Spiral ligament fibrocyte-derived MCP-1/CCL2 contributes to inner ear inflammation secondary to nontypeable H. influenzae-induced otitis media

Abstract

Background: Otitis media (OM), one of the most common pediatric infectious diseases, causes inner ear inflammation resulting in vertigo and sensorineural hearing loss. Previously, we showed that spiral ligament fibrocytes (SLFs) recognize OM pathogens and up-regulate chemokines. Here, we aim to determine a key molecule derived from SLFs, contributing to OM-induced inner ear inflammation.

Methods: Live NTHI was injected into the murine middle ear through the tympanic membrane, and histological analysis was performed after harvesting the temporal bones. Migration assays were conducted using the conditioned medium of NTHI-exposed SLFs with and without inhibition of MCP-1/CCL2 and CCR2. qRT-PCR analysis was performed to demonstrate a compensatory up-regulation of alternative genes induced by the targeting of MCP-1/CCL2 or CCR2.

Results: Transtympanic inoculation of live NTHI developed serous and purulent labyrinthitis after clearance of OM. THP-1 cells actively migrated and invaded the extracellular matrix in response to the conditioned medium of NTHI-exposed SLFs. This migratory activity was markedly inhibited by the viral CC chemokine inhibitor and the deficiency of MCP-1/CCL2, indicating that MCP-1/CCL2 is a main attractant of THP-1 cells among the SLF-derived molecules. We further demonstrated that CCR2 deficiency inhibits migration of monocyte-like cells in response to NTHI-induced SLF-derived molecules. Immunolabeling showed an increase in MCP-1/CCL2 expression in the cochlear lateral wall of the NTHI-inoculated group. Contrary to the in vitro data, deficiency of MCP-1/CCL2 or CCR2 did not inhibit OM-induced inner ear inflammation in vivo. We demonstrated that targeting MCP-1/CCL2 enhances NTHI-induced up-regulation of MCP-2/CCL8 in SLFs and up-regulates the basal expression of CCR2 in the splenocytes. We also found that targeting CCR2 enhances NTHI-induced up-regulation of MCP-1/CCL2 in SLFs.

Conclusions: Taken together, we suggest that NTHI-induced SLF-derived MCP-1/CCL2 is a key molecule contributing to inner ear inflammation through CCR2-mediated recruitment of monocytes. However, deficiency of MCP-1/CCL2 or CCR2 alone was limited to inhibit OM-induced inner ear inflammation due to compensation of alternative genes.

Figure 1

Inner ear inflammation secondary to NTHI-induced middle ear infection. Live NTHI was inoculated into the murine middle ear through the tympanic membrane and temporal bones were harvested at 7 days after bacterial inoculation. H & E staining shows that middle ear infection results in serous labyrinthitis (A) and purulent labyrinthitis (B). Note accumulation of serous substances with hemorrhage (arrow) and massive infiltration of inflammatory cells (arrowhead) in the cochlear spaces. Original magnification: ×50.

Figure 2

Attraction of THP-1 cells by SLFs-derived molecules. The culture medium of SLFs was collected after 6, 12 and 24 h with or without exposure to the NTHI lysate, respectively. Migration assays show that the conditioned medium of NTHI-exposed SLFs highly attracts THP-1 cells compared to that of NTHI-unexposed SLFs (A). RFU: relative fluorescence unit. Invasion assays show that NTHI-induced SLF-derived molecules enhance invasion of THP-1 cells (dark and solid) to the extracellular matrix on the semi-permeable membrane with pores (light and open) (B). It is noted that NTHI lysate per se does not affect migration and invasion of THP-1 cells, compared to the NTHI-induced SLF-derived molecules (C). *: p < 0.05. The experiments were performed in triplicate and repeated twice. Values are given as the mean ± standard deviation (n = 3).

Figure 3

Identification of the main molecule attracting THP-1 cells among NTHI-induced molecules derived from SLFs. Viral CC chemokine inhibitor (CCI) blocks migration of THP-1 cells responding to the NTHI-induced SLF-derived molecules (A). Migration assays were performed using THP-1 cells and the conditioned medium of NTHI-exposed SLFs with and without treatment of CCI (0.02, 0.1 and 0.5 μg/ml) for 12 h and 36 h. Silencing of MCP-1/CCL2 inhibits migration of THP-1 cells attracted by the conditioned medium of NTHI-exposed SLFs, which is restored by addition of the recombinant MCP-1/CCL2 (rMCP-1, 0.1 and 1 ng/ml) (B). SLFs were transfected with either non-specific siRNA (NC) or MCP-1-specific siRNA (MCP-1) and culture medium was collected after 12 h with and without exposure to the NTHI lysate. Results were expressed relative to the fold change of mRNA levels, taking the value of the NC siRNA-treated group as 1. THP-1 cell migration in response to the conditioned medium of NTHI-exposed SLFs is inhibited more than 60% by MCP-1/CCL2 deficiency, but not by TNF-α deficiency (C). Primary SLFs were cultured in the lower compartment of the migration chamber and were exposed to the NTHI lysate. THP-1 cells were co-cultured in the upper compartment, and migrated cells to the lower chamber were quantitated using a hemocytometer. The experiments were performed in triplicate and repeated twice. Values are given as the mean ± standard deviation (n = 3). *: p < 0.05.

Figure 4

SLF-derived MCP-1/CCL2 is associated with inner ear inflammation secondary to NTHI-induced OM in vivo. Murine temporal bones were harvested at 7 days after transtympanic inoculation of saline (A) and live NTHI (B). Immunolabeling shows MCP-1/CCL2 is highly expressed in the spiral ligaments (white arrows) and spiral limbus as well as the serous substances (black arrow) of the NTHI-inoculated mice. In contrast, it is noted that the cochlea of the saline-inoculated group weakly expresses MCP-1/CCL2 except the stria vascularis (arrowheads). Original magnification: ×50.

Figure 5

Requirement of CCR2 for migration of monocytes in response to NTHI-induced SLF-derived molecules. Migration assays show that the CCR2 inhibitor (RS 102895) markedly inhibits migration of THP-1 cells attracted by NTHI-induced SLF-derived molecules, in a dose-dependent manner (A). Giemsa staining of the migrated splenocytes shows that CCR2 deficiency inhibits migration of monocyte-like cells in response to the NTHI-induced SLF-derived molecules (B).

Figure 6

Deficiency of MCP-1/CCL2 and CCR2 affects NTHI-induced regulation of other CC chemokines. qRT-PCR shows that NTHI-induced up-regulation of MCP-2/CCL8 is markedly enhanced in SLFs of MCP-1^-/- mice compared to the wild type mouse (A). It is noted that the regulation of MCP-3/CCL7 is not affected. CCR2 deficiency enhances NTHI-induced up-regulation of MCP-1/CCL2, but not MCP-2/CCL8 (B). Primary SLFs derived from the wild type, MCP-1-deficient and CCR2-deficient mice were exposed to the NTHI lysate for 3 h. Results were expressed relative to the fold change of mRNA levels, taking the value of the non-treated group as 1. MCP-1/CCL2 deficiency up-regulates CCR2 expression in the splenocytes, but CCR1 expression is not affected (C). mRNA levels of CCR1 and CCR2 were relatively shown, taking the CCR2 level of the wild type splenocytes as 1. *: p < 0.05. The experiments were performed in triplicate and repeated three times. Values are given as the mean ± standard deviation.