Bacterial and viral penetration of blood, tissues and interstitial fluids are typically detected by Toll-like receptors (TLRs) that trigger inflammatory signaling cascades to induce an overwhelming immune response to reduce the risk of pathogenic infection. TLRs are highly conserved pattern-recognition receptors present in diverse cell types, including immune, endothelial, epithelial, and fibrocytes (Atkinson, 2008). There are currently 11 human (and 13 mammalian) TLRs that share common transmembrane domains with leucine-rich repeats that bind to an overlapping array of extracellular (or endosomal) ligands, and a cytosolic signaling domain – the Toll-IL-1 Receptor (TIR) domain (Figure 3A). Here, we briefly review the signaling cascades activated by TLR4, the most studied TLR, and also TLR3.

TLR4 (a.k.a CD284, cluster of differentiation 284), was the first to have its specific ligand defined–lipopolysaccharides (LPS) from the cell wall of Gram-negative bacteria (Poltorak et al., 1998). TLR4 is constitutively expressed on the plasma membranes of monocytes, T cells, B cells, and dendritic cells, with induced expression in non-hematopoietic cells (Chakravarty and Herkenham, 2005). Extracellular, soluble LPS-binding protein (LBP) extracts LPS monomers from aggregates released from lyzed bacteria (Schumann et al., 1990). Bound LPS then complexes with CD14 (cluster of differentiation 14), a membrane-anchored glycoprotein, and extracellular lymphocyte antigen 96 (also known as MD2) to activate TLR4 (Shimazu et al., 1999). The complex facilitates picomolar detection of LPS, otherwise millimolar levels of LPS are required to activate TLR4 directly.

Activated TLR4 triggers one or more TIR domain-containing signaling adaptors: Myeloid Differentiation Primary Response Gene 88 (MyD88), TIR Domain-Containing Adaptor Protein (TIRAP), TIR-domain-containing adaptor inducing interferon-β (TRIF), and TRIF-related Adaptor Molecule (TRAM) that activate individual signaling cascades (Kawai and Akira, 2010; Kim and Sears, 2010; Juskewitch et al., 2012; Hamerman et al., 2016). These cascades are divided into MyD88-dependent (MyD88 and TIRAP), and MyD88-independent (TRIF and TRAM) signaling cascades (Figure 3B). The MyD88-dependent pathway signals through IL-1 receptor-associated kinase (IRAK)-4, transforming growth factor-β-activated kinase (TAK) 1, and TAK-binding protein 2 or 3 (TAB2/3) to activate mitogen-activated protein kinase (MAPK) downstream. This in turn leads to the transcription and expression of pro-inflammatory cytokines, such as TNFα, IL-1α, IL-1β, IL-6, or alternatively apoptosis (Takeuchi and Akira, 2009; Guo and Friedman, 2010). The MyD88-independent pathway activates IκB kinase (IKK) complex, releasing NF-κB for translocation to the nucleus and transcription of genes for expression of type 1 interferons (O’Neill et al., 2013). Another, complex TIR-domain-containing adaptor protein called Sterile α and HEAT (Armadillo motif; SARM) inhibits the TRIF-mediated (MyD88-independent) pathway, and attenuates LPS-mediated signaling to dampen inflammation and abrogate septic shock and multiple organ dysfunction syndrome (Aird, 2003). The time-course of plasma/serum expression profiles for individual acute phase inflammatory (API) cytokines and chemokines changes over hours and days following induction of the inflammatory response (Allan and Rothwell, 2001; Juskewitch et al., 2012).

Viral double-stranded DNA (dsDNA), mRNA, ionizing radiation or hypoxia can activate the ubiquitously expressed TLR3, located on both cell and endosomal membranes, to activate an adaptor protein called TRIF (Figure 3B) (Zarember and Godowski, 2002; Kawai and Akira, 2010). TRIF initiates two pathways via IKKα,β and TRAF-3. IKKα,β activates NF-κB subunits that translocate to the nucleus to initiate the transcription of genes for API cytokines and chemokines induced by MyD88 signaling, with differing expression profiles over time (Alexopoulou et al., 2001; Lien and Zipris, 2009; Kawai and Akira, 2010; Kishimoto, 2010). TRAF3 activates TBK1/IKKi to phosphorylate and homodimerize the transcription factors IRF-3 and IRF-7. These dimers then translocate to the nucleus to induce the expression of type I interferon-α (IFN-α [13 subtypes]) and IFN-β. After secretion, these interferons induce the expression of pro-inflammatory cytokines (Assmann et al., 2015). The IFN-α family, and IFN-β, influence a vast spectrum of biological functions, including inhibition of viral replication (Borden et al., 2007), and regulating the homeostatic differentiation of natural killer cells, dendritic cells, B cells, T cells, and osteoclasts (Farrar and Murphy, 2000). Activated IFN-β also phosphorylates the signal transducers and activators of transcription 1 (STAT1) protein (Imaizumi et al., 2016a). IFN-stimulated genes (ISGs) then induce and modulate various biological processes, especially anti-viral activities that target almost all steps in the lifecycle of a virus (Imaizumi et al., 2016a,b).

Aminoglycosides are readily endocytosed by specific and non-specific mechanisms (Myrdal et al., 2005). Megalin and cubulin are apical membrane receptors that can bind to aminoglycosides to induce endocytosis and are expressed in renal and cochlear epithelia, but not in hair cells (Tauris et al., 2009; Nagai and Takano, 2014). Mice lacking megalin show reduced renal uptake of aminoglycosides and attenuated aminoglycoside-induced nephrotoxicity (Nagai et al., 2001; Schmitz et al., 2002), and may represent a partial otoprotective mechanism by sequestering aminoglycosides from endolymph (Tauris et al., 2009). However, blocking endocytosis did not reduce hair cell death in vitro (Alharazneh et al., 2011). Blocking trafficking of aminoglycoside-laden endosomes to lysosomes exacerbates hair cell death suggesting that aminoglycoside-induced cytotoxicity proceeds upstream of endosomal and lysosomal activity, which may be partially cytoprotective (Esterberg et al., 2014; Hailey et al., 2017). Although inflammation enhances cochlear uptake of aminoglycosides across the BLB of cochlear endothelial cells, it remains to be determined if inflammation modulates transcytosis of aminoglycosides, especially when LPS exposure can reduce caveolin-mediated endocytosis in lung endothelial cells (Singla et al., 2011).

Endothelial cells and macrophages readily endocytose pathogens and particulates which induce inflammatory responses that further induce endocytotic processes (Majkova et al., 2010; Utech et al., 2010). Binding of the LPS-binding proteins complex to TLR4 induces endocytosis and induction of cytokine expression, as described above. Furthermore, this ligand-receptor binding is also endocytosed with downstream production of cytokines (Tan et al., 2015). In preclinical models, treatment with antibodies to TLR2 and TLR4 attenuate the inflammatory response and promote survival of severe experimental sepsis; however, side-effects include delayed healing from infection (Lima et al., 2015; Gao et al., 2017). Etanercept, an antibody that attenuates the TNFα-mediated inflammation triggered by TLR4, can acutely maintain cochlear blood flow and preserve hearing following acoustic overstimulation that typically induces cochlear inflammation (Arpornchayanon et al., 2013), and also cisplatin-induced cochleotoxicity (Kaur et al., 2011). Investigation of off-target side-effects will be crucial to determine the efficacy and safety of these approaches.

Any non-selective cation channel on the apical plasma membrane of hair cells (and supporting cells) bathed by endolymph, with a pore diameter larger than the maximum cross-sectional diameter of aminoglycosides (∼0.8–0.9 nm), is a candidate ion channel permeant to these drugs. These include the mechanoelectrical transducer (MET) channel of hair cells, and a variety of Transient Receptor Potential (TRP) channels, expressed by hair cells and supporting cells. There are seven subfamilies of TRP channels (TRPC, TRPM, TRPV, TRPA, TRPP, TRPML, and TRPN; all of which are found in mammals except for TRPN). At least four subfamilies are expressed in the cochlea, of which three subfamilies (TRPA, TRPC, TRPV) have a pore diameter larger than the maximum cross-sectional diameter of aminoglycosides, but not the fourth subfamily (TRPML). The very low concentration of calcium ions in endolymph increases the open probability of these non-selective cation channels, enhancing their permeability to aminoglycosides (Marcotti et al., 2005; Myrdal and Steyger, 2005; Karasawa et al., 2008; Banke, 2011). Furthermore, TRP channels can mediate inflammatory responses through multiple mechanisms, including interactions with other TRP channels, immunological receptors (e.g., TLR4) and signaling molecules such as pro-inflammatory cytokines (Numata et al., 2011). These are discussed below.

MET channels are big, multi-subunit complexes, including TMC1 and TMC2 (Kawashima et al., 2011), whose interactions are currently being unraveled, and subject to much debate. Nonetheless, their electrophysiological properties are well-characterized and many accessory components identified (Farris et al., 2006). The MET channels are permeable to a variety of aminoglycosides, including fluorescently tagged aminoglycosides (Marcotti et al., 2005; Coffin et al., 2009; Alharazneh et al., 2011; Vu et al., 2013). Genetic disruptions of essential components of the MET complex, e.g., myosin VIIa, or cadherin-23, reduce aminoglycoside uptake (Richardson et al., 1997; Vu et al., 2013). The conductance of MET channels, and therefore aminoglycoside permeation, can readily be modulated by extracellular cations, and permeant or impermeant MET channel blockers, e.g., tubocurarine, quinine (Farris et al., 2004; Coffin et al., 2009; Alharazneh et al., 2011), and are discussed elsewhere in this Research Topic (Kirkwood et al., 2017; O’Sullivan et al., 2017). The intracellular modulation of the MET channel current by inflammatory signaling (or by any other factors) remains to be determined and, if present, will have wider functional implications besides drug permeation into hair cells.

Transient Receptor Potential Ankyrin 1 (TRPA1) is an inflammatory, irritant, and oxidative stress sensor and has been indirectly localized to the basolateral membrane of outer hair cells (Kwan et al., 2006; Stepanyan et al., 2011). TRPA1 has a pore diameter of 1.1 nm, is dilatable to ∼1.4 nm, and is permeable to organic cations under the effect of agonists, see Tables 1 and 2 (Chen et al., 2009; Karashima et al., 2010; Banke, 2011). TRPA1 channels are required for the release of inflammatory neuropeptides and are activated by inflammatory agents released by damaged or diseased non-neuronal cells (Bautista et al., 2013). TRPA1 channels can be sensitized by inflammatory signals such as protein kinase A (PKA) and phospholipase C (PLC), which can include translocation of TRPA1 from vesicular stores to the plasma membrane (Schmidt et al., 2009). Endogenous TRPA1 agonists, such as methylglyoxal, 4-hydroxynonenal (4-HNE, a product and inducer of oxidative stress), 12-lipoxygenase-derived hepoxilin A3, 5,6-epoxyeicosatrienoic acid and reactive oxygen species (Table 2), are generated under various pathophysiological conditions activate TRPA1, contributing to peripheral neurogenic inflammation (Koivisto et al., 2014). In vitro experiments show that TRPA1 agonists, cinnamaldehyde, and 4-HNE increase outer hair cell uptake of fluorescent gentamicin (Myrdal and Steyger, 2005; Stepanyan et al., 2011). Thus, insults that induce oxidative stress in outer hair cells could potentially activate basolateral TRPA1 channels to enhance aminoglycoside uptake from the perilymphatic scala tympani, another depository of aminoglycosides in vivo (Tran Ba Huy et al., 1986; Ohlemiller et al., 1999). A cochlear expression map for TRPA1 is required to determine its potential involvement in inflammation enhanced cochlear uptake of aminoglycosides.

The Transient Receptor Potential Vanilloid (TRPV) subfamily includes TRPV1, the first TRP channel to be identified as candidate aminoglycoside-permeant channel (Myrdal and Steyger, 2005). TRPV1 has a pore diameter of ∼1 nm (Chung et al., 2008; Jara-Oseguera et al., 2008) and can be dilated by agonists (Bautista and Julius, 2008; Moiseenkova-Bell et al., 2008). TRPV1 is activated by high temperatures (>43°C), capsaicin, and protons, see Table 2 (Caterina et al., 1997; Vellani et al., 2001). Cell lines expressing TRPV1 co-incubated with capsaicin and streptomycin undergo rapid cell death (Caterina et al., 1997), suggestive of TRPV1-facilitation of aminoglycoside-induced cytotoxicity. TRPV1 is expressed in the cuticular plate, stereocilia, and cell bodies of hair cells and selected adjacent supporting cells (Zheng et al., 2003), as well as in marginal cells of the stria vascularis (Jiang et al., 2015). Thus, TRPV1 is expressed at key locations along the strial and endolymphatic trafficking route (Figure 4).

Involvement of TRPV1 in inflammation is well documented (Davis et al., 2000). Pro-inflammatory mediators up-regulate TRPV1 expression in chronic inflammatory diseases (Engler et al., 2007; Akbar et al., 2008; Cho and Valtschanoff, 2008). Inflammation can also mobilize the translocation of TRPV1 channels from the vesicular reservoir to the plasma membrane via exocytosis (Planells-Cases et al., 2011). Sensitization and translocation of TRPV1 to plasma membrane can also be induced by pro-inflammatory mediators, nerve growth factor and ATP released from damaged cells following tissue trauma (Julius and Basbaum, 2001; Ji et al., 2002; Zhang et al., 2005). Cytokines such as IL-1β, IL-6, and TNFα increase neuronal excitability via TRPV1 (Schafers and Sorkin, 2008; Miller et al., 2009). After kanamycin challenge, TRPV1 expression is unregulated in cochlear and vestibular sensory cells and neuronal ganglia (Kitahara et al., 2005; Ishibashi et al., 2009), and both native and fluorescently tagged gentamicin can permeate TRPV1 (Jiang et al., 2015).

These data suggest that acoustic overstimulation, or systemic inflammation, that induces cochlear expression of cytokines and chemokines, could sensitize or enhance the expression of TRPV1 at key locations to facilitate trafficking of systemically administered aminoglycosides across the stria vascularis into endolymph, as well as into hair cells independently of the MET channel (Li and Steyger, 2011; Li et al., 2011, 2015). Notably, TRPV1 plays a major role in cellular inflammation during cisplatin-induced ototoxicity, as described elsewhere in this Research Topic (Sheth et al., 2017). Whether an intracellular inflammatory-TRPV1 signaling pathway in hair cells occurs during systemic inflammation and/or aminoglycoside cytotoxicity remains to be determined.

TRPV4 is temperature-sensitive (25–34°C), and mechanically activated by osmotic swelling of cells, as well as by chemically agonists (see Table 2), like 4α-phorbol 12,13-didecanoate (Liedtke et al., 2000; Strotmann et al., 2000; Vriens et al., 2004). TRPV4 is expressed by hair cells in the region of the cuticular plate, stereocilia, and cell bodies of hair cells, as well as marginal cells and intermediate cells in the stria vascularis; in addition, TRPV4 is permeable to fluorescently tagged gentamicin (Karasawa et al., 2008). Thus, TRPV4 is expressed at key locations along the strial trafficking route into endolymph and hair cells (Figure 4). After kanamycin challenge, the expression of TRPV4 is downregulated in the inner ear sensory cells, neuronal ganglia and stria vascularis (Kitahara et al., 2005; Ishibashi et al., 2009), suggesting that TRPV4 does not enhance cochlear uptake of aminoglycosides during insult, and may represent an otoprotective response. Thus, sepsis-enhanced cochlear uptake of aminoglycosides must overcome any decreased trafficking resulting from inflammatory down-regulated expression of individual aminoglycoside-permeant ion channels.

TRPC3 and TRPC6 are canonical TRP channels expressed by hair cells, with a large (∼6 nm diameter) inner chamber (Mio et al., 2007; Goel and Schilling, 2010; Quick et al., 2012). Endothelial cells also express TRPC6, and activation by phosphoinositides or products downstream of reactive oxygen species induce translocation from the vesicular reservoir to the plasma membrane via exocytosis (Chaudhuri et al., 2016). This results in endothelial inflammation, increased cellular permeability and disrupted barrier function (Tauseef et al., 2012). Similar translocation and activation has been reported for other members of TRP channels too. For example, TRPC4 phosphorylation by Src family tyrosine kinases (STKs) following epidermal growth factor receptor stimulation, induces exocytotic insertion of TRPC4 into the plasma membrane (Odell et al., 2005) and TRPV4 translocation happens after shear stress in primary vascular endothelial cells (Baratchi et al., 2016). Thus, the roles of these TRP channels, and their permeability to aminoglycosides and trafficking across the BLB, especially during inflammation remain to be determined.

Vasodilation is a primary consequence of inflammation in order to facilitate extravasation of plasma (i.e., increased paracellular flux) into the interstitial space of tissues. However, in the tight junction-coupled blood-brain barrier and BLB, vasodilation occurs without major increases in paracellular flux. When inflammation-induced vasodilation in the BLB was abrogated in TLR4-hyporesponsive mice, aminoglycoside-uptake by the cochlea was also attenuated (Koo et al., 2015). Conversely, vasodilators like serotonin and ginkgo biloba enhance cochlear uptake of aminoglycosides and cochleotoxicity (Didier et al., 1996; Miman et al., 2002; Koo et al., 2011). Although, these vasodilators have other confounding cochlear effects, it is intriguing that downstream products of reactive oxygen species (e.g., 4-HNE, peroxidized lipids) also dilated cerebral arterioles via activation of TRPA1 (Sullivan et al., 2015). Thus, it will be important to untangle which feature of these polymodal events directly contributes to the increased strial endothelial uptake of aminoglycosides (Koo et al., 2015).