cell wall component, or the fungal cell wall extract zymosan both lead to rapid and comparable intravitreal accumulation of blood-derived myeloid cells. However, when combined with retro-orbital optic nerve crush injury, lengthy growth of severed retinal ganglion cell (RGC) axons occurs only in zymosan-injected mice, and not in LPS-injected mice. In mice deficient for the pattern recognition receptor dectin-1 but not Toll-like receptor-2 (TLR2), zymosan-mediated RGC regeneration is greatly reduced. The combined loss of dectin-1 and TLR2 completely blocks the proregenerative effects of zymosan. In the retina, dectin-1 is expressed by microglia and dendritic cells, but not by RGCs. Dectin-1 is also present on blood-derived myeloid cells that accumulate in the vitreous. Intraocular injection of the dectin-1 ligand curdlan [a particulate form of β(1, 3)-glucan] promotes optic nerve regeneration comparable to zymosan in WT mice, but not in dectin-1(-/-) mice. Particulate β(1, 3)-glucan leads to increased Erk1/2 MAP-kinase signaling and cAMP response element-binding protein (CREB) activation in myeloid cells in vivo. Loss of the dectin-1 downstream effector caspase recruitment domain 9 (CARD9) blocks CREB activation and attenuates the axon-regenerative effects of β(1, 3)-glucan. Studies with dectin-1(-/-)/WT reciprocal bone marrow chimeric mice revealed a requirement for dectin-1 in both retina-resident immune cells and bone marrow-derived cells for β(1, 3)-glucan-elicited optic nerve regeneration. Collectively, these studies identify a molecular framework of how innate immunity enables repair of injured central nervous system neurons.

Keywords: dectin-1; mouse; neuroinflammation; optic nerve; regeneration.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Zymosan, but not LPS, enables immune-mediated axon regeneration. (A) Flow cytometric analysis of immune cells accumulating in the eye of WT mice at 7 d post-ONC and i.o. zymosan (5 µL, 12.5 µg/µL) injection (n = 5 mice), i.o. LPS (3 µL, 5 µg/µL) injection (n = 3 mice), or i.o. PBS (5 μL) injection. (B–D) Longitudinal sections of WT mouse optic nerves at 2 wk after ONC and i.o. injection. Regenerating axons are visualized by anti-GAP43 immunofluorescence labeling. The injury site is marked with an asterisk. (Scale bar: 200 µm.) (B) WT mice with i.o. zymosan (n = 6) show robust axon regeneration. No significant regeneration is observed in WT mice with i.o. LPS (n = 4) (C) or WT mice with i.o. PBS (n = 5) (D). (E) Quantification of the number of GAP43+ axons per nerve at 0.2–1.6 mm distal to the injury site. Asterisks indicate a significant difference from zymosan-induced regeneration. Results are presented as mean ± SEM. ***P < 0.001; **P < 0.01; *P < 0.05, one-way ANOVA, Tukey’s post hoc test.
Fig. 2.
Fig. 2.
Dectin-1 and TLR2 operate as partially redundant zymosan receptors. (A–E) Longitudinal sections of mouse optic nerves at 2 wk after ONC and i.o. injection of zymosan stained with anti-GAP43. The injury site is marked with an asterisk. (Scale bar: 200 µm.) (A and B) WT mice with i.o. zymosan (n = 5) (A) and complement receptor 3 null mice (CR3−/−) with i.o. zymosan (n = 3) (B) show robust and comparable axon regeneration. (C) In dectin-1−/− mice, i.o. zymosan (n = 5) results in significantly reduced regeneration. (D–E) The i.o. zymosan fails to induce axon regeneration in dectin-1−/−;MyD88−/− compound mutants (n = 7) and dectin-1−/−;TLR2−/− compound mutants (n = 6). (F) Quantification of the number of GAP43+ axons per nerve at 0.2–1.6 mm distal to the injury site. Data are presented as mean ± SEM. Asterisks directly above individual bars indicate a significant difference compared with WT + zymosan. ***P < 0.001; **P < 0.01; *P < 0.05, one-way ANOVA, Tukey’s post hoc test. (G) Comparison of the cellular composite of zymosan-induced inflammation in WT (n = 6 mice), dectin-1−/−;MyD88−/− (n = 6 mice), and dectin-1−/−;TLR2−/− (n = 3 mice) compound mutants. Independent of mouse genotype, similar numbers of macrophages/monocytes, neutrophils, DCs, B cells, and CD4+ and CD8+ T cells, but not of NK cells, were identified in the vitreous.
Fig. 3.
Fig. 3.
β(1, 3)-glucan promotes dectin-1–dependent axon regeneration. (A–C) Longitudinal sections of mouse optic nerves at 2 wk after ONC and i.o. curdlan (5 µL, 25 µg/µL) stained with anti-GAP43. The injury site is marked with an asterisk. (Scale bar: 200 µm.) (A) WT mice with i.o. curdlan (n = 7) show robust axon regeneration. (B) In dectin-1−/− mice (n = 12), i.o. curdlan fails to elicit a regenerative response. (C) In CARD9−/− mice with i.o. curdlan (n = 9), axon regeneration is significantly reduced, yet increased compared with dectin-1−/− mice at 0.2–0.6 mm distal to the injury site. (D) Quantification of GAP43+ axons at 0.2–1.6 mm distal to the injury site. Results are presented as mean ± SEM. Asterisks directly above individual bars indicate significance compared with WT + curdlan. **P < 0.001; *P < 0.05, one-way ANOVA, Tukey’s post hoc test. (E) Western blot analysis of adult mouse eye extracts at 6 h after ONC and i.o. injection of PBS or curdlan. (F) Quantification of Western blot band intensity relative to respective PBS-injected eyes. Compared with PBS-injected eyes, curdlan induces a significant increases in pERK and pSyk levels in WT and CARD9−/− eyes, but not in dectin-1−/− eyes. Curdlan significantly increases pCREB (S133) levels in WT eyes, but not in dectin-1−/− or CARD9−/− eyes. Curdlan does not increase phosphorylation of the NF-κB subunit p65 (S536) in any of the genotypes examined. Between three and five eyes from two separate experiments were analyzed for each condition and genotype. Data shown are mean ± SEM. **P < 0.01; *P < 0.05.
Fig. 4.
Fig. 4.
Dectin-1 is expressed on retina-resident and blood-derived myeloid cells. (AC) Flow cytometry analysis of dectin-1 expression in the eye (blue line), compared with an isotype control antibody (red line). (A and B) Histogram of dectin-1+ microglia and DCs in the eyes of naïve mice (A) and 7 d post-ONC in the absence of zymosan (B). (C) Analysis of dectin-1+ cells in the eye at 7 d post-ONC and i.o. zymosan. (D) Cross-section through the eye at 14 d post-ONC and i.o. zymosan stained with anti–dectin-1 (red) and DAPI (blue). Many dectin-1+ cells are found in the vitreous (4), but not in the retina, including the outer nuclear layer (1), the inner nuclear layer (2), or the RGC layer (3). (E) Anti–dectin-1 immunolabeling is not observed on GFAP+ retinal cells.
Fig. 5.
Fig. 5.
Dectin-1 expression is necessary on both radioresistant retinal cells and BM-derived infiltrating cells for curdlan-induced axon regeneration. Reciprocal BM chimeric mice were subjected to i.o. curdlan injection (5 µL, 25 µg/µL), and axon regeneration was assessed 2 wk later by anti-GAP43 labeling. The injury site in the optic nerve is marked with an asterisk. (Scale bar: 200 µm.) (A) WT mice that received WT donor BM (WT→WT) show robust curdlan-induced axon regeneration. (B–D) In contrast, WT mice that received dectin-1−/− BM (KO→WT) (B) show significantly less regeneration, comparable to that in dectin-1−/− mice that received WT BM (WT→KO) (C) and dectin-1−/− mice that received dectin-1−/− BM (KO→KO) (D). (E) Quantification of GAP43+ axons at 0.2–1.6 mm distal to the injury site (WT→WT + curdlan; n = 6 nerves, 6 mice; KO→WT + curdlan, n = 8 nerves, 5 mice; WT→KO + curdlan, n = 12 nerves, 8 mice; KO→KO + curdlan, n = 6 nerves, 4 mice). (F) Flow cytometry analysis of intraocular inflammation at 7 d after i.o. curdlan and ONC. Inflammation in WT→WT and WT→KO mice is comparable. Significantly decreased inflammation is observed in KO→WT and KO→KO mice. Results are presented as mean ± SEM. Asterisks indicate a significant difference from WT→WT mice. ***P < 0.001; **P < 0.01; *P < 0.05, one-way ANOVA, Tukey’s post hoc test.

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