Contribution of circulating monocytes in maintaining homeostasis of resident macrophages in postnatal and young adult mouse cochlea

Abstract

The percentage of macrophage subpopulations based on their origins in the adult cochlea remains unclear. This study aimed to elucidate the origins of cochlear macrophages during the onset phase and development of auditory function. We used three types of mice: wildtype ICR mice, colony-stimulating factor 1 receptor (Csf1r)-deficient mice, and Ms4a3Cre-Rosa tdTomato (Ms4a3^tdT) transgenic mice. Macrophages were labeled with ionized calcium-binding adapter molecule 1 (Iba1), which is specific to more mature macrophages, and CD11b, which is specific to monocyte lineage. We investigated the spatial and temporal distribution patterns of resident macrophages in the cochlea during the postnatal and early adult stages. During the adult stages, the rate of monocytes recruited from the systemic circulation increased; moreover, Iba1⁺/CD11b^- cochlear macrophages gradually decreased with age. Fate mapping of monocytes using Ms4a3^tdT transgenic mice revealed an increased proportion of bone marrow-derived cochlear macrophages in the adult stage. Contrastingly, the proportion of yolk sac- and fetal liver-derived tissue-resident macrophages decreased steadily with age. This heterogeneity could be attributed to differences in environmental niches within the tissue or at the sub-tissue levels. Future studies should investigate the role of cochlear macrophages in homeostasis, inflammation, and other diseases, including infection, autoimmune, and metabolic diseases.

Figure 1

Origin and renewal of tissue-resident macrophages, schematic representation of the origins and distribution of resident macrophages in embryonic and adult cochleae, and distribution of tissue macrophages in the cochlea. Two subtypes of resident macrophages (Mϕ) are present within the embryonic cochlea: Csf1r-dependent Mϕ, which originate from the YS, and Csf1r-independent Mϕ, which migrate from the FL via systemic circulation. A large proportion of the cochlear-resident Mϕ population was derived from the YS since Iba1-positive Mϕs reside in the mesenchyme surrounding the otocyst as early as E10.5. Csf1r-independent Mϕ expressing CD11b migrate as early as E14.5 and reside only in specific components of the cochlea. In the adult cochlea, the density of Mϕs expressing CD11b increases, suggesting that FL and BM contribute to the repopulation of cochlear-resident Mϕ. YS, yolk sac; FL, fetal liver; BM, bone marrow; Mϕ, macrophage; Csf1r, colony-stimulating factor 1 receptor; EMP, erythro-myeloid progenitor.

Figure 2

Immunostaining for Iba1 and CD11b and the time course of density in wildtype mice. (a) Density of Iba1 + cells at each stage. (b) Immunostaining of wildtype mice cochlea of Iba1 and CD11b antibodies at each stage. (Left) Whole cochlea section. (Right) Cochlea sections in the middle turn. Immunostaining for Iba1 and CD11b showed a rapid decrease from P0 to P14 followed by a gradual decrease from P14 to P30 in the number of Iba1⁺/CD11b⁻ cells. In contrast, there was an increase in the number of Iba1⁻/CD11b⁺ and Iba1⁺/CD11b⁺ cells with age. Red, Iba1; green, CD11b; blue, Hoechst nuclei; yellow, merged images. (c) Density at each stage. (d) Percentages of Iba1⁺/CD11b⁻, Iba1⁻/CD11b⁺, and Iba1⁺/CD11b⁺ cells at each stage. (e) Density in each organ at each stage. (f) Percentages of Iba1⁺/CD11b⁻, Iba1⁻/CD11b⁺, and Iba1⁺/CD11b⁺ cells in each organ at each stage. Red, Iba1; green, CD11b; yellow, double-positive cells. Asterisks represent statistical significance when p-values are < .05 (two-way ANOVA). Scale bars: 100 µm. Iba1, ionized calcium-binding adapter molecule 1. SG, spiral ganglion; SLi, spiral ligament, SV, stria vascularis; OC, organ of Corti.

Figure 3

Immunostaining for Iba1 and time course of density in Ms4a3Cre-Rosa tdTomato-transgenic mice. (a) Immunostaining of the Iba1 antibody and tdTomato reporter in Ms4a3Cre-Rosa tdTomato (Ms4a3^tdT) transgenic mice cochlea at each stage. (Left) Whole cochlea section. (Right) Cochlea sections in the middle turn. Fate mapping of monocytes labeled with Ms4a3 clearly revealed that the proportion of bone marrow-derived cochlear macrophages increased in the adult stage, whereas the proportion of yolk sac- and fetal liver-derived tissue-resident macrophages decreased steadily with age. Red, tdTomato; green, Iba1; blue, Hoechst nuclei; yellow, merged images. (b) Density at each stage. (c) Percentages of Iba1⁺/Ms4a3⁻, Iba1⁻/Ms4a3⁺, and Iba1⁺/Ms4a3⁺ cells at each stage. (d) Density in each organ at each stage. (e) Percentages of Iba1⁺/Ms4a3⁻, Iba1⁻/Ms4a3⁺, and Iba1⁺/Ms4a3⁺ cells in each organ at each stage. Red, tdTomato; green, Iba1; yellow, double-positive cells. Asterisks represent statistical significance when p-values are < .05 (two-way ANOVA). Scale bar: 100 µm. Iba1, ionized calcium-binding adapter molecule 1. SG, spiral ganglion; SLi, spiral ligament, SV, stria vascularis; OC, the organ of Corti.

Figure 4

Immunostaining for Iba1 and CD11b and the time course of density in Csf1r-deficient mice. (a) Immunostaining with Iba1 and CD11b antibodies in Csf1-deficient mice cochlea at each stage. (Left) Whole cochlea section. (Right) Cochlea sections in the middle turn. Hematopoiesis in the fetal liver and bone marrow contributes to the repopulation of cochlear macrophages with age despite depletion of yolk sac-derived macrophage in the cochlea of Csf1-deficient mice. Red, Iba1; green, CD11b; blue, Hoechst nuclei; yellow, merged images. (b) Density at each stage. (c) Percentages of Iba1⁺/CD11b⁻, Iba1⁻/CD11b⁺, and Iba1⁺/CD11b⁺ cells at each stage. (d) Density in each organ at each stage. (e) Percentages of Iba1⁺/CD11b⁻, Iba1⁻/CD11b⁺, and Iba1⁺/CD11b⁺ cells in each organ at each stage. Red, Iba1; green, CD11b; yellow, double-positive cells. Asterisks represent statistical significance when p-values are < .05 (two-way ANOVA). Scale bars: 100 µm. Csf1r, colony-stimulating factor 1 receptor; Csf1r-KO, Csf1r-deficient; Iba1, ionized calcium-binding adapter molecule 1; SG, spiral ganglion; SLi, spiral ligament, SV, stria vascularis; OC, the area beneath the organ of Corti.