Exosomes mediate sensory hair cell protection in the inner ear
Andrew M. Breglio, … , Matthew J.A. Wood, Lisa L. Cunningham
Andrew M. Breglio, … , Matthew J.A. Wood, Lisa L. Cunningham
Published February 6, 2020
Citation Information: J Clin Invest. 2020;130(5):2657-2672. https://doi.org/10.1172/JCI128867.
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Research Article Cell biology Neuroscience

Exosomes mediate sensory hair cell protection in the inner ear

Abstract

Hair cells, the mechanosensory receptors of the inner ear, are responsible for hearing and balance. Hair cell death and consequent hearing loss are common results of treatment with ototoxic drugs, including the widely used aminoglycoside antibiotics. Induction of heat shock proteins (HSPs) confers protection against aminoglycoside-induced hair cell death via paracrine signaling that requires extracellular heat shock 70-kDa protein (HSP70). We investigated the mechanisms underlying this non–cell-autonomous protective signaling in the inner ear. In response to heat stress, inner ear tissue releases exosomes that carry HSP70 in addition to canonical exosome markers and other proteins. Isolated exosomes from heat-shocked utricles were sufficient to improve survival of hair cells exposed to the aminoglycoside antibiotic neomycin, whereas inhibition or depletion of exosomes from the extracellular environment abolished the protective effect of heat shock. Hair cell–specific expression of the known HSP70 receptor TLR4 was required for the protective effect of exosomes, and exosomal HSP70 interacted with TLR4 on hair cells. Our results indicate that exosomes are a previously undescribed mechanism of intercellular communication in the inner ear that can mediate nonautonomous hair cell survival. Exosomes may hold potential as nanocarriers for delivery of therapeutics against hearing loss.

Authors

Andrew M. Breglio, Lindsey A. May, Melanie Barzik, Nora C. Welsh, Shimon P. Francis, Tucker Q. Costain, Lizhen Wang, D. Eric Anderson, Ronald S. Petralia, Ya-Xian Wang, Thomas B. Friedman, Matthew J.A. Wood, Lisa L. Cunningham

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Figure 8

The protective effect of exosomes requires interaction of exosomal HSP70 with TLR4 on hair cells.

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The protective effect of exosomes requires interaction of exosomal HSP70...
(A) Exosomes improved hair cell survival in neomycin-exposed utricles from control (WT) littermates (red) but not from hair cell–specific TLR4-cKO mice (blue). Data indicate the mean ± SEM (n = 12 utricles per condition). **P < 0.01 and ****P < 0.0001, by 2-way ANOVA with Holm-Šídák multiple comparisons test. (B and C) A PLA was performed to detect interaction between exosomal HSP70 and HSP40 or HSP70 and TLR4. (B) Two different fbAbs against HSP70 (HS) abolished the interaction between HSP70 and TLR4, whereas IgG had no effect. SC, Santa Cruz Biotechnology; TF, Thermo Fisher Scientific. (C) Heat shock increased the interaction between HSP70 and HSP40 and between HSP70 and TLR4 in WT utricles. Hair cell–specific deletion of TLR4 abolished the PLA signal in heat-shocked utricles from TLR4-cKO mice. Data in B and C indicate the mean ± SEM and are shown as the average number of puncta per 1000 μm2 (n = 4–12 utricles per condition). **P < 0.01 and ***P < 0.001, by Brown-Forsythe and Welsh ANOVA followed by Dunnett’s T3 multiple comparisons test. (D–G) Confocal images of PLA signals in utricles from WT (D–F) or TLR4-cKO (G) mice under control or heat shock conditions. Top row, F-actin (green) and PLA signal (white); bottom row, PLA signal only (white). (D) Negative control (no primary Ab). (E) Heat shock induced HSP70 interaction with HSP40 in WT utricles. (F) Heat shock increased HSP70 interaction with TLR4 in WT utricles. HSP70 fbAbs inhibited interaction between HSP70 and TLR4 in WT utricles, independently of heat shock, whereas control IgG had no effect. (G) Hair cell–specific deletion of TLR4 abolished the HSP70-TLR4 interaction in TLR4-cKO mice. Scale bar: 20 μm.