Fractalkine Signaling Regulates Macrophage Recruitment into the Cochlea and Promotes the Survival of Spiral Ganglion Neurons after Selective Hair Cell Lesion

Abstract

Macrophages are recruited into the cochlea in response to injury caused by acoustic trauma or ototoxicity, but the nature of the interaction between macrophages and the sensory structures of the inner ear remains unclear. The present study examined the role of fractalkine signaling in regulating the injury-evoked behavior of macrophages following the selective ablation of cochlear hair cells. We used a novel transgenic mouse model in which the human diphtheria toxin receptor (huDTR) is selectively expressed under the control of Pou4f3, a hair cell-specific transcription factor. Administration of diphtheria toxin (DT) to these mice resulted in nearly complete ablation of cochlear hair cells, with no evident pathology among supporting cells, spiral ganglion neurons, or cells of the cochlear lateral wall. Hair cell death led to an increase in macrophages associated with the sensory epithelium of the cochlea. Their numbers peaked at 14 days after DT and then declined at later survival times. Increased macrophages were also observed within the spiral ganglion, but their numbers remained elevated for (at least) 56 d after DT. To investigate the role of fractalkine signaling in macrophage recruitment, we crossed huDTR mice to a mouse line that lacks expression of the fractalkine receptor (CX3CR1). Disruption of fractalkine signaling reduced macrophage recruitment into both the sensory epithelium and spiral ganglion and also resulted in diminished survival of spiral ganglion neurons after hair cell death. Our results suggest a fractalkine-mediated interaction between macrophages and the neurons of the cochlea.

Significance statement: It is known that damage to the inner ear leads to recruitment of inflammatory cells (macrophages), but the chemical signals that initiate this recruitment and the functions of macrophages in the damaged ear are unclear. Here we show that fractalkine signaling regulates macrophage recruitment into the cochlea and also promotes the survival of cochlear afferents after selective hair cell lesion. Because these afferent neurons carry sound information from the cochlea to the auditory brainstem, their survival is a key determinant of the success of cochlear prosthetics. Our data suggest that fractalkine signaling in the cochlea is neuroprotective, and reveal a previously uncharacterized interaction between cells of the cochlea and the innate immune system.

Keywords: cochlea; fractalkine; hair cells; macrophages; neuroprotection; spiral ganglion neurons.

Figure 1.

Pou4f3^DTR/+ mice show extensive loss of hair cells after a single DT injection. A–A″, Cochlear whole mounts labeled for Myosin VIIa (red, hair cells) from Pou4f3^+/+ mice at 7 d following DT injection. Hair cells are intact in all the regions of the cochlea, suggesting that DT treatment does not affect the inner ear. B–B″, Myosin VIIa immunolabeling (red) of cochleae of Pou4f3^DTR/+ revealed no clear hair cell pathology at 1 d after DT. C–C″, Severe loss of hair cells was first evident in the cochleae of Pou4f3^DTR/+ mice at 3 d after DT injection. D–D″, Loss of cochlear hair cells in Pou4f3^DTR/+ mice was nearly complete at 7 d after DT injection. Note the complete ablation of hair cells in the apical and middle region of the cochlea of Pou4f3^DTR/+ mice, with some remnants of dead hair cells in the basal region. E, Quantification of the percentage of surviving hair cells in Pou4f3^DTR/+ mice at 1, 3, 7, and 14 d after DT injections (dpi). Complete loss of hair cells was observed at 14 d after DT. Data are mean ± SD; n = 5 or 6 cochleae per time point. ****p < 0.0001, 1 dpi versus all other time points at respective turn of the cochlea (two-way ANOVA followed by Tukey's multiple comparison post hoc tests). F, Plastic-embedded section from DT-treated Pou4f3 ^+/+ mouse cochlea (lower apical turn) displaying well-defined nuclei of inner and outer hair cells, and as nuclei of Dieter cells and inner and outer pillar cells. G, Plastic-embedded section from DT-treated Pou4f3 ^DTR/+ mouse cochlea (lower apical turn) showing the loss of both outer and inner hair cells (arrows). OHC, Outer hair cell; IHC, inner hair cell; D1–3, Deiter's cells; OPC, outer pillar cell; IPC, inner pillar cell. Scale bars: A–A‴, B–B‴, C–C‴, D–D‴, 30 μm; F–G, 55 μm.

Figure 2.

Cochlear supporting cells and SGNs are not affected by DT-mediated hair cell ablation. Top row, Cochlear whole mounts immunolabeled for SOX2 (green, supporting cells) from (A) Pou4f3^+/+ and Pou4f3^DTR/+ mice at (B) 7 d, (C) 14 d, and (D) 56 d after DT injection. We observed no evidence for the loss of supporting cells, despite the complete ablation of both inner and outer hair cells. Supporting cell nuclei change their shape and frequently redistribute after hair cell loss. E, Average number of SOX2-positive supporting cells at various times after DT injection. Bottom row, Mid-modiolar sections labeled for neurofilament and β-III tubulin (red, neurons) from (F) Pou4f3^+/+ and (G–I) Pou4f3^DTR/+ mice at 7, 14, and 56 d following DT injection. No loss of spiral ganglion cell bodies was evident at any survival time. J, Average SGN density from the middle region of the cochlea at various survival times. Data are mean ± SD; n = 4–6 per group. Scale bar, 30 μm.

Figure 3.

Loss of hair cells is sufficient to recruit macrophages toward the cochlear sensory epithelium. Pou4f3^+/+ (control) and Pou4f3^DTR/+ mice received a single DT injection, and cochleae were examined at 1, 3, 7, 14, 28, and 56 d recovery. A–D, Cochlear whole mounts showing GFP-labeled macrophages (green) and neurons (Neurofilament and β-III tubulin, red) demonstrate increased numbers of macrophages within the sensory regions of lesioned (e.g., Pou4f3^DTR/+) mice (B–D), compared with controls (A). Elevated macrophage numbers were first observed at 3 d after DT. E, The 3D renderings of confocal image stacks of lesioned cochleae show that macrophages (green) are typically located below the basilar membrane among the tympanic mesothelial cells and in the osseous spiral lamina. F, Quantification of macrophages per 100 μm of sensory epithelium. Macrophage numbers near the sensory epithelium peak at 14 dpi and diminish by 56 dpi. G, The area in the sensory epithelium (white box) of the cochlea from which macrophages were counted. H, I, The 3D renderings obtained from confocal image stacks of lesioned cochleae show that macrophages (green) are often in close contact with neurons (red) crossing the tunnel of Corti (white arrows) and extended processes toward hair cells (position shown with white arrowheads). Data are mean ± SD; n = 6 per group. dpi, days post injection; NF, neurofilament; IHC, inner hair cell; OHC; outer hair cell; ISB; inner spiral bundle; ToC; tunnel of Corti Statistical significance was computed using two-way ANOVA followed by Tukey's multiple comparison tests. **p < 0.01 (Pou4f3^+/+ vs Pou4f3^DTR/+ mice at 3 dpi). ***p < 0.0001 (Pou4f3^+/+ vs Pou4f3^DTR/+ mice at 7, 14, 28, and 56 dpi). ^#p < 0.001 (1 dpi vs all others). Scale bar, 30 μm.

Figure 4.

Loss of hair cells leads to increased numbers of macrophages in the spiral ganglion. Pou4f3^+/+ and Pou4f3^DTR/+ mice were injected with DT, and cochleae were examined at 1, 3, 7, 14, 28, and 56 d recovery. A, Mid-modiolar frozen section from a DT-treated Pou4f3^+/+ (control) mouse, showing GFP-expressing macrophages (green) and neurons (Neurofilament/TUJ-1, red). B–D, Comparable images taken from mid-modiolar sections of DT-treated Pou4f3^DTR/+ mice at various recovery times. E, Quantification of macrophage numbers per 1000 μm² of spiral ganglia (middle region of the cochlea) of control and Pou4f3^DTR/+ mice. Increased numbers of macrophages were observed in mice that had DT-induced hair cell lesions. Notably, macrophage numbers remained elevated as late as 56 d after DT. Data are mean ± SD; n = 3–7 per group. **p < 0.01, 1 dpi vs 7, 14, and 28 dpi (two-way ANOVA followed by Bonferroni's multiple comparisons post hoc tests). ***p < 0.001, 1 dpi vs 56 dpi (two-way ANOVA followed by Bonferroni's multiple comparisons post hoc tests). ^#p < 0.001, Pou4f3^+/+ and Pou4f3^DTR/+ mice at respective days after DT injection (two-way ANOVA followed by Bonferroni's multiple comparisons post hoc tests). dpi, days post DT injection. Scale bar, 30 μm.

Figure 5.

Fractalkine (CX₃CL1) is expressed in the adult mouse cochlea. A, Mid-modiolar section from mature C57BL/6 mouse immunolabeled for fractalkine (red, antibody against the chemotactic domain of CX₃CL1) and neurons (green) showing expression of CX₃CL1 in certain cell types within the sensory epithelium. B–D, Mid-modiolar section, showing the spiral ganglion and labeled for fractalkine (red) and neurons (green). Immunoreactivity for CX₃CL1 was observed on SGNs. E–G, Higher-magnification image reveals that CX₃CL1 immunoreactivity was confined to SGN somata, and was not present in surrounding Schwann and satellite cells. OHC, Outer hair cell; IHC, inner hair cell; D1–3, Dieter cell; OPC, outer pillar cell; IPC, inner pillar cell; BC, Boettcher cells. Scale bars: A, 55 μm; B–D, 20 μm; E, F, G, 15 μm.

Figure 6.

Genetic deletion of CX₃CR1 led to a reduction in macrophages near the sensory epithelium after hair cell ablation. The effects of CX₃CR1 on macrophage recruitment were assessed using mice of four genotypes: Pou4f3^DTR/+ and either heterozygous or homozygous for CX₃CR1 (Pou4f3^DTR/+:CX₃CR1^+/− and Pou4f3^DTR/+:CX₃CR1^−/−), and Pou4f3^+/+ and either heterozygous or homozygous for CX₃CR1 (Pou4f3^+/+:CX₃CR1^+/− and Pou4f3^+/+:CX₃CR1^−/−). Cochlear whole mounts and mid-modiolar sections were processed to image macrophages (CX3CR1^+/GFP, green) and neurons (neurofilament, red). We observed a significant reduction in the numbers of macrophages near the sensory region of the cochlea (B′) and in the spiral ganglion (E′) in CX₃CR1^−/− mice, compared with CX₃CR1^+/− mice (A′, D′) at both 14 d (top) and 56 d (bottom) after DT injection. C, F, G, Quantitative data on macrophages in sensory epithelium at 14 dpi (C) and in the spiral ganglia at 56 dpi (F). Quantification of macrophages within the spiral ganglia in the middle region of the cochlea at all time points after DT-induced hair cell lesion (G). Data are mean ± SD; n = 3–7 for 7 dpi per group; n = 6–7 for 14 dpi per group; n = 3–5 for 28 dpi per group; and n = 9–11 for 56 dpi per group. dpi, days post DT injection; NF, Neurofilament. Statistical significance was computed using two-way ANOVA followed by Tukey's multiple comparison tests. *p < 0.0001 (Pou4f3^+/+:CX₃CR1^+/− vs Pou4f3^DTR/+:CX₃CR1^+/−). ^#p < 0.0001 (Pou4f3^DTR/+:CX₃CR1^+/− vs Pou4f3^DTR/+:CX₃CR1^−/−). p < 0.05 (Pou4f3^+/+:CX₃CR1^−/− vs Pou4f3^DTR/+:CX₃CR1^−/− in C, for middle region of the cochlea in G, and at 56 d post DT in F. Scale bar, 30 μm.

Figure 7.

Genetic deletion of CX₃CR1 results in significant loss of SGNs after hair cell ablation. Cochlear mid-modiolar sections labeled for neurofilament (neurons, red) revealed significant loss of spiral ganglion cell bodies in the CX₃CR1^−/− mice (B′), compared with CX₃CR1^+/− (A′) mice, at 56 d after DT-induced hair cell lesion. We observed no evidence for SGN loss in Pou4f3^+/+ mice that were either CX₃CR1^−/− (B) or CX₃CR1^+/− (A). C, Quantitative data on SGN density (per 1000 μm²) for the four genotypes. D, E, Plastic sections from the basal cochlear turn of Pou4f3^+/+mice at 56 d after DT injection. Both CX₃CR1^+/− (D) and CX₃CR1^−/− (E) possessed normal numbers of SGNs. D′, E′, Plastic sections from the basal cochlear turn of Pou4f3 ^DTR/+ mice at 56 d after DT injection. Spiral ganglia of CX₃CR1^+/− mice (D′) contained normal numbers of auditory neurons after hair cell lesions, whereas the ganglia of CX₃CR1^−/− mice (E′) showed diminished neuronal survival. F, Density of SGNs (per 1000 μm²) from the basal cochlear turn at all time points after DT-induced hair cell lesion. Data are mean ± SD; n = 5–10 per group. dpi, days post-DT injection. Statistical significance was computed using two-way ANOVA followed by Tukey's multiple comparison post hoc tests. *p < 0.05. **p < 0.01. ***p < 0.001 (Pou4f3^DTR/+:CX₃CR1^+/− vs Pou4f3^DTR/+:CX₃CR1^−/−). ^#p < 0.05 (Pou4f3^+/+:CX₃CR1^−/− vs Pou4f3^DTR/+:CX₃CR1^−/− and Pou4f3^+/+:CX₃CR1^+/− vs Pou4f3^DTR/+:CX₃CR1^−/−). Scale bars: A, A′, B, B′, 30 μm; D, D′, E, E′, 10 μm.