Article18 March 2021Open Access Source DataTransparent process Toll-like receptor 4 is activated by platinum and contributes to cisplatin-induced ototoxicity Ghazal Babolmorad Ghazal Babolmorad Department of Medical Microbiology and Immunology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Asna Latif Asna Latif Department of Medical Microbiology and Immunology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Ivan K Domingo Ivan K Domingo Department of Medical Microbiology and Immunology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Niall M Pollock Niall M Pollock Department of Biological Sciences, Faculty of Science, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Cole Delyea Cole Delyea Department of Medical Microbiology and Immunology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Aja M Rieger Aja M Rieger Department of Medical Microbiology and Immunology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author W Ted Allison W Ted Allison Department of Biological Sciences, Faculty of Science, University of Alberta, Edmonton, AB, Canada Department of Medical Genetics, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Amit P Bhavsar Corresponding Author Amit P Bhavsar [email protected] orcid.org/0000-0003-3336-9288 Department of Medical Microbiology and Immunology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada Department of Medical Genetics, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Ghazal Babolmorad Ghazal Babolmorad Department of Medical Microbiology and Immunology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Asna Latif Asna Latif Department of Medical Microbiology and Immunology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Ivan K Domingo Ivan K Domingo Department of Medical Microbiology and Immunology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Niall M Pollock Niall M Pollock Department of Biological Sciences, Faculty of Science, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Cole Delyea Cole Delyea Department of Medical Microbiology and Immunology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Aja M Rieger Aja M Rieger Department of Medical Microbiology and Immunology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author W Ted Allison W Ted Allison Department of Biological Sciences, Faculty of Science, University of Alberta, Edmonton, AB, Canada Department of Medical Genetics, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Amit P Bhavsar Corresponding Author Amit P Bhavsar [email protected] orcid.org/0000-0003-3336-9288 Department of Medical Microbiology and Immunology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada Department of Medical Genetics, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada Search for more papers by this author Author Information Ghazal Babolmorad1, Asna Latif1, Ivan K Domingo1, Niall M Pollock2, Cole Delyea1, Aja M Rieger1, W Ted Allison2,3 and Amit P Bhavsar *,1,3 1Department of Medical Microbiology and Immunology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada 2Department of Biological Sciences, Faculty of Science, University of Alberta, Edmonton, AB, Canada 3Department of Medical Genetics, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada *Corresponding author. Tel: +780 492 0904; E-mail: [email protected] EMBO Reports (2021)22:e51280https://doi.org/10.15252/embr.202051280 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Toll-like receptor 4 (TLR4) recognizes bacterial lipopolysaccharide (LPS) and can also be activated by some Group 9/10 transition metals, which is believed to mediate immune hypersensitivity reactions. In this work, we test whether TLR4 can be activated by the Group 10 metal platinum and the platinum-based chemotherapeutic cisplatin. Cisplatin is invaluable in childhood cancer treatment but its use is limited due to a permanent hearing loss (cisplatin-induced ototoxicity, CIO) adverse effect. We demonstrate that platinum and cisplatin activate pathways downstream of TLR4 to a similar extent as the known TLR4 agonists LPS and nickel. We further show that TLR4 is required for cisplatin-induced inflammatory, oxidative, and cell death responses in hair cells in vitro and for hair cell damage in vivo. Finally, we identify a TLR4 small molecule inhibitor able to curtail cisplatin toxicity in vitro. Thus, our findings indicate that TLR4 is a promising therapeutic target to mitigate CIO. SYNOPSIS Cisplatin is invaluable in cancer treatment, but its use is limited due to cisplatin-induced ototoxicity. TLR4 is activated by cisplatin, contributing to cisplatin-induced hair cell death, but genetic and small molecule inhibition of TLR4 curtails ototoxicity. Platinum and the platinum-based chemotherapeutic cisplatin activate Toll-like receptor 4 (TLR4). TLR4 signaling is required for cisplatin-induced responses in hair cells and for hair cell damage. Genetic inhibition of TLR4 protects against cisplatin-induced toxicity in vitro and reduces hair cell death in zebrafish. A TLR4 inhibitor prevents ototoxic cisplatin responses in vitro. Introduction Toll-like receptor 4 (TLR4) is a membrane-bound pattern recognition receptor that is best characterized for its ability to initiate innate immune signaling upon detection of the Gram-negative bacterial surface component lipopolysaccharide (LPS) (Kawasaki & Kawai, 2014). LPS detection requires the TLR4 co-receptor MD-2. Structural analyses have revealed that the LPS binding pocket is comprised of both MD-2 and TLR4 on the external face of the membrane, with MD-2 making a major contribution to agonist binding (Park et al, 2009; Park & Lee, 2013). LPS binding to the TLR4/MD-2 complex induces TLR4 dimerization and signal propagation through adapter protein recruitment on the cytoplasmic face of the membrane (Park & Lee, 2013). Two canonical signaling pathways are activated through TLR4. The TLR4 adapter protein TIRAP engages the MyD88-dependent signaling pathway that culminates in NF-κB nuclear translocation and pro-inflammatory cytokine production. TRAM, the alternate TLR4 adapter protein, engages TRIF resulting in IRF3 translocation to the nucleus and stimulation of type I interferon response (Kawasaki & Kawai, 2014). It is also widely accepted that TLR4 is activated by other agonists including damage-associated molecular patterns (DAMPs, e.g., HMGB1 and HSP70) and the fusion protein from respiratory syncytial virus (Kurt-Jones et al, 2000; Lee & Seong, 2009; Rallabhandi et al, 2012; Gaikwad et al, 2017; Yuan et al, 2018). TLR4 was also found to mediate immune hypersensitivity reactions to the Group 9/10 transition metals nickel, cobalt, and palladium (Schmidt et al, 2010; Raghavan et al, 2012; Rachmawati et al, 2013). Mechanistically, metal binding to TLR4 induces receptor dimerization to activate downstream signaling (Raghavan et al, 2012). Platinum is a Group 10 transition metal that shares chemical properties with nickel and palladium but it is unknown whether it can activate TLR4. Cisplatin is a platinum-based, highly effective chemotherapeutic frequently used to treat solid tumors in children. In adults, it is used to treat ovarian, testicular, cervical, lung, head and neck, and bladder cancers (Dasari & Tchounwou, 2014). Cisplatin-containing regimens contribute to a 5-year survival rate that approaches 80% in childhood cancer patients and has become an asset to cancer therapy (American Childhood Cancer Organization, 2017). The anti-tumor activity of cisplatin is based on its formation of intra-strand and inter-strand guanine crosslinks in DNA that prevent the strands from separating, or it alkylates DNA bases causing DNA miscoding (Siddik, 2003). This DNA modification activates multiple signal transduction pathways leading to cell-cycle arrest and programmed cell death (Sorenson et al, 1990; Sarin et al, 2017; Maekawa et al, 2019). Despite its effectiveness, cisplatin use is limited by the development of several toxicities that include nephrotoxicity, neurotoxicity, and ototoxicity. Although nephrotoxicity can be reversed by saline hydration and mannitol diuresis, there is no treatment for cisplatin-induced neurotoxicity or ototoxicity (Rybak et al, 2009). The ototoxic effect of cisplatin leads to permanent bilateral hearing loss and is estimated to affect 26-90% of children treated with cisplatin where age, treatment regimen, and concomitant factors also influence susceptibility (Skinner et al, 1990; Brock et al, 1991; Blakley & Myers, 1993; Li et al, 2004; Brock et al, 2012). Cisplatin-induced ototoxicity (CIO) can have significant life-long consequences in children by impairing speech and language development, impairing social-emotional development, and increasing the risk of learning difficulties (Gurney et al, 2007; Gurney et al, 2009). Moreover, the likelihood of developing ototoxicity increases in a dose-dependent manner, with nearly 100% of patients receiving high dosages of cisplatin (150–225 mg/m2) showing some degree of ototoxicity. This compromises anti-cancer treatment, potentially impacting overall survival as cisplatin dose reduction or discontinuation is required to mitigate this ototoxicity (Kopelman et al, 1988; Chang & Chinosornvatana, 2010). CIO is perhaps exacerbated because cisplatin accumulates preferentially in the cochlea of the inner ear (Breglio et al, 2017), and more particularly in the outer hair cells of the Organ of Corti, which are terminally differentiated mechanotransducers and the site of the first steps in sound perception (Lim, 1986). The cochlea is considered a closed system due to its isolated anatomical position and structure and, as such, is not able to rapidly flush out cisplatin and the metabolites generated in response (Lim, 1986). Apoptotic damage in the hair cells of the cochlea is the primary mechanism of cisplatin-induced hearing loss (Rybak et al, 2007). In the current study, we sought a mechanistic understanding of the signaling pathway activated by cisplatin to enable mitigation of its adverse long-term effects. We found that cisplatin activates TLR4, independently of the MD-2 co-receptor. Further, deletion of Tlr4 in a murine inner ear cell line reduced cisplatin-induced ototoxicity. Similarly, knockdown of Tlr4 homologs in zebrafish protected against cisplatin-induced hair cell death. Moreover, we attenuated cisplatin ototoxic responses with the TLR4 chemical inhibitor, TAK-242. These findings provide key insights into the etiology of cisplatin-induced ototoxicity and are crucial to developing protective therapies against CIO, thereby improving the prognosis and long-term health outcomes of cancer patients. Results Platinum and cisplatin activate TLR4 in vitro Nickel, palladium, and cobalt (Group 9 and 10 transition metals) have been well characterized as TLR4 ligands that induce contact hypersensitivity (Rachmawati et al, 2013). Given that platinum is a Group 10 transition metal, we were interested in determining whether it also could serve as a TLR4 ligand. We investigated this using reporter cell lines that did (HEK-hTLR4) or did not (HEK-null2) stably express human TLR4 and its MD-2/CD14 co-receptors. These isogenic human embryonic kidney (HEK) cell lines also express a reporter of NF-κB activation, where NF-κB induces transcription of a secreted alkaline phosphatase (SEAP) reporter; these cells have been used previously to identify TLR4 ligands (Schmidt et al, 2010). We treated HEK-hTLR4 and HEK-null2 cells with platinum (both platinum (II) and (IV) as chloride salts), or LPS or nickel as positive control TLR4 agonists, and monitored NF-κB activation. As expected, we saw significant activation of NF-κB in HEK-hTLR4 cells treated with LPS or nickel compared to media-only controls (Fig 1A). By contrast, HEK-null2 cells showed no significant change in NF-κB activation, demonstrating the effects were dependent on TLR4. HEK-hTLR4 cells treated with platinum(II) or platinum(IV) also showed significant induction of SEAP activity compared to HEK-null2 cells that was intermediate between nickel and LPS (Fig 1A). Separately, we assessed TLR4 activity by measuring its downstream induction of IL-8 cytokine secretion in the same cells. IL-8 secretion increased significantly in the HEK-hTLR4 cells for LPS, nickel chloride, and platinum(IV) chloride (Fig 1B). Platinum(II) chloride induced IL-8 secretion 10-fold, though this was not statistically significant. Again, no IL-8 secretion was observed in cells where hTLR4 was absent. Figure 1. Platinum and cisplatin activate TLR4 in vitro Activity of an NF-κB reporter relative to vehicle control in human embryonic kidney cells that express TLR4 (hTLR4) or an isogenic control cell line that does not express TLR4 (null2) and were stimulated with vehicle (veh), 1 ng/ml LPS, 400 µM nickel chloride, or 25, 50, or 100 µM platinum(II) chloride, and platinum(IV) chloride (n = 3 independent biological replicates). As per panel (A), secreted IL-8 was monitored as a metric of TLR4 activation upon stimulation with 50 pg/ml LPS, 200 µM nickel chloride, 100 µM platinum(II) chloride, or 100 µM platinum(IV) chloride (n = 4 independent biological replicates). IL-8 secretion in HEK-hTLR4 and HEK-null2 cells following treatment with cisplatin at the indicated concentrations (n = 3 independent biological replicates). IL-8 secretion in HEK-hTLR4 cells pre-treated with 4 µ M TAK242 (TLR4 inhibitor) or vehicle, and subsequent treatment with 50 pg/ml LPS, 200 µM nickel chloride, or 25 µM cisplatin (n = 3 independent biological replicates). Mock cells were not subject to pre-treatment prior to agonist addition. Data Information: For all panels, actual individual data from each experiment are plotted as box (25th and 75th percentile borders; median central band) with Tukey whiskers. Statistical analyses were assessed by 2-way ANOVA: in (A) hTLR4 compared to null2 cells; in (B) agonist treatment compared to non-treated (nil); in (C) comparisons between successive concentrations; and in (D) comparisons between vehicle and TAK-242 treatments. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (Dunnett's test, A, B, D; Tukey test, C). Source data are available online for this figure. Source Data for Figure 1 [embr202051280-sup-0002-SDataFig1.xlsx] Download figure Download PowerPoint We next tested whether cisplatin, a platinum-based chemotherapeutic, could also activate TLR4 considering its highly similar composition to platinum chloride. We found that cisplatin also induced IL-8 secretion in HEK-hTLR4 cells more than 100-fold, but not in HEK-null2 cells. Furthermore, cisplatin activation of TLR4 was dose-dependent up to 50 μM, with pronounced toxicity limiting assessments at higher concentrations (Fig 1C). HEK-null2 cells remained largely unresponsive at higher cisplatin concentrations. To further examine cisplatin activation of TLR4, we studied MyD88-dependent and MyD88-independent signaling events downstream of TLR4 in response to cisplatin treatment. To this end, we monitored phospho-signaling events and reporter assays in the mouse inner ear Organ of Corti cell line HEI-OC1, which provides a popular in vitro model of drug-induced hearing loss (Kalinec et al, 2016). We observed rapid phosphorylation of the MyD88-dependent signaling components NF-κB and p42/44 within 30 min of treatment (Fig EV1A). By contrast, we did not observe phosphorylation of IRF3 over the same time course. However, using a luciferase-based IRF3 reporter we observed significant induction of this TRAM-dependent signaling pathway 24 h after cisplatin treatment (Fig EV1B). The kinetics of signaling that we observed are consistent with those reported in the literature for TLR4 activation and for cisplatin treatment of HEI-OC1 cells (Kawai & Akira, 2006; So et al, 2007, 2008; Chung et al, 2008). Click here to expand this figure. Figure EV1. Cisplatin induces rapid MyD88-dependent signaling and slower MyD88-independent signaling downstream of Tlr4 Phospho-signaling in HEI-OC1 cells following treatment with 20 µM cisplatin or 10 ng/ml LPS at the indicated time points (representative of 3 independent biological replicates). IRF3 reporter activity in HEI-OC1 cells treated with and without 20 µM cisplatin after 24 h (n = 4 independent biological replicates). Data information: In (B), data are shown as mean and standard deviation. ****P < 0.0001 (unpaired Student's t-test). Source data are available online for this figure. Download figure Download PowerPoint To independently assess the requirement for TLR4 in cisplatin-induced IL-8 secretion in HEK-hTLR4 cells, we repeated our treatments in the presence of a small molecule TLR4 inhibitor (TAK-242) that binds to the intracellular domain of TLR4, disrupting its interactions with cytosolic adaptor proteins (Matsunaga et al, 2011). Chemical inhibition with TAK-242 mitigated the effect of cisplatin on TLR4 activation similarly to nickel and LPS in HEK-hTLR4 cells (Fig 1D). Taken together, these data demonstrate that platinum and cisplatin behave similarly to nickel and LPS with respect to their ability to activate TLR4. Cisplatin activation of TLR4 does not require its co-receptor, MD-2 We next sought to appreciate the mechanisms of TLR4 activation upon cisplatin activation, as this would influence potential therapeutic design. Canonical TLR4 signaling after binding LPS requires the TLR4 co-receptor MD-2, whereas their requirements for metal-based activation of TLR4 are less well defined (Nagai et al, 2002; Schmidt et al, 2010; Raghavan et al, 2012). To examine the role of the MD-2 co-receptor in TLR4 activation, we used an HEK cell background that stably expresses TLR4 but not the MD-2 co-receptor (HEK-isoTLR4). HEK-isoTLR4 cells were transfected with an empty vector control, or with a human MD-2 expressing plasmid, and assayed for IL-8 secretion upon treatment with TLR4 agonists. As expected, treatment with LPS did not yield a significant increase in secreted IL-8 unless HEK-isoTLR4 cells were transfected with MD-2 (Fig 2A). This result confirmed that TLR4 was active in these cells and that they were MD-2-deficient. We obtained similar findings with nickel chloride, which is consistent with reports that nickel activation of TLR4 is MD-2-dependent (Raghavan et al, 2012; Oblak et al, 2015). By contrast, we observed significant secretion of IL-8 in response to platinum(II) chloride, platinum(IV) chloride, and cisplatin in HEK-isoTLR4 cells transfected with the empty vector. Although transfection of MD-2 further enhanced IL-8 secretion in response to these agonists, these findings indicate that unlike LPS and nickel, the platinum-containing agonists are not strictly MD-2 dependent. We also tested the DAMP, HMGB1 in this assay and observed that significant increases in IL-8 secretion elicited by this TLR4 agonist also required MD-2. These data suggest that cisplatin activation of TLR4 can occur independently of this DAMP, potentially mediated by its platinum constituent (Fig 2A). Figure 2. Cisplatin activation of TLR4 is independent of the TLR4 co-receptor, MD-2 IL-8 secretion in HEK cells stably expressing hTLR4 but not MD-2 (HEK-isoTLR4), transfected with empty vector (EV) or MD-2 and left untreated (nil) or treated with 1 ng/ml LPS, 200 µM nickel chloride, 2 µg/ml HMGB1, 100 µM platinum(II) chloride, 100 µM platinum(IV) chloride, or 25 µM cisplatin (n = 3 or 4 independent biological replicates). Fold IL-8 secreted (relative to nil treatment) in MD-2-deficient HeLa cells treated with 10 or 100 ng/ml LPS or 25 µM cisplatin (n = 4 independent biological replicates). IL-8 secretion in HeLa cells transfected with non-targeting (siNT) or TLR4-targeting (siTLR4) siRNA and left untreated (nil), or treated with 30 µM cisplatin (n = 3 independent biological replicates). Mock cells were not subject to siRNA treatment prior. Data Information: Actual individual data are plotted as box (25th and 75th percentile borders; median central band) with Tukey whiskers (A, C) or mean and standard deviation (B). Statistical analyses were determined in comparison to nil treatments using 2-way (A, C) or one-way (B) ANOVA. ns, not significant; *P < 0.05; ****P < 0.0001 (Dunnett's test). Source data are available online for this figure. Source Data for Figure 2 [embr202051280-sup-0003-SDataFig2.xlsx] Download figure Download PowerPoint To further investigate the requirement of the MD-2 co-receptor for cisplatin activation of TLR4, we used HeLa cells that have been reported to lack MD-2 expression (Wyllie et al, 2000). We treated HeLa cells with cisplatin and LPS and observed that cisplatin, but not LPS, induced significant IL-8 secretion (Fig 2B). To confirm that IL-8 secretion elicited by cisplatin in HeLa cells was dependent on TLR4, we repeated cisplatin treatments in HeLa cells transfected with non-targeting or TLR4-targeting siRNA. We determined that siRNA treatment reduced TLR4 expression by > 75% (Fig EV2). Following TLR4 knockdown, we observed 70% lower cisplatin-induced IL-8 secretion indicating that secretion of this cytokine is mediated by TLR4 (Fig 2C). Taken together, these data indicate that TLR4 co-receptors are dispensable for cisplatin activation of TLR4. Click here to expand this figure. Figure EV2. TLR4 expression in HeLa cells is significantly reduced by transient silencing TLR4 expression levels (relative to nil treatment) in HeLa cells transfected with non-targeting (siNT) or TLR4-targeting (siTLR4) siRNA molecules and treated with 30 µM cisplatin. Data information: Actual individual data from 2 independent experiments are plotted with mean and standard deviation indicated. Source data are available online for this figure. Download figure Download PowerPoint Tlr4 deletion mitigates cisplatin ototoxic responses in a murine inner ear cell line Having shown that cisplatin can act as an agonist of TLR4 to induce a pro-inflammatory response in vitro, we next asked whether TLR4 plays a role in mediating the molecular events that contribute to cisplatin-induced ototoxicity. Cisplatin treatment induces the generation of reactive oxygen species (ROS) in the cochlea, which appear to be critical mediators of CIO (Clerici et al, 1996; van Ruijven et al, 2004). Hallmarks of in vitro cisplatin ototoxic responses, as modeled by Organ of Corti cell lines, include increased pro-inflammatory IL-6 signaling, which can upregulate ROS generation that in turn influence morphological and functional alterations leading to apoptotic cell death (Ravi et al, 1995; So et al, 2007; Kim et al, 2010). We mutated Tlr4 in the mouse inner ear hair cell line HEI-OC1 by CRISPR/Cas9. We established single-cell clones of Tlr4-edited cells, along with non-targeting guide RNA-edited control cells and conducted a primary screen to identify clones with diminished LPS responses. Sanger sequencing at the Tlr4 locus identified a clone with frame-shift mutations in exon 1 (one adenine insertion or a four nucleotide deletion; Fig EV3A). Compared to control cells, the deletion clone exhibited decreased Tlr4 protein abundance (Fig EV3B), significantly reduced binding/internalization of a fluorescent LPS analog (Fig EV3C), and significantly reduced LPS-induced cytokine secretion (Fig EV3D). Importantly, LPS-induced IL-6 secretion was enhanced 4-fold upon complementation with ectopically expressed Tlr4 in the deletion cells, compared to less than 2-fold in control cells (Fig EV3D, inset). Taken together with the genetic data, these results confirm a Tlr4 deletion in CRISPR-targeted HEI-OC1 cells. Click here to expand this figure. Figure EV3. Tlr4−/− HEI-OC1 cells show diminished LPS-responsiveness unless complemented with Tlr4 Comparison of genomic DNA at the Tlr4 locus from Tlr4−/− HEI-OC1 and wild-type cells. Sequences from the Tlr4−/− cell line contained a single nucleotide insertion or four nucleotide deletion and summarized below. Anti-TLR4 staining in Tlr4−/− and control HEI-OC1 cells. Bars (lower right) are 50 µm. Flow cytometric analysis of conjugated LPS internalization in Tlr4−/− and control HEI-OC1 cells (n = 4 independent biological replicates). IL-6 secretion in Tlr4−/− and control HEI-OC1 cells transfected with empty vector (EV), Tlr4 (pTlr4), or left untransfected (−) and subsequently treated with 100 ng/ml LPS (n = 4 independent biological replicates). Inset, fold induction of IL-6 secretion was determined relative to the untransfected cells treated with LPS. Data information: In (C and D), data are presented as mean and standard deviation. Statistical comparisons were assessed by 2-way ANOVA (D). **P < 0.01; ****P < 0.0001 (unpaired Student's t-test, C; Bonferroni test, D). Source data are available online for this figure. Download figure Download PowerPoint To examine the impact of the Tlr4 deletion on cisplatin ototoxic responses, we treated Tlr4 deletion and control HEI-OC1 cells with cisplatin for 24 h to measure apoptosis, pro-inflammatory cytokine secretion, and intracellular ROS generation. With increasing cisplatin concentrations, Tlr4-deleted HEI-OC1 cells had a higher level of viability than control, with a concomitant decrease in AnxV+/PI- cells suggesting that the cells were dying apoptotically (Fig 3A and B). Similarly, we observed a significant decrease in cisplatin-induced ROS formation in Tlr4 deletion cells (Fig 3C). Moreover, Tlr4-deleted cells had reduced IL-6 secretion in response to cisplatin treatment compared to control cells (Fig 3D). Taken together, these data indicate that TLR4 is an important mediator of cisplatin ototoxic responses in inner ear hair cells. Figure 3. Deletion of Tlr4 in a murine ear outer hair cell line (HEI-OC1) reduces cisplatin-induced ototoxic responses A–D. HEI-OC1 cells containing a Tlr4 deletion (Tlr4−/−) were compared to HEI-OC1 non-targeting (NT) control cells and assessed for cell viability (A), Annexin V/propidium iodide staining (B), ROS generation (C), and IL-6 secretion (D) following cisplatin treatment at the indicated concentrations (n = 3 independent biological replicates). Data Information: In all panels, data are presented as mean and standard deviation. Statistical comparisons to NT at the same cisplatin concentration were assessed by 2-way (A, B) or one-way (C, D) ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (Bonferroni test). Source data are available online for this figure. Source Data for Figure 3 [embr202051280-sup-0004-SDataFig3.xlsx] Download figure Download PowerPoint Cisplatin-induced toxicity is consistent with its primary activation of TLR4 It has been previously reported that cisplatin induces the expression of Tlr4 leading to subsequent activation by LPS to potentiate cisplatin ototoxicity (Oh et al, 2011). This model describes a secondary effect of cisplatin on TLR4 activation. While our data demonstrating that cisplatin toxicity responses depend, at least in part, on Tlr4 could be consistent with this model, our observations in the HEK-hTLR4 system suggest that cisplatin has a primary effect on TLR4 activation (e.g., co-receptor-independent TLR4 activation). To further characterize the effect of cisplatin in an ear outer hair cell line, we conducted kinetic analyses of Tlr4 activation to distinguish between primary (early) and secondary (later) effects. We examined IL-6 secretion over time in HEI-OC1 cells stimulated by the TLR4 agonists, cisplatin, and LPS. We observed that cisplatin- and LPS-induced IL-6 secretion followed similar kinetics for 4 h (Fig 4A). We confirmed that IL-6 secretion induced by cisplatin at early time points was dependent on Tlr4 by performing similar experiments in our Tlr4-deleted HEI-OC1 cells, complemented with either Tlr4 or an empty vector. Here we observed no significant IL-6 secretion over 8 h in this cell line unless complemented with Tlr4 (Fig 4B). Together, these data suggest that cisplatin is activating TLR4 in a primary manner. Figure 4. Cisplatin has a primary role in Tlr4 activation in HEI-OC1 cells IL-6 secretion in HEI-OC1 cells treated with 100 pg/ml LPS or 20 µM cisplatin (n = 3 or 4 independent biological replicates). IL-6 secretion in TLR4−/− cells transfected with empty vector (EV) or mouse Tlr4 (mTlr4) following treatme