Toll-like receptor 4 is activated by platinum and contributes to cisplatin-induced ototoxicity

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.

Keywords: TLR4; chemotherapy; cisplatin; hearing loss; ototoxicity.

Figure 1. Platinum and cisplatin activate TLR4 in vitro

1. 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).

2. 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).

3. IL‐8 secretion in HEK‐hTLR4 and HEK‐null2 cells following treatment with cisplatin at the indicated concentrations (n = 3 independent biological replicates).

4. 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 (25^th and 75^th 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.

Figure EV1. Cisplatin induces rapid MyD88‐dependent signaling and slower MyD88‐independent signaling downstream of Tlr4

1. 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).

2. 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.

Figure 2. Cisplatin activation of TLR4 is independent of the TLR4 co‐receptor, MD‐2

1. 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).

2. 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).

3. 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 (25^th and 75^th 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.

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.

Figure EV3. Tlr4 ^−/− HEI‐OC1 cells show diminished LPS‐responsiveness unless complemented with Tlr4

1. 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.

2. Anti‐TLR4 staining in Tlr4 ^−/− and control HEI‐OC1 cells. Bars (lower right) are 50 µm.

3. Flow cytometric analysis of conjugated LPS internalization in Tlr4 ^−/− and control HEI‐OC1 cells (n = 4 independent biological replicates).

4. 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.

Figure 3. Deletion of Tlr4 in a murine ear outer hair cell line (HEI‐OC1) reduces cisplatin‐induced ototoxic responses

1. 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.

Figure 4. Cisplatin has a primary role in Tlr4 activation in HEI‐OC1 cells

1. IL‐6 secretion in HEI‐OC1 cells treated with 100 pg/ml LPS or 20 µM cisplatin (n = 3 or 4 independent biological replicates).

2. IL‐6 secretion in TLR4^−/− cells transfected with empty vector (EV) or mouse Tlr4 (mTlr4) following treatment with 20 µM cisplatin (n = 6 independent biological replicates).

3. Il6 and Tlr4 transcript levels in HEI‐OC1 cells following treatment with 20 µM cisplatin for the indicated times (n = 3 independent biological replicates).

Data information: In all panels, data are presented as mean and standard deviation. Statistical comparisons to 0 h time point were assessed by 2‐way ANOVA. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (Dunnett’s test). Source data are available online for this figure.

Figure 5. Zebrafish Tlr4 mediates cisplatin‐induced ototoxicity in vivo

1. Hair cell viability in larval zebrafish (assessed by DASPEI staining) following cisplatin treatment at the indicated concentrations.

2. Hair cell viability in larval zebrafish pre‐treated with control‐, tlr4ba‐, and/or tlr4bb‐targeting morpholino oligonucleotides (MO) and subsequently treated with 15 µM cisplatin.

Data information: In both panels, each data point represents a score of hair cell integrity in an individual animal (taken from multiple samples per animal) with lines representing mean and standard deviation. Statistical comparisons between cisplatin concentrations (A) or to control morpholino (B, except as indicated in blue) were assessed by one‐way ANOVA. *P < 0.05; **P < 0.01; ****P < 0.0001 (Tukey test). Source data are available online for this figure.

Figure EV4. Cisplatin‐induced neuromast toxicity is reduced in independent tlr4bb knockdowns

Hair cell viability in larval zebrafish pre‐treated with control‐, splice‐targeting tlr4bb (MO1) or translation‐targeting tlr4bb (MO2) morpholinos prior to treatment with 15 µM cisplatin. Data information: Each data point represents a score of hair cell integrity in an individual animal (taken from multiple samples per animal) with lines representing mean and standard deviation. Statistical comparisons to control morpholino were assessed by one‐way ANOVA. ***P < 0.001 (Tukey test). Source data are available online for this figure.

Figure 6. Small molecule inhibition of Tlr4 mitigates cisplatin‐induced ototoxic responses in HEI‐OC1 cells

1. IL‐6 secretion in HEI‐OC1 cells pre‐treated with DMF vehicle (veh), 4 µM TAK242 (TAK), or left untreated (nil) following treatment with 10 ng/ml LPS or 20 µM cisplatin (n = 5–7 independent biological replicates).

2. ROS generation in HEI‐OC1 cells pre‐treated with DMF vehicle (veh), 4 µM TAK242 (TAK), or left untreated (nil) following treatment with 20 µM cisplatin (n = 3 independent biological replicates).

Data Information: In all panels, data are presented as mean and standard deviation. Statistical comparisons to nil treatment were assessed by 2‐way (A) or one‐way (B) ANOVA. *P < 0.05; **P < 0.01; ****P < 0.0001(Dunnett’s test). Source data are available online for this figure.