The Significance of TLR4 in Cisplatin-Induced Ototoxicity

Pediatric Cancer: Pediatric CancerCancer treatment is associated with a host of side effects and long-term health considerations; trauma associated with radiation therapy, surgery, and adverse drug reactions remains a hurdle in providing the best long-term health outcomes (Science 2019;363:1166-1169). Hearing loss in particular has been a side effect of cancer therapy since the discovery of platinum-based chemotherapeutic agents like cisplatin, which has been an indispensable tool in treating a multitude of cancers, including head and neck, cervical, ovarian, and lung cancers, among others. Cisplatin also contributes to an 80 percent survival rate in childhood cancer (Eur J Pharmacol 2014;740:364-378). However, this chemotherapeutic comes with its own subset of adverse drug reactions, of which the most prevalent and debilitating is irreversible hearing loss. The most affected demographic of cisplatin-induced hearing loss, or ototoxicity, is childhood cancer patients, with over half and up to 77 percent of children being impacted in some cases (Front Cell Neuro 2017;11:338; J Ped Hematol Oncol 2007;29:355-360). While it's recognized that cisplatin leads to the death of inner ear cells that are responsible for audio signal transduction, it's still unclear what specific factors are involved in initiating this phenomenon. Interestingly, the immune system may be a unique instigator of this adverse drug reaction. An immune system protein complex, Toll-like receptor 4 (TLR4), is usually involved in helping the body recognize pathogens and damage; however, it has also been shown to interact with metals like nickel and cobalt to mediate allergic reactions (Nat Immunol 2010;11:814; PLoS One 2015;10(3):e0120583). Cisplatin is a platinum-based drug, and because platinum shares chemical properties with nickel, this suggests that cisplatin may interact with TLR4 to mediate aberrant immune activation in the inner ear to initiate cell death. We conducted a study that sought to understand the significance of TLR4 in cisplatin-induced ototoxicity (CIO) and the potential of blocking cisplatin and TLR4 interactions for prospective therapies for childhood cancer patients (EMBO Reports 2021;e51280). Study Highlights To isolate the role of TLR4 in mediating CIO, we used an in vitro cell culture system of cells that either expressed the human TLR4 complex (HEK-hTLR4) or did not express the human TLR4 complex (HEK-null2). By using isogenic cell lines, we were able to attribute any discrepancies in responses directly to the presence of TLR4. We tested downstream markers of both TLR4 activation and cell stress with NF–κB and IL-8. NF–κB is an intracellular protein that upregulates immune proteins involved in inflammation, including IL-8, and can be stimulated by TLR4 activation. First, to examine if the TLR4 complex increases cell responsiveness to platinum, HEK-hTLR4 and HEK-null2 were both treated with platinum (II) and platinum (IV) alongside nickel and LPS, the canonical ligand of TLR4 (Front Immunol 2014;5:461). It was found that HEK-hTLR4 cells responded to platinum treatment with increased NF–κB and IL-8, indicating TLR4 activation, while HEK-null2 cells did not respond significantly to any of the treatments; this thereby indicated the involvement of TLR4 in these responses. Furthermore, HEK-hTLR4 cells also produced significant IL-8 in response to increasing cisplatin treatment while HEK-null2 once again did not respond, suggesting the TLR4 complex is also involved in responding to cisplatin, and this responsiveness could be attributed to its platinum-based structure. While the HEK cell isogenic models work well to demonstrate the specific contribution of TLR4 to cellular responses to cisplatin, inflammatory protein secretion was also measured in a more relevant model of hearing loss. Specifically, HEI-OC1 cells are a mouse inner ear cell line and a well-established model of CIO (Hear Res 2016;335: 105-117). TLR4 was genetically knocked down in HEI-OC1 cells, and its responses under cisplatin treatment were assessed in parallel to regular HEI-OC1 cells that did express TLR4. Similar to the experiments in HEK cells, regular HEI-OC1 cells had increased cell stress signals compared to the knockdowns, and there was also increased cell death in comparison to HEI-OC1 cells with a TLR4 deletion. These results corroborate the finding that TLR4 has a significant role in CIO in a site-specific model while further evidencing that their interaction might be more direct than previously thought. To determine whether cisplatin and TLR4 interactions are distinct from interactions of TLR4 with its canonical ligand, LPS, we investigated specific structural properties of the TLR4 signaling complex. Of the TLR4 receptor complex, one particular protein called MD2 works in conjunction with TLR4 to render full responsiveness to LPS in physiological systems. Based on this, we sought to understand if cisplatin exhibited similar characteristics, and intriguingly, when HEK cell lines expressing only TLR4 protein alone were treated with cisplatin, cells responded by secreting IL-8—as compared to stimulation with LPS, which could not elicit a response without MD2. Similarly, cell lines that express non-functional forms of MD2, like HeLa cells, respond to cisplatin but do not respond to LPS. This evidence demonstrates that cisplatin interacts with TLR4 in an MD2-independent way that is distinct from TLR4 interactions with ligands like LPS. Finally, to investigate the potential of protective therapies, we examined the effect of small molecule inhibitors on blocking cisplatin-TLR4 interactions and rescuing cells from stress and death. A commercially available TLR4 inhibitor, TAK242, was used to interrupt intracellular TLR4 complex activity in HEI-OC1 cells before treating with cisplatin or LPS. Inhibition of the TLR4 complex suppressed IL6 secretion from the treatment of both cisplatin and LPS, and production of reactive oxygen species (ROS) induced under cisplatin treatment was also effectively diminished with TAK242. Not only do these results substantiate the involvement of TLR4 in CIO, they also demonstrate that inhibition of TLR4 by small molecules can rescue cells from CIO and could be candidates for prospective otoprotective therapies.cisplatin with TLR4: Overview of proposed interaction of cisplatin with TLR4 to induce pro-inflammatory cytokine secretion and reactive oxygen species production. Cisplatin activation of TLR4 does not require the MD-2 co-receptor and can be inhibited by the TLR4 antagonist, TAK-242. Created with BioRender.Future Directions This study effectively demonstrates the substantial role that TLR4 plays in mediating CIO, and the interactions between cisplatin and TLR4 can be attributed to the platinum-based structure of cisplatin. Furthermore, it demonstrates the prospect of using small molecule inhibitors of TLR4 to interrupt cisplatin-TLR4 interactions. Being able to distinguish TLR4 interactions with cisplatin from TLR4 interactions with canonical ligands like LPS suggests that it might be possible to develop small molecule therapies that interrupt TLR4-cisplatin processes without interfering with natural TLR4 activity that is required for healthy immune functions. This opens up avenues into the application of small-molecule inhibitors as otoprotective therapies to potentially improve long-term health care outcomes for childhood cancer patients. ASNA LATIF, BSC, is a Master's student in immunology at the University of Alberta in Edmonton, Canada. AMIT P. BHAVSAR, PHD, is Assistant Professor and Tier 2 Canada Research Chair in the Department of Medical Microbiology & Immunology at the University of Alberta.Asna Latif, BSc: Asna Latif, BScAmit P. Bhavsar, PhD: Amit P. Bhavsar, PhD

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