JARO

JARO (2014) DOI: 10.1007/s10162-014-0491-7 D 2014 Association for Research in Otolaryngology (outside the USA)

Research Article

Journal of the Association for Research in Otolaryngology

Heat Shock Protein-Mediated Protection Against Cisplatin-Induced Hair Cell Death TIFFANY G. BAKER,1 SOUMEN ROY,2 CARLENE S. BRANDON,1 INGA K. KRAMARENKO,1 SHIMON P. FRANCIS,1 MONA TALEB,1 KEELY M. MARSHALL,1 RETO SCHWENDENER,3 FU-SHING LEE,4 AND LISA L. CUNNINGHAM2 1

Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 165 Ashley Ave, Charleston, SC 29425, USA

2

National Institute on Deafness and other Communication Disorders, National Institutes of Health, 35A Convent Drive, Bethesda, MD 20892, USA 3 Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland 4

Department of Otolaryngology-Head and Neck Surgery, Medical University of South Carolina, 165 Ashley Ave, Charleston, SC 29425, USA

Received: 11 July 2014; Accepted: 16 September 2014

ABSTRACT Cisplatin is a highly successful and widely used chemotherapy for the treatment of various solid malignancies in both adult and pediatric patients. Side effects of cisplatin treatment include nephrotoxicity and ototoxicity. Cisplatin ototoxicity results from damage to and death of cells in the inner ear, including sensory hair cells. We showed previously that heat shock inhibits cisplatin-induced hair cell death in whole-organ cultures of utricles from adult mice. Since heat shock protein 70 (HSP70) is the most upregulated HSP in response to heat shock, we investigated the role of HSP70 as a potential protectant against cisplatin-induced hair cell death. Our data using utricles from HSP70−/− mice indicate that HSP70 is necessary for the protective effect of heat shock against cisplatin-induced hair cell death. In addition, constitutive expression of inducible HSP70 offered modest protection against cisplatin-induced hair cell death. We also examined a second heat-inducible protein, heme oxygenase-1 (HO-1, also called HSP32). HO-1 is an enzyme responsible for the catabolism of free heme. We previously showed that induction of HO-1 using cobalt protoporphyrin IX (CoPPIX) inhibits aminoglycoside-induced hair cell death. Here, we show that HO-1 also offers significant protection against cisplatin-induced hair cell death. HO-1 inducCorrespondence to: Lisa Cunningham & National Institute on Deafness and other Communication Disorders & National Institutes of Health & 35A Convent Drive, Bethesda, MD 20892, USA. Telephone: 301-4432766; email: [email protected]

tion occurred primarily in resident macrophages, with no detectable expression in hair cells or supporting cells. Depletion of macrophages from utricles abolished the protective effect of HO-1 induction. Together, our data indicate that HSP induction protects against cisplatin-induced hair cell death, and they suggest that resident macrophages mediate the protective effect of HO-1 induction. Keywords: ototoxicity, HSPs, cisplatin, utricles

INTRODUCTION Cisplatin causes significant permanent hearing loss in 20–80 % of treated patients (Fausti et al. 1994; Knight et al. 2005; Coradini et al. 2007; Lewis et al. 2009, reviewed in Neuwelt and Brock 2010). Risk factors that increase the likelihood of cisplatin-induced hearing loss include patient age, gender, cumulative cisplatin dose, certain genetic alleles, and radiation to the base of the skull (Schaefer et al. 1985; Schell et al. 1989; Li et al. 2004; Huang et al. 2007; Caronia et al. 2009; Ross et al. 2009; Yancey et al. 2012). As cisplatininduced hearing loss is irreversible, it is imperative that therapies become available to prevent this serious and currently unavoidable side effect. The heat shock protein (HSP) family includes both constitutive and inducible proteins; the constitutive forms are involved in a variety of critical cell functions, whereas the inducible forms are transcrip-

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tionally upregulated in response to stress. Many HSPs act as molecular chaperones, promoting the proper folding, transport, subcellular localization, and activity of client proteins. Nonlethal heat shock preconditioning results in upregulation of inducible HSP family members, which are protective against a wide variety of cellular stresses (Martindale and Holbrook 2002; Richter et al. 2010; Rampelt et al. 2012). Previously, we showed that heat shock inhibits both cisplatin- and aminoglycoside-induced hair cell death (Cunningham and Brandon 2006). In addition to heat shock, noise exposure has also been shown to induce HSPs in the inner ear (Matsunobu et al. 2009; Roy et al. 2013). HSP70 is the most ubiquitous and evolutionarily conserved member of the HSP family (Karlin and Brocchieri 1998; Sharma and Masison 2009). At least 13 HSP70 genes exist in mice. Most of these HSP70s are constitutively expressed and function as molecular chaperones that regulate the folding and trafficking of client proteins in the cytoplasm and other organelles. Two of these HSP70 genes (Hspa1a and Hspa1b) are rapidly induced in response to a variety of cellular stresses. Stress-induced HSP70 inhibits apoptotic cell death in a large number of systems. HSP70 inhibits multiple pro-apoptotic signals, including oligomerization of Bax, release of both cytochrome c and second mitochondria-derived activator of caspases (Smac) from mitochondria, formation of a functional apoptosome, and even cell death subsequent to caspase 3 activation (Jaattela et al. 1998; Beere et al. 2000; Tsuchiya et al. 2003; Stankiewicz et al. 2005; Jiang et al. 2009; Evans et al. 2010). Moreover, HSP70 inhibits aminoglycosideinduced hair cell death in vitro and hearing loss in vivo (Taleb et al. 2008, 2009; May et al. 2013). Our previous data indicate that HSP70 is the most highly induced HSP in response to heat shock in whole-organ cultures of utricles from mature mice (Cunningham and Brandon 2006; Taleb et al. 2008; Francis et al. 2011; May et al. 2013). Our more recent studies indicate that supporting cells mediate the protective effect of HSP70 induction by secreting HSP70 (May et al. 2013). Another HSP with the potential to protect against cisplatin ototoxicity is heme oxygenase-1 (HO-1, also called HSP32). Like other inducible heat shock proteins, HO-1 is upregulated in response to a variety of stressors, including heat shock (Shibahara et al. 1987; Fairfield et al. 2004; Wegiel et al. 2013). However, unlike some HSPs, HO-1 does not have chaperone activity. Instead HO-1 is an enzyme responsible for heme catabolism, the products of which include bilirubin, carbon monoxide (CO), and free iron (Tenhunen et al. 1968, 1969; Wegiel et al. 2013). Bilirubin and CO each have antioxidant and antiinflammatory properties (Stocker et al. 1987; Hayashi et al. 1999; Otterbein et al. 2000; Kirkby and Adin 2006; Wegiel et al. 2013). Pharmacological induction

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of HO-1 is protective against multiple stresses in many tissue types, including ischemia-reperfusion injury in liver and retina (Tsuchihashi et al. 2007; Sun et al. 2010). Several studies have demonstrated that inducers of HO-1 protect against cisplatin-induced death in the HEI-OC1 auditory cell line (Kim et al. 2006a, 2009; So et al. 2006, 2008; Choi et al. 2007, 2008, 2011; Gao et al. 2010). We have shown that HO1 induction inhibits hearing loss and hair cell death caused by aminoglycoside antibiotics (Francis et al. 2011). HO-1 also protects neonatal rat cochlear explants from cisplatin-induced hair cell death (Kim et al. 2006a, 2009). In addition, ebselen, an HO-1 inducer, modestly reduces hearing loss in mice treated with cisplatin (Kim et al. 2009). The current study was designed to examine the protective effects of HSP70 and HO-1 against cisplatin-induced hair cell death. In addition, we examined the mechanism(s) underlying the protective effect of HO-1.

MATERIALS AND METHODS Animals All mice were maintained in the central animal care facility at the Medical University of South Carolina (Charleston, SC, USA) or at the NIDCD Division of Intramural Research animal care facility. Mice were euthanized via carbon dioxide asphyxiation and then decapitated. All animal protocols were approved by the MUSC Institutional Animal Care and Use Committee or by the NIDCD Animal Care and Use Committee.

CBA/J Mice Adult CBA/J mice (4 to 6 weeks old) were obtained from Harlan Laboratories, Inc. (Indianapolis, IN, USA).

C57Bl/6J Mice Adult C57Bl/6J mice (4–6 weeks old) were obtained from The Jackson Laboratory (Bar Harbor, ME).

HSP70 Knockout Mice HSP70.1/3−/− mice have inactivated Hspa1b (a.k.a. Hsp70.1) and Hspa1a (a.k.a. Hsp70.3) genes due to the insertion of a single neo gene on chromosome 17 (Hunt et al. 2004). These mice are viable and fertile; however, they exhibit increased susceptibility to a variety of stresses, including cardiac ischemia (Hunt et al. 2004; Kim et al. 2006b). Mating pairs were obtained from the Mutant Mouse Regional Resource Center at the University of California at Davis and

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consisted of male and female HSP70.1/3−/− mice. Since all offspring were knockouts, wild-type B6129SF2/J mice, obtained from the Jackson Laboratory, were used as strain controls.

rHSP70i CE Mice Transgenic mice that constitutively express rHSP70i (rHSP70i CE) were the kind gift of Dr. Wolfgang Dillmann (University of California San Diego). rHSP70i CE mice constitutively express rat inducible HSP70 (rHSP70i) under the control of the chicken βactin promoter (Marber et al. 1995). rHSP70i CE mice are on a BALB/c×C57Bl/6 background. Wild-type female CB6F1 mice (The Jackson Laboratory) were mated with male rHSP70i transgenic mice, resulting in litters consisting of ~50 % transgenic and ~50 % wild-type mice. Wild-type littermates served as controls. Genotyping was performed as previously described (Taleb et al. 2008).

CX3CR1GFP/+ Mice CX3CR1 is a seven-transmembrane G-proteincoupled receptor for the chemokine fractalkine which is expressed by monocytic, natural killer, dendritic, and microglial cells in mice (Jung et al. 2000). CX3CR1GFP/GFP mice have an EGFP gene in place of the CX3CR1 gene; therefore, they express GFP instead of CX3CR1 (Jung et al. 2000). Mice with GFP in place of both CX3CR1 alleles express no CX3CR1 protein (Jung et al. 2000). CX3CR1GFP/GFP mice exhibit normal development and fertility. CX3CR1GFP/GFP male mice were acquired from The Jackson Laboratory and bred with C57Bl/6 females to produce CX3CR1GFP/+ mice. These heterozygous mice have been used in previous studies of inner ear macrophages, which indicated that CX3CR1GFP/+ mice have normal macrophage function in the inner ear and express GFP exclusively in macrophages (Hirose et al. 2005; Sautter et al. 2006; Sato et al. 2008, 2010).

Utricle Culture Culture of utricles from adult mice has been previously described (Cunningham 2006; Brandon et al. 2012). Culture media consisted of either BME/EBSS or DMEM/F12 supplemented with 5 % fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 50 U/mL penicillin G. Utricles were maintained in an incubator at 37 °C in a 5 % CO2/95 % air environment. Cisplatin was supplied as a 1 mg/mL stock solution (Teva Parenteral Medicine, Inc., Irvine, CA) and diluted in culture medium. Final cisplatin concentrations ranged from 10 to 60 μg/mL. Cisplatin was not

added to untreated control cultures. Heat shock was performed by transferring utricles and surrounding media from each relevant treatment well to microcentrifuge tubes and placed in a 43 °C water bath for 30 min, after which the utricles were returned to a 37 °C incubator and their respective culture wells (Cunningham and Brandon 2006). Utricles that were not heat shocked were transferred to tubes, but they remained at 37 °C for the 30-min time period in order to serve as heat shock controls. Cisplatin was added to utricle cultures 6 h after the utricles were removed from the 43 °C water bath. The HO-1 inducer Co (III) protoporphyrin IX chloride (CoPPIX) was provided in powder form (Frontier Scientific, Inc., Logan, UT, USA). A 1-mM CoPPIX solution was prepared by dissolving the powder in 0.2 M NaOH, lowering the pH below 8 with 1 M HCl, and bringing the solution to volume with 1× phosphate-buffered saline (PBS). The 1-mM solution of CoPPIX was then filter sterilized with a 0.22-μm filter. The CoPPIX stock solution was further diluted in culture medium resulting in final concentrations of 10 or 20 μM. CoPPIX-treated utricles were cultured in CoPPIX for 12 h (Kim et al. 2006a). Utricles treated with CoPPIX alone were returned to culture media following CoPPIX incubation. For utricles undergoing both CoPPIX and cisplatin treatments, the CoPPIX media was replaced with cisplatin-containing culture media and incubated in cisplatin for 24 h. The HO-1 inhibitor Zn (II) protoporphyrin IX (ZnPPIX) was provided in powder form (Frontier Scientific, Inc.) and prepared as previously described (Francis et al. 2011). Briefly, ZnPPIX was dissolved with DMSO and 0.2 M NaOH to make a 50-mM stock solution. The stock solution was diluted to 100 μM in culture medium and filter sterilized. Liposomal clodronate (LC) was made as previously described (Seiler et al. 1997) and stored at −80 °C. LC or control liposomes were thawed and added to culture medium for incubation with utricles from CX3CR1GFP/+ mice. Utricles were treated with 0, 0.1, 0.5, 1, 2, 4, or 8 mM LC for 48 h. Confocal z-stack images were acquired for each utricle and macrophage counts were performed on maximum intensity projection images from each z-stack.

Immunofluorescence Immunofluorescence was performed similarly to previously described protocols (Shibahara et al. 1987; Cunningham and Brandon 2006). Utricles were fixed in 4 % paraformaldehyde overnight at 4 °C. They were then washed in 0.1 M Sorenson’s phosphate buffer (SPB). Otoconia were dissolved by incubating utricles in Cal-Ex decalcifying solution for 2 min (Fisher Scientific, Fair Lawn, NJ, USA). Otoconia

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removal was followed by multiple washes in 0.1 M SPB. Utricles were then incubated in 0.1 M sodium borohydride followed by multiple washes in 0.1 M SPB. Utricles were incubated in blocking solution (2 % bovine serum albumin, 0.8 % normal goat serum, 0.4 % Triton X-100 in 1× phosphate buffered saline (PBS)) at room temperature for 3 h. Utricles were incubated in primary antibodies diluted in blocking solution overnight at 4 °C. Primary antibodies included mouse monoclonal anti-calmodulin (Sigma C3545, St. Louis, MO, USA; 1:150), mouse monoclonal anti-myosin-VIIa (Developmental Studies Hybridoma Bank, MYO7A 138-1, University of Iowa, Iowa City, USA; 1:250), rabbit polyclonal anti-myosin 7a (Proteus Biosciences 25-6790, Ramona, CA, USA; 1:100), rabbit polyclonal anti-HO-1 (Hsp32 Enzo ADISPA-894-D, Farmingdale, NY 11735, USA; 1:250), and rat monoclonal anti-mouse CD68: Alexa Fluor 488 (MCA1957A488T, AbD Serotec, Bio-Rad, Kidlington Oxford OX5 1GE; 1:300). Following primary antibody incubation, utricles were washed with 1× PBS prior to incubation with secondary antibodies: Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen A11001; 1:500), Alexa Fluor 647-conjugated goat anti-mouse IgG (Invitrogen A21236, 1:500), and Alexa Fluor 594-conjugated goat anti-rabbit IgG (Invitrogen A11012; 1:500). Utricles were incubated in secondary antibody (diluted in blocking solution) for 3 h in the dark at RT. After three washes in PBS, utricles were mounted on glass slides using Fluoromount G (Southern Biotech, Birmingham, AL, USA). Utricles were visualized using a Zeiss Axioplan 2 fluorescent microscope and a monochrome digital camera (Zeiss Axiocam MR). Imaging software was used for hair cell counts (AxioVision 40 V 4.6.3.0). Hair cells were counted in each of five 900 μm2 areas. Cell counts from the five regions were averaged. Hair cell density is reported as the mean number of hair cells per unit area (±SEM) for each utricle. Confocal images were acquired using a Zeiss 780 Laser-scanning confocal microscope. 3D reconstructions of the utricles were made by using Volocity (×64) 3D image analysis software (Version: 6.3 PerkinElmer Inc., USA).

Western Blotting Cultured utricles were homogenized in a 0.1-mL dounce homogenizer containing radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1 mM EDTA, 1 % NP40, 0.25 % Na-deoxycholate, 0.1 % SDS), 1 mM sodium orthovanadate, and 1 mM sodium fluoride. The resultant supernatants were resuspended in 5× SDS Laemmli sample loading buffer. The samples were subjected to SDS PAGE using 4–15 or 4–20 % Tris-HCl minigels (BioRad, Hercules, CA, USA). The proteins were then transferred to a 0.45-μm pore Immobilon-P PVDF

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membrane (Millipore, Billerica, MA, USA). Membranes were blocked in 5 % milk in 1× PBS with 0.1 % Tween 20 (PBST). Protein bands were visualized by chemiluminescence using SuperSignal® West Dura Extended Duration Substrate (Pierce Biotechnology, Rockford, IL, USA) and developed using CL-XPosure™ Film (Pierce Biotechnology). All antibodies were diluted in 5 % milk PBST. Primary antibodies are as follows: HSP27 (Upstate Biotechnology 06-517, Lake Placid, NY, USA, 1:1000), HO-1 (R & D Systems MAB3776, Minneapolis, MN, USA, 1:1000), HSP40, HSP60, HSP70, HSP90 (Cell Signaling Technology 4868, 4870, 4872, 4874, respectively, Danvers, MA, USA, 1:1000), and actin (Sigma A2066, 1:1000). HRP-conjugated secondary antibodies included antimouse IgG (Cell Signaling Technology 7076), antirabbit IgG (Cell Signaling Technology 7074), and antirat IgG (Cell Signaling Technology 7077). Membranes were stripped between antibodies using Re-Blot Plus Strong Solution (2504, Millipore). Results were quantified by densitometry using ImageJ software (Image Processing and Analysis in Java, http://rsb.info.nih.gov/ ij/index.html).

Statistical Analyses Results are presented as mean±standard error of the mean (SEM). Data were analyzed using statistical software SYSTAT 8.0 (San Jose, CA, USA) and GraphPad 3.0 (La Jolla, CA, USA). Tests included 12- and 3-way analyses of variance (ANOVA). Post hoc testing included Tukey’s and Dunnett’s multiple comparisons. Results were considered significant when the p value was less than 0.05.

RESULTS Heat Shock Inhibits Cisplatin-Induced Hair Cell Death We previously showed that heat shock inhibits hair cell death caused by treatment with a moderate cisplatin concentration (Cunningham and Brandon 2006). In order to examine the protective effect of heat shock across the cisplatin dose-response curve, heat-shocked and control (not heat-shocked) utricles from wild-type CBA/J mice were treated with cisplatin at a range of concentrations for 24 h. Following cisplatin treatment, the utricles were fixed, labeled with anti-calmodulin (hair cell marker), and hair cells were counted. In control utricles, cisplatin treatment resulted in a dose-dependent loss of hair cells (Fig. 1A). Utricles that were heat shocked 6 h prior to cisplatin treatment had significantly greater hair cell survival across the dose-response curve (2-way ANOVA: F6,160 =5.78, p=0.0000) (Fig. 1A).

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Heat Shock Protein-Mediated Protection Against Hair Cell Death

HSP70 Is Required for the Protective Effect of Heat Shock Against Cisplatin-Induced Hair Cell Death

HSP70 provides partial protection against cisplatininduced hair cell death.

HSP70 is required for the protective effect of heat shock against aminoglycoside-induced hair cell death (Taleb et al. 2008; May et al. 2013). In order to determine if HSP70 is also necessary for the protective effect of heat shock against cisplatin-induced hair cell death, we used utricles from HSP70.1/3−/− and wildtype mice. Utricles of both genotypes were either maintained at 37 °C or heat shocked. Heat shock had a significant protective effect against cisplatin-induced hair cell death in wild-type utricles (2-way ANOVA: F1,54 =8.98, p=0.004) (Fig. 1B). However, there was no significant protective effect of heat shock in utricles from HSP70.1/3−/− mice (2-way ANOVA: F1,39 =0.03, p=0.865), indicating that HSP70 is necessary for the protective effect of heat shock against cisplatininduced hair cell death.

HO-1 Induction Inhibits Cisplatin-Induced Hair Cell Death

HSP Levels in Utricles from rHSP70i CE Mice and Their Wild-Type Littermates In order to determine if HSP70 is sufficient to protect hair cells against cisplatin-induced death, we utilized utricles from mice that constitutively express ratinducible HSP70 (rHSP70i CE) (Marber et al. 1995; Kim et al. 2006b; Taleb et al. 2008, 2009). We first examined HSP induction in response to heat shock in utricles from rHSP70i CE mice and their wild-type littermates (Fig. 1C). In the absence of heat shock, rHSP70i CE utricles expressed approximately 2.3-fold higher levels of HSP70 (Fig. 1C). Heat shock resulted in induction of HSP70, HSP40, HO-1 (HSP32), and HSP27 in utricles of both genotypes.

Constitutive Expression of HSP70 Inhibits Cisplatin-Induced Hair Cell Death In order to determine whether HSP70 is sufficient to account for the protective effect of heat shock against cisplatin-induced hair cell death, utricles from rHSP70i CE mice and their wild-type littermates were treated with cisplatin for 24 h. Constitutive rHSP70i expression had a modest but significant effect relative to wild-type (2-way ANOVA: F1,116 =9.60, p=0.0024). However, baseline HSP70 expression in rHSP70i CE utricular explants was not sufficient for complete rescue from cisplatin-induced hair cell death (2-way ANOVA: F4,116 =1.28, p=0.2827) (Fig. 1D). Despite the significant overall effect of genotype, post hoc testing indicated that protection by HSP70 was not significant at any individual dose of cisplatin. These results demonstrate that constitutive expression of

Since HSP70 provided only partial protection against cisplatin-induced hair cell death, we then examined whether HO-1 induction is protective in this system. Induction of HO-1 inhibits hair cell death and hearing loss caused by exposure to the other major class of ototoxic drugs, the aminoglycoside antibiotics (Francis et al. 2011). We utilized the HO-1 inducer cobalt protoporphyrin IX (CoPPIX) (Drummond and Kappas 1982; Ferrandiz and Devesa 2008) to determine if HO-1 induction is protective against cisplatininduced hair cell death. We first examined HSP induction by CoPPIX using Western blotting (Fig. 2A) and qRT-PCR (data not shown). HO-1 was robustly upregulated in response to CoPPIX treatment at both the protein (Fig. 2A) and mRNA levels (data not shown), while other HSPs were not induced (Fig. 2A). Utricular explants were treated with CoPPIX for 12 h and then treated with cisplatin for 24 h. Hair cell counts indicate a significant protective effect of CoPPIX treatment against cisplatin-induced hair cell death across the dose-response relationship (2-way ANOVA: F4,69 =4.24, p=0.004) (Fig. 2B). In order to confirm that the protective effect of CoPPIX is mediated by induction of HO-1, we utilized the HO1 inhibitor zinc protoporphyrin IX (ZnPPIX) (Maines 1981; Wong et al. 2011). Utricles were pretreated with 20 μM CoPPIX in the presence or absence of 10 μM ZnPPIX for 12 h prior to cisplatin treatment. ZnPPIX remained in the culture medium while the utricles were incubated in 25 μg/mL cisplatin for 24 h. Again, cisplatin resulted in significant hair cell death relative to control, and CoPPIX resulted in significant protection against cisplatin-induced hair cell death (Fig. 2C) (1-way ANOVA: F 6,72 = 22.53, p G0.0001). ZnPPIX abolished the protective effect of CoPPIX (Tukey’s multiple comparison test: pG0.001) (Fig. 2C), confirming that HO-1 is required for the protection conferred by CoPPIX against cisplatin-induced hair cell death.

Combined Expression of Both HSP70 and HO-1 Does Not Offer Additional Protection In order to investigate whether HSP70 and HO-1 might work additively or synergistically to protect against cisplatin-induced hair cell death, utricles from rHSP70i CE mice and their wild-type littermates were exposed to the HO-1 inducer CoPPIX for 12 h,

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FIG. 1. HSP70 is necessary but not sufficient for the protective effect of heat shock against cisplatin-induced hair cell death. A Control and heat-shocked utricles were exposed to cisplatin for 24 h. Heat shock resulted in significant protection against cisplatininduced hair cell death (2-way ANOVA: F6,160 =5.78, p=0.0000). Data points represent the mean±SEM for n=5–40 utricles per condition. Asterisks denote significant differences in hair cell density between utricles that were heat shocked and controls. B Control and heat-shocked utricles from adult wild-type and Hsp70−/− mice were exposed to cisplatin (20 μg/mL) for 24 h. Heat shock inhibited cisplatin-induced hair cell death in HSP70+/+ utricles (2-way ANOVA: F1,54 =8.98, p=0.004) but not in utricles from HSP70−/− mice (2-way ANOVA: F1,39 =0.03, p=0.865). Bars represent the mean±SEM for n=5–18 utricles per condition. Asterisks denote significant differences in hair cell density in the extrastriolar region. N/S not significant. C Utricles from rHsp70i CE transgenic mice and their wild-type littermates were heat shocked and then recovered for

6 h prior to being processed for Western blotting. Heat shock resulted in upregulation of HSPs 27, 32, 40, and 70 in utricles of both genotypes. In the absence of heat shock, utricles from mice that constitutively overexpress HSP70 had 2.3-fold higher levels of HSP70 than wild-type utricles. D Utricles from rHsp70i CE mice and their wild-type littermates were treated with cisplatin for 24 h. Results reveal a main effect of genotype, indicating that constitutive expression of rHSP70i has an effect against cisplatin-induced hair cell death (2-way ANOVA: F1,116 =9.60, p= 0.0024); however, rHSP70i CE genotype did not rescue the epithelium from cisplatininduced hair cell death (2-way ANOVA: F4,116 =1.28, p=0.2827). Furthermore, post hoc testing indicated that protection by HSP70 was not significant at any individual dose of cisplatin. Data points represent the mean ± SEM for n = 7–24 utricles per condition. Asterisks denote significant differences in hair cell density between rHSP70i CE and wild-type utricles.

followed by treatment with cisplatin for 24 h. Hair cell counts again revealed that CoPPIX was protective against cisplatin-induced hair cell death (3-way ANOVA: F1,106 =7.19, p=0.009) (Fig. 2D). In addition, rHSP70i CE utricles had significantly more hair cells surviving following treatment with 25 μg/mL cisplatin when compared to wild-type utricles (3-way ANOVA: F2,106 =5.06, p=0.008). However, CoPPIX pretreatment of rHSP70i CE utricles did not offer greater protec-

tion than either rHSP70i or induction of HO-1 (via CoPPIX treatment in wild-type (WT) utricles) alone (3-way ANOVA: F1,106 =0.74, p=0.39).

CoPPIX Upregulates HO-1 in Resident Macrophages In order to investigate the mechanism(s) by which HO-1 inhibits cisplatin-induced hair cell death, we

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FIG. 2. HO-1 protects against cisplatin-induced hair cell death. A Utricles from CBA/J mice were heat shocked or treated with 20 μM CoPPIX prior to being processed for Western blotting. CoPPIX treatment resulted in upregulation of HO-1, with no upregulation of other HSPs. B Control and CoPPIX-treated utricles were exposed to cisplatin for 24 h. CoPPIX resulted in robust protection against cisplatin-induced hair cell death (2-way ANOVA: F4,69 = 4.24, p=0.004). Data points represent the mean± SEM for n=5–7 utricles per condition. Asterisks denote significant differences in hair cell density between utricles that received CoPPIX and those that did not. C Utricles were treated with the HO-1 inhibitor ZnPPIX for 36 h in the presence of CoPPIX and/or cisplatin. ZnPPIX abolished the protective effect of CoPPIX against cisplatin-induced hair cell death (Tukey’s

multiple comparison test: pG0.001). Bars represent the mean± SEM for n=7–12 utricles per condition. Asterisks denote significant differences between groups indicated. D Utricles from rHSP70i CE mice and their wild-type littermates were treated with CoPPIX for 12 h, followed by treatment with cisplatin for 24 h. No synergistic effect was noted between rHSP70i CE and CoPPIX (3-way ANOVA: F1,106 = 0.74, p = 0.39). Data points represent the mean±SEM for n=4–14 utricles per condition. Asterisks denote significant differences in hair cell density between HSP70 WT utricles that received CoPPIX and those that did not. Number sign denotes significant differences between hair cell density of HSP70 WT versus rHSP70i CE utricles.

next sought to identify the specific cell type(s) in the utricle that upregulate HO-1 in response to CoPPIX. Utricles were treated with CoPPIX for 12 h and processed for HO-1 immunolocalization (Fig. 3). HO-1 immunoreactivity was not detected in supporting cells or hair cells after CoPPIX treatment. Instead, CoPPIX treatment resulted in increased HO1 immunoreactivity in resident macrophages (Fig. 3). These data indicate that CoPPIX results in HO-1 induction in resident macrophages, and they suggest that macrophages play a role in mediating the

protective effect of HO-1 against cisplatin-induced hair cell death.

Liposomal Clodronate Depletes Macrophages Without Killing Hair Cells In order to examine the roles of macrophages in mediating the protective effect of HO-1 induction, we utilized liposomal clodronate (LC, a.k.a. liposomal dichloromethylene-bisphosphonate) to ablate resident macrophages in utricles. LC has been used

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to selectively ablate macrophages in a variety of systems, including the inner ear (Van Rooijen and Sanders 1994; van Rooijen et al. 1996; van Rooijen and Hendrikx 2010; Warchol et al. 2012). We first examined the effect of LC on macrophage survival by treating utricles from CX3CR1GFP/+ mice, which express GFP exclusively in macrophages in the

FIG. 3. HO-1 is upregulated in resident macrophages. Utricles were treated with or without CoPPIX. Utricles were stained for the hair cell marker myosin 7a, the macrophage marker CD68, and HO1. Hair cells and supporting cells did not exhibit HO-1 immunoreactivity in control or CoPPIX-treated utricles. CD68+ macrophages did exhibit HO-1 immunoreactivity in response to CoPPIX treatment. Arrows indicate cells that are both CD68 and HO-1 positive. The orthogonal views (E–N) demonstrate that the hair cell and supporting cell layers are HO-1 negative, and the CD68+/HO-1+ doublelabeled cells lie beneath the sensory epithelium. Confocal micrographs are presented. Scale bars represent 50 μm.

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inner ear (Hirose et al. 2005). Utricles from CX3CR1GFP/+ mice were treated with various doses of LC for 48 h (Fig. 4A, B). GFP-positive macrophages and myosin 7a-positive hair cells were counted from maximum intensity projections of confocal z-stacks taken at 1.0-μm intervals through the tissue. Liposomal clodronate (2–8 mM) resulted

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FIG. 4. Macrophages are required for HO-1-mediated protection. A CX3CR1GFP/+ utricles were treated with LC for 48 h. Liposomal clodronate (2, 4, and 8 mM) depleted macrophages from the utricular epithelium (1-way ANOVA: F6,30 =6.82, p=0.0001). B Liposomal clodronate had no significant effect on hair cell density (1-way ANOVA: F6,31 =0.16, p=0.99). Bars represent the mean±SEM for n=4–10 utricles per condition. Asterisks denote significant differences in cell numbers compared to control. C Utricles were treated with LC to deplete macrophages, and then treated with CoPPIX to induce HO-1. LC did not result in hair cell death (Tukey’s multiple comparison test: p90.05), but cisplatin resulted in significant hair cell death (Tukey’s multiple comparison test: pG0.001). LC did not alter cisplatin-induced hair cell death (Tukey’s multiple comparison test: p90.05). CoPPIX inhibited cisplatin-induced hair cell death (Tukey’s multiple comparison test: pG0.01), and depletion of macrophages using LC abolished the protective effect of CoPPIX (Tukey’s multiple comparison test: pG0.01). All statistical analyses are 1-way ANOVA with Tukey’s multiple comparison test: p90.05. Bars represent the mean±SEM for n=4–6 utricles per condition. Asterisks denote significant differences in hair cell density between the groups indicated by the horizontal line.

in significant depletion of macrophages (1-way ANOVA: F6,30 =6.826, p=0.0001) (Fig. 4A) without loss of hair cells (1-way ANOVA: F6,31 =0.16, p=0.99) (Fig. 4B). Subsequent macrophage depletion experiments were performed using 4 mM LC, a dose at which approximately 75 % of macrophages were removed from the epithelium without loss of hair cells (Fig. 4A, B).

Depletion of Resident Macrophages Abolishes the Protective Effect of HO-1 Against Cisplatin-Induced Hair Cell Death In order to determine if macrophages are required for the protective effect of HO-1 against cisplatininduced hair cell death, we used LC to deplete macrophages from CX3CR1GFP/+ utricles prior to CoPPIX treatment (Fig. 4C). Cisplatin resulted in significant hair cell death, and CoPPIX was protective (1-way ANOVA: F5,27 =22.74, pG0.0001). Again, we saw that LC alone was not toxic to hair cells (Fig. 4C) (Tukey’s multiple comparison test: p90.05). Depletion of resident macrophages using LC abolished the protective effect of CoPPIX treatment (Tukey’s multiple comparison test: pG0.01) (Fig. 4C). These data indicate that macrophages are required for the protective effect of HO-1 induction by CoPPIX.

DISCUSSION Our data indicate that HSP induction inhibits cisplatin-induced hair cell death in vitro. We previously reported that heat shock inhibits hair cell death caused by exposure to a moderate dose of cisplatin (Cunningham and Brandon 2006). In addition, heat

shock is protective against hair cell death caused by the other major class of ototoxic drugs, the aminoglycoside antibiotics (Cunningham and Brandon 2006). Here, we expanded our previous study to examine a range of cisplatin doses, and we found that heat shock provides robust protection against cisplatin-induced hair cell death at multiple cisplatin doses (Fig. 1A). We confirmed (Fairfield et al. 2004) that heat shock of mouse utricular explants from more than one genetic background results in upregulation of multiple HSPs, including HSP27, HO-1 (HSP32), and HSP70 (Cunningham and Brandon 2006; Taleb et al. 2008; Francis et al. 2011) (Figs. 1C and 2A). The increased expression of individual HSPs, HSP 70 (Fig. 1C, D), and HO-1 (Fig. 2), also protected hair cells from cisplatin-induced death. Interestingly, HO-1 induction with CoPPIX resulted in upregulation of HO-1 in resident macrophages of mouse utricles, and HO-1 was not upregulated in hair cells or supporting cells (Fig. 3). Depletion of macrophages from utricular explants with liposomal clodronate abolished the protective effect of HO-1 induction (Fig. 4C), suggesting a novel protective role for macrophages against cisplatin-induced hair cell death. Previous studies of cisplatin-induced ototoxicity have implicated DNA damage (Zhang et al. 2003; van Ruijven et al. 2005; Li et al. 2006), generation of reactive oxygen species (Ravi et al. 1995; Rybak et al. 1995; Clerici et al. 1996), and inflammation (So et al. 2007, 2008; Kim et al. 2008) as mediators of the apoptotic death of hair cells. Similarly, multiple mechanism(s) may underlie HSPmediated inhibition of cisplatin ototoxicity. In other systems, HSPs protect cells against damage-induced death by preventing the accumulation and aggregation of damaged proteins, as well as by promoting proper re-folding of denatured proteins (Jolly and Morimoto 2000; Yamamoto et al. 2000; Martindale and Holbrook 2002; Mayer and Bukau 2005; Kastle and Grune 2012). The protection conferred by HSPs is not limited to these “chaperone” activities; HSPs also inhibit lipid peroxidation and oxidative damage to DNA (Park et al. 1998; Su et al. 1999; Martindale and Holbrook 2002). In addition, HSPs have been shown to inhibit multiple pro-apoptotic signaling events (Jaattela et al. 1998; Beere et al. 2000; Pandey et al. 2000a, b; Concannon et al. 2001, 2003; Tsuchiya et al. 2003; Stankiewicz et al. 2005; Rodina et al. 2007; Jiang et al. 2009; Evans et al. 2010; Pasupuleti et al. 2010). HO-1 is a heat shock-inducible protein that lacks intrinsic chaperone activity; however, it inhibits oxidative stress, inflammation, and apoptosis in multiple tissue types (Kirkby and Adin 2006; Ryter et al. 2006; Gozzelino et al. 2010; Paine et al. 2010; Blancou et al. 2011). Our data indicate that HO-1 induction confers significant protection against cisplatininduced hair cell death.

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HSP70 is the most highly upregulated HSP in response to heat shock (Fig. 1C). Our data using utricles from HSP70−/− mice indicate that HSP70 is required for the protective effect of heat shock against cisplatin-induced hair cell death (Fig. 1B). These data are in agreement with our previously reported findings regarding the role of HSP70 as a mediator of the protective effect of heat shock against aminoglycosideinduced hair cell death (Taleb et al. 2008; May et al. 2013). However, constitutive expression of HSP70 conferred robust protection against aminoglycosideinduced hair cell death (Taleb et al. 2008, 2009) and only modest protection against cisplatin-induced hair cell death (Fig. 1D). This difference in effectiveness of rHSP70i against hair cell death caused by aminoglycosides versus cisplatin may be indicative of different underlying mechanisms of hair cell death caused by these two ototoxic agents. Because rHSP70i constitutive expression provided only modest protection against cisplatin-induced hair cell death relative to heat shock, we also examined the effect of HO-1, which is also induced in response to heat shock (Fig. 1C). HO-1 is a biologically protective enzyme induced in the face of multiple stressors that catabolizes free heme to produce known antiinflammatory and antioxidant molecules, CO, and bilirubin (Tenhunen et al. 1968, 1969; Stocker et al. 1987; Hayashi et al. 1999; Otterbein et al. 2000; Kirkby and Adin 2006; Wegiel et al. 2013). We utilized the pharmacological agent CoPPIX to specifically induce HO-1 expression in utricles. HO-1 induction by CoPPIX offered robust protection against cisplatininduced hair cell death (Figs. 2B, C). Our data using the HO-1 inhibitor ZnPPIX confirm that the protective effect of CoPPIX is mediated by HO-1 activity (Fig. 2C), indicating that HO-1 is required for the protective effect of CoPPIX. Since both HSP70 and HO-1 were independently protective, we examined the possibility that combined upregulation of these two proteins would confer additive or synergistic protection against cisplatin-induced hair cell death. In order to achieve increased expression of both HSP70 and HO-1, we treated rHSP70i CE utricles with CoPPIX prior to cisplatin treatment. Results indicate that there is no additive or synergistic effect against cisplatin-induced hair cell death when both HSPs are expressed (Fig. 2D). CoPPIX inhibits cisplatin-induced death in the HEI-OC1 auditory-derived cell line (Kim et al. 2006a, 2009; So et al. 2006, 2008; Choi et al. 2007, 2008, 2011; Gao et al. 2010). HO-1 expression has been reported in cochlear hair cells and stria vascularis following a variety of stressors, including noise stress, heat stress, and pharmacological induction (Fairfield et al. 2004; Lopez et al. 2008; Kim et al. 2009; Matsunobu et al. 2009; Fetoni et al. 2010). In the current study, CoPPIX

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treatment resulted in HO-1 induction primarily in resident macrophages without induction in hair cells or supporting cells (Fig. 3). It is not clear why CoPPIX did not result in HO-1 induction in hair cells or supporting cells in mouse utricles in vitro. The localization of HO-1 expression in macrophages observed herein is consistent with reports that HO-1 is expressed in macrophages of other organs, including liver and lung (Devey et al. 2009; Obata et al. 2011). Depletion of 75 % of the macrophages from utricular explants using LC abolished the protective effect of HO-1 induction (Fig. 4A, C), indicating that a critical number of macrophages is required for the protective effect of HO-1 against cisplatin-induced hair cell death. These data suggest a novel protective role for macrophages as important mediators of hair cell survival in response to cisplatin. The finding that macrophages may be able to act as mediators of hair cell survival is consistent with recent reports from our lab and others regarding the importance of other (non-hair cell) cell types as critical regulators of hair cell survival and death (Lahne and Gale 2008; Bird et al. 2010; May et al. 2013). Furthermore, protection conferred from the HO-1/macrophage axis has been demonstrated in other systems, particularly in response to ischemiareperfusion injury in the liver (Devey et al. 2009). In that system, HO-1 appears to regulate the cytokine secretion profile of the macrophages in a manner in which increased HO-1 expression results in a switch from pro- to anti-inflammatory cytokine secretion (Weis et al. 2009; Wegiel et al. 2013). Thus, HO-1 levels may influence whether resident macrophages exhibit a pro-inflammatory phenotype that promotes cell death versus a non-inflammatory phenotype that promotes cell survival. CoPPIX is a potent inducer of HO-1, and our data indicate that HO-1 induction may hold potential as a co-therapy aimed at prevention of cisplatin-induced ototoxicity. Systemic CoPPIX administration results in significant toxic side effects (Smith et al. 1987; Galbraith and Kappas 1989; Muhoberac et al. 1989; Rosenberg and Kappas 1995); however, local administration of CoPPIX to the ear should be examined to determine if HO-1 induction can reduce cisplatin-induced hearing loss. Local administration of CoPPIX (or another HO-1 inducer) would reduce the potential for HO1 to inhibit the therapeutic efficacy of cisplatin. Encouraging advances have been made in the field of inner ear drug delivery, including the use of hydrogels and nanoparticles (Pritz et al. 2013; Hutten et al. 2014; Yu et al. 2014), which may be able to be targeted to macrophages (Lameijer et al. 2013).

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Cisplatin-induced hair cell death is a complex process that involves not only signal transduction pathways within hair cells themselves but also interactions between hair cells and other cell types, including resident macrophages. Heat shock preconditioning, as well as induction of individual heat shock proteins, is protective against cisplatininduced hair cell death. The results of the current study imply that HSPs, particularly HO-1, are candidate targets for the rational design of co-therapies aimed at preventing cisplatin-induced hearing loss. In addition, our data indicate that macrophages can offer protection against cisplatin-induced hair cell death, a finding that is consistent with our recent data indicating the importance of non cellautonomous signals in determining whether a hair cell under stress ultimately lives or dies (May et al. 2013). Finally, these data illustrate an advantage of whole-organ cultures over monolayer cells for studies of hair cell survival and death following ototoxic damage. Organ culture model systems allow us to examine hair cell death and survival in the context of these important signals from surrounding cell types.

ACKNOWLEDGMENTS Our thanks to Dr. Mark Warchol for many helpful conversations, for participation in pilot studies using CX3CR1GFP/+ utricles and for guidance regarding the liposomal clodronate for these studies. Thanks to Lindsey May for help with qRT-PCR experiments. Thanks to Dr. Suhua Sha for graciously sharing her lab space during the completion of these studies. This work was s upp orted by NIH/NIDCD R0 1 D C0 076 13, F30DC010522, and NIH/NCRR extramural research facilities construction (C06) grants C06 RR015455 and C06 RR14516 from the National Center for Research Resources, as well as the MUSC GAANN fellowship in Cell and Neurobiology. Imaging facilities for this research were supported by Cancer Center Support Grant P30 CA138313 to the Hollings Cancer Center, Medical University of South Carolina. Additional support for this work was provided by the NIDCD Intramural Research Program (ZIA DC00079).

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