Chemokine Receptor CCR2 Is Protective toward Outer Hair Cells in Chronic Suppurative Otitis Media

Abstract

Chronic suppurative otitis media (CSOM) is a neglected disease that afflicts 330 million people worldwide and is the most common cause of permanent hearing loss among children in the developing world. Previously, we discovered that outer hair cell (OHC) loss occurred in the basal turn of the cochlea and that macrophages are the major immune cells associated with OHC loss in CSOM. Macrophage-associated cytokines are upregulated. Specifically, CCL-2, an important member of the MCP family, is elevated over time following middle ear infection. CCR2 is a common receptor of the MCP family and the unique receptor of CCL2. CCR2 knockout mice (CCR2-/-) have been used extensively in studies of monocyte activation in neurodegenerative diseases. In the present study, we investigated the effect of CCR2 deletion on the cochlear immune response and OHC survival in CSOM. The OHC survival rate was 84 ± 12.5% in the basal turn of CCR2+/+ CSOM cochleae, compared with was 63 ± 19.9% in the basal turn of CCR2-/- CSOM cochleae (p ≤ 0.05). Macrophage numbers were significantly reduced in CCR2-/- CSOM cochleae compared with CCR2+/+ CSOM cochleae (p ≤ 0.001). In addition, CCL7 was upregulated, whereas IL-33 was downregulated, in CCR2-/- CSOM cochleae. Finally, the permeability of the blood-labyrinth barrier in the stria vascularis remained unchanged in CCR2-/- CSOM compared with CCR2+/+ CSOM. Taken together, the data suggest that CCR2 plays a protective role through cochlear macrophages in the CSOM cochlea.

FIGURE 1.

CCR2 deletion leads to increased OHC loss in CSOM. Representative whole-mount sections of the cochlea (basal, middle) in CSOM at 10 d from CCR2^+/+ CSOM (A and B) and CCR2^−/− (C and D) mice. Arrows show OHC loss. (E) The OHC survival rate as a percentage in the cochlear basal turn of both groups (*p = 0.036). Red, myosin VIIa. The number of mice per group is shown in brackets. Data are shown as mean ± SD. Scale bar, 100 μm.

FIGURE 2.

Macrophages are reduced in CCR2^−/− CSOM. (A–F) Cryosections of the cochlea (basal, middle, and apical turn) are shown in CCR2^+/+ CSOM (left) and CCR2^−/− CSOM (right) mice at 10 d following middle ear infection. (G) A significant decrease in the number of macrophages per turn (***p < 0.001). The number of mice per group is shown in parentheses. The data represent mean ± SD. Green, F4/80 (arrows). Scale bar, 100 µm.

FIGURE 3.

A broad range of cytokines are downregulated at 10 d in immunoassay analysis. The average expression ratio of CCR2^−/−/CCR2^+/+ in the left. Thirty-one of 44 target cytokines were downregulated, whereas 13 of 44 were upregulated, in CCR2^−/− cochleae. The dotted box shows the significant downregulation of IL-33 and upregulation of CCL7 in CCR2^−/− cochleae. Represents mean ± SD.

FIGURE 4.

The dextran-GFP leakage measurement. Dextran-GFP is shown in blood vessels surrounding the cochleae (arrows, A–C). The dextran-GFP was not visible inside the cochleae. Scale bar, 100 µm.