Two cell populations participate in clearance of damaged hair cells from the sensory epithelia of the inner ear

Abstract

The cochlea and the vestibular organs are populated by resident macrophages, but their role in inner ear maintenance and pathology is not entirely clear. Resident macrophages in other organs are responsible for phagocytosis of injured or infected cells, and it is likely that macrophages in the inner ear serve a similar role. Hair cell injury causes macrophages to accumulate within proximity of damaged regions of the inner ear, either by exiting the vasculature and entering the labyrinth or by the resident macrophages reorganizing themselves through local movement to the areas of injury. Direct evidence for macrophage engulfment of apoptotic hair cells has been observed in several conditions. Here, we review evidence for phagocytosis of damaged hair cells in the sensory epithelium by tissue macrophages in the published literature and in some new experiments that are presented here as original work. Several studies also suggest that macrophages are not the only phaogocytic cells in the inner ear, but that supporting cells of the sensory epithelium also play an important role in debris clearance. We describe the various ways in which the sensory epithelia of the inner ear are adapted to eliminate damaged and dying cells. A collaborative effort between resident and migratory macrophages as well as neighboring supporting cells results in the rapid and efficient clearance of cellular debris, even in cases where hair cell loss is rapid and complete.

Keywords: Hair cell; Inflammation; Macrophage; Phagocytosis; Supporting cell.

Figure 1

E10 mouse embryo. Macrophages are observed adjacent to the otic vesicle at embryonic day 10 suggesting that macrophages may play a significant role during this period of inner ear development. Selection and elimination of unwanted cells may be undertaken by these local tissue macrophages. Green: CX3CR1^GFP(macrophages), Red: phalloidin (f-actin). Scale bar: 50μm.

Figure 2

Schematic figure shows the typical location of macrophages in the mammalian cochlea. A. Control mouse cochlea shows tissue macrophages in the spiral ligament and ganglion in the resting ear. B. Cochlear macrophages migrate from the vasculature after noise exposure. Mononuclear phagocytes typically accumulate in large numbers in the spiral ligament, spiral limbus, and in the spiral ganglion. They also enter the fluid-filled scalae and assume a rounded and less ramified shape when they migrate out of the spiral ligament.

Figure 3

Macrophages reach into the sensory epithelium in situ. Macrophage extending a process from the osseous spiral lamina through the habenula perforata towards the inner hair cell. The cochlea was fixed in vivo and then sectioned for light microscopy. Green: CX3CR1^GFP (macrophage), Red: CD36 (scavenger receptor B). Scale bar: 10μm.

Figure 4

Time-lapse images from newborn mouse cochlear cultures. A–A′″. Still images from time-lapse experiments highlight processes and displacement of cochlear macrophages. Mouse cochlear cultures were treated with kanamycin and hair cells were identified with FM464. Macrophages (white) are observed among the spiral ganglion neurons (magenta), locating injured hair cells and extending processes to engulf them. Arrows demonstrate processes extending around hair cells. Scale bar: 50μm. B–B′″. Organotypic cultures harvested from CD36 null, CX3CR1^GFP reporter mice. CD36 null macrophages were mobile, identified damaged hair cells readily and were active in phagocytosis. Arrows demonstrate processes surrounding and engulfing hair cells. Red: FM464 (hair cells), green: CX3CR1^GFP (macrophages). C. Example of cell length measurement in macrophages in culture. D. Representative image of technique used to assess macrophage movement: all pixels that corresponded to individual macrophages were identified and followed over time. Displacement of macrophages in CD36KO mice were not different from displacement of CD36 WT mice (data not shown). E. Numbers of macrophage types in CD36KO and CD36WT mice, where “movers” freely displaced greater than the length of a cell soma with X minutes, “shakers” extended processes while remaining in one location, and “eaters” consumed red material. There was no significant difference in numbers of movers, shakers, or eaters observed in CD36KO mice when compared to WT mice (two-tailed T test, n=220 macrophages assessed, compilations of videos from 8 preparations of CD36KO and 7 from CD36WT cochleae). Abbreviations: CD36KO: CD36 null mice, CD36WT: CD36 wild type mice.

Figure 5

Confocal images of cochlear macrophages, whole mount preparation. A. Control mouse organotypic cochlear culture: macrophages are concentrated among the spiral ganglion neurons. Some macrophages extend processes towards the hair cells along the afferent neurons (arrow). Red: FM464 (hair cells), Green: CX3CR1-GFP (macrophages), Magenta: thy-1 (neurons). B. Kanamycin-treated organotypic cultures with internalization of hair cell debris. Red: FM464 (hair cells), Green: CX3CR1-GFP (macrophages). C. Fixed whole organ preparation: 48 hours after kanamycin, hair cells are mostly absent, and hair cell debris is visible within the phagosomes of macrophages. Red: FM464 (hair cells), Green: CX3CR1-GFP (macrophages). Scale bar: 50μm.

Figure 6

Dying cells exhibit surface molecules that are identified as “find me” cues and summon macrophages to target these damaged cells. Other leukocytes, such as neutrophils are repelled from these dying cells in order to limit resultant inflammation and adjacent injury. Other molecules then signal to the macrophage that the cell is suitable for digestion by exhibiting “eat me” cues. From (Munoz et al., 2010).

Figure 7

Macrophage response to hair cell injury in the chick basilar papilla. A sizable population of macrophages resides in the hyaline/cuboidal cell region of the papilla, which adjoins the inferior (abneural) border of the sensory epithelium (left). Hair cell injury leads to an apparent redistribution of these macrophages, which migrate toward the sensory region (right). Such macrophages remain below the basilar membrane and are normally not observed within the sensory epithelium. Labels: Green: KUL01 (macrophages); red: phalloidin (f-actin). Modified from: ME Warchol, RA Schwendener, K Hirose, PLoS One: 7(12): e51574 (2012).

Figure 8

Macrophage activity in response to hair cell injury in lateral line neuromasts of larval zebrafish. A: Macrophages (green, YFP) are present in the near vicinity of uninjured neuromasts. Image shows the distal-most neuromasts (P9-11) of the posterior lateral line of a 7 dpf zebrafish. Red: HCS-1 immunoreactivity (hair cells). B: Neomycin-induced hair cell death causes nearby macrophages to enter the injured neuromasts. Note that the number of macrophages near each neuromasts appears nearly unchanged. C: Quantification of macrophages located near lateral line neuromasts at various time points after ototoxic injury. Fish were incubated for 30 min in 50 μM neomycin and then fixed at 0, 1, 2 or 24 hr recovery. Following fixation and immunoprocessing, the number of YFP-labeled macrophages within a 25μm radius of neuromasts P9, P10 and P11 was quantified. The numbers of macrophages associated with the lesioned neuromasts were relatively constant at all recovery times and also unchanged from the numbers near unlesioned (control) fish (n=10 fish/time point; mean±SD). D: Time-lapse imaging reveals macrophage phagocytosis of dying hair cells. Hair cells were loaded with FM464 (red) and zebrafish were incubated in neomycin. Fish were then anesthetized and imaged by time-lapse microscopy. Time stamps on each image refer to elapsed time after initiation of time-lapse recording. At early times (1 min, 19 min), cup-shaped macrophage phagosomes were observed in contact with hair cell debris (arrows, red). Such debris was then internalized and consolidated within the macrophage cytoplasm (arrowheads, red). Such observations confirm that macrophages actively participate in the removal of dying hair cells in zebrafish.