Cochlear Immune Response in Presbyacusis: a Focus on Dysregulation of Macrophage Activity

Abstract

Age-related hearing loss, or presbyacusis, is a prominent chronic degenerative disorder that affects many older people. Based on presbyacusis pathology, the degeneration occurs in both sensory and non-sensory cells, along with changes in the cochlear microenvironment. The progression of age-related neurodegenerative diseases is associated with an altered microenvironment that reflects chronic inflammatory signaling. Under these conditions, resident and recruited immune cells, such as microglia/macrophages, have aberrant activity that contributes to chronic neuroinflammation and neural cell degeneration. Recently, researchers identified and characterized macrophages in human cochleae (including those from older donors). Along with the age-related changes in cochlear macrophages in animal models, these studies revealed that macrophages, an underappreciated group of immune cells, may play a critical role in maintaining the functional integrity of the cochlea. Although several studies deciphered the molecular mechanisms that regulate microglia/macrophage dysfunction in multiple neurodegenerative diseases, limited studies have assessed the mechanisms underlying macrophage dysfunction in aged cochleae. In this review, we highlight the age-related changes in cochlear macrophage activities in mouse and human temporal bones. We focus on how complement dysregulation and the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 inflammasome could affect macrophage activity in the aged peripheral auditory system. By understanding the molecular mechanisms that underlie these regulatory systems, we may uncover therapeutic strategies to treat presbyacusis and other forms of sensorineural hearing loss.

INTRODUCTION

Presbyacusis is a chronic multidimensional disorder that affects approximately 50% of people who are 75 years and older in the USA. This number is expected to grow as the population continues to age (National Academy of Science, 2016). Presbyacusis is defined by reduced hearing sensitivity and speech comprehension, especially in noisy environments, which diminishes central processing of acoustic information (Gates and Mills, 2005). Although hearing loss affects each person differently, adults experiencing hearing loss often report symptoms such as isolation, social withdrawal, fatigue, and depression (Kaland and Salvatore, 2002). In the past seven decades, characterization of presbyacusis pathophysiology has demonstrated that degeneration and dysfunction of multiple cochlear cell types are associated with the onset and/or progression of declines in auditory function. These cell types include sensory hair cells of the organ of Corti, spiral ganglion neurons and myelinating glial cells of the peripheral auditory nerve (AN), and non-sensory cells in the stria vascularis and spiral ligament of the cochlear lateral wall (Gratton et al. 1996; Gratton et al. 1997; Spicer et al. 1997; Spicer and Schulte 2002; Merchant 2010; Schuknecht and Gacek 1993; Schuknecht 1955, 1964; Fleischer 1972; Saitoh et al. 1995; Sha et al. 2008; Hao et al. 2014; Hequembourg and Liberman 2001; Kidd Iii and Bao 2012; Kusunoki et al. 2004; Liu et al. 2019a, 2019b; Makary et al. 2011; Ohlemiller et al. 2008; Shi 2016; Wu et al. 2020; Hilding 1953; Xing et al. 2012; Eckert et al. 2020). Although multiple factors and pathways contribute to presbyacusis, we do not fully understand the main causes of the condition.

Inflammaging refers to chronic and low-grade inflammation of tissues and/or organs. This common form of immune dysfunction occurs increasingly with age (Franceschi and Campisi 2014). Dysregulation of the immune response and inflammation (such as inflammaging) play critical roles in age-related neurodegenerative disorders (Verschuur et al. 2014; Watson et al. 2017). Macrophages are a key part of the innate immune system that have important roles in inflammaging and cochlear tissue degeneration in presbyacusis (Watson et al. 2017; Keithley 2019). In animal models, macrophages are present in several areas of the adult cochlea, including the organ of Corti (Frye et al. 2017), the AN (within the osseous spiral lamina, Rosenthal’s canal and modiolus) (Kaur et al. 2015, 2018; Lang et al. 2016; Brown et al. 2017; Panganiban et al. 2018), and the spiral ligament of the cochlear lateral wall (Hirose et al. 2005; Tornabene et al. 2006). Macrophages also closely interact with the strial microvasculature, a major part of the blood-labyrinth barrier that governs cochlear homeostasis. These innate immune cells direct the immune response as part of the neurovascular unit (Shi 2016; Neng et al. 2013; Zhang et al. 2013). The interactions of macrophages with other non-sensory cells in the stria regulate permeability of the blood-labyrinth barrier. This relationship is highly sensitive to injury, such as noise exposure that changes macrophage numbers, morphology, and activation states (see reviews by Shi 2016; Presta et al. 2018). Macrophage activation and changes in the innate immune system can lead to chronic inflammation that contributes to age-related changes in the cochlear microenvironment. This process plays a central role in degeneration and dysfunction of cochlear cells in presbyacusis.

Several reviews comprehensively described the macrophage-associated inflammatory response and inflammation in sensorineural hearing loss (Hirose et al. 2017; Jiang et al. 2017; Kalinec et al. 2017; Wood and Zuo 2017; Watson et al. 2017; Frye et al. 2019; Warchol 2019). In this review, we focus on changes in cochlear macrophage activities in mouse models of age-related hearing loss and human cochleae obtained from older donors. We will discuss two key molecular mechanisms that regulate the innate immune response and macrophage activity: the complement system and the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome. Both mechanisms are involved in various age-related neurogenerative disorders, such as age-related macular degeneration (Gao et al. 2015), cardiovascular disease (North and Sinclair 2012), and Alzheimer’s disease (Halle et al. 2008)^.. Thus, these mechanisms could be targeted to create therapies that prevent or slow disease progression. This review focuses on how complement dysregulation and the NLRP3 inflammasome may contribute to changes in macrophage activity and the loss or dysfunction of cochlear cells in presbyacusis.

IDENTIFICATION AND CHARACTERIZATION OF MACROPHAGES IN HUMAN COCHLEA

Although early studies of human temporal bone found resident macrophages in the endolymphatic sac (Altermatt et al. 1990; Rask-Anderson et al. 1991), around the vestibular dark cell region (Masuda et al. 1997), and in the middle ear mucosa (Lim 1976), it has been thought that the human cochlea is immune-privileged. At least two subtypes of the cochlear macrophages are present in embryonic cochlea of mice (Kishimoto et al. 2019): one that depends on Csf1r and originates from the yolk sac, and another that does not depend on Csf1r and originates from the fetal liver via the systemic blood circulation. Researchers have used several well-established animal models, during development and in response to cochlear injury, to identify and characterize macrophage/microglial cells in multiple regions of the cochlea. These studies have deepened our understanding of how these innate immune cells affect the development and maintenance of cochlear cells, and repair/regeneration after cochlear injury (Fredelius et al. 1990; Bhave et al. 1998; Sekiya et al. 2001; Hirose et al. 2005; Frye et al. 2017; Kishimoto et al. 2019; Kaur et al. 2015, 2018; Brown et al. 2017; Panganiban et al. 2018; Noble et al. 2019; Warchol 2019). To translate macrophage studies in animal models and develop therapeutic targets and diagnostic tools for people with presbyacusis, we must integrate studies of human and animal cochlear tissues.

Human and rodent macrophages/microglia share common proteins, including ionized calcium-binding adapter molecule 1 (IBA1). In humans, IBA1 is encoded by the allograft inflammatory factor 1 (AIF1) gene (Autieri et al. 1996). IBA1⁺ macrophages were found throughout the peripheral AN within the osseous spiral lamina and modiolus, and in Rosenthal’s canal of the human temporal bones (Fig. 1 (a); O’Malley et al. 2016; Liu et al. 2019b; Noble et al. 2019). IBA1-expressing cells were present in the outer sulcus and root cells of the cochlear lateral wall, adjacent to perivascular cells within the intermediate cell layer of the stria vascularis, and in the spiral ligament (Fig. 1 (b–d); O’Malley et al. 2016; Liu et al. 2019b; Noble et al. 2019). In the organ of Corti, IBA1⁺ macrophages were located below Hensen cells (O`Malley et al. 2016).

Fig. 1.

Location of macrophages in multiple areas of adult human cochlea. (a) IBA1⁺ macrophages are present in the auditory nerve (the eighth nerve; AN) within the osseous spiral lamina (OSL) and modiolus, and within the spiral ganglion (SG). IBA1-expressing cells are also located in the organ of Corti (arrows), within the stria vascularis (SV) and the outer sulcus and root cell (RC) regions, below the basilar membrane (M), and in the scala vestibuli on the surface of the bone (arrowheads). Images in (a) and (e) were modified from O’Malley et al. 2016. (b) IBA1⁺ cells are present in the SV and spiral ligament of the cochlear lateral wall in a whole-mount preparation. The images of a single macrophage on the right were taken from the suprastrial (I), strial (II), and substrial (III) regions. (c, c″) A super-resolution image of a macrophage within the SV showing a three-dimensional version of macrophages with ramified processes in the strial region. Images in (b, c, c″) were modified from Noble et al. 2019. The image in c″ is the enlarged image of the boxed area in c. (d, d”) An IBA⁺ macrophage within the SV was stained for major histocompatibility complex class type II (MHCII). The image in d″ is the enlarged image of the boxed area in d. Nu, nucleus. Images in (d, d″) were modified from Liu et al. (2019a, 2019b). CD68⁺ (e) macrophages in the SV within the intermediate cell layer (*) show ramified processes (arrows). The specimen in (b, c, c″) was obtained from a 57-year-old donor. Specimens in (a, e) were from donors ranging from 52 to 88 years old. Specimens in (d, d″) were from donors aged 40 to 70 years. BV blood vessel

In addition to IBA1, other macrophage/microglial markers have been used successfully in human temporal bone, including cluster of differentiation 163 (CD163), CD68, and major histocompatibility complex class type II (MCHII) (Fig. 1 (d–f); O’Malley et al. 2016; Liu et al. 2019b; Noble et al. 2019). CD163 is a transmembrane protein, and scavenger receptor expressed in the monocytic lineage, including monocytes, macrophages, and microglia (Lau et al. 2004). CD163 is a widely accepted marker for microglia with an activated state (Jurga et al. 2020). Similar to IBA1, CD163-expressing cells were found in several cochlear regions, such as in the SV (Fig. 1 (e)) and spiral ligament of the cochlear lateral wall, in the organ of Corti, along the AN within the modiolus, and around and within the spiral ganglia, endolymphatic duct, and several other cochlear regions (O’Malley et al. 2016). CD68 is a general microglial marker and is significantly upregulated in monocyte lineage cells during inflammation (Holness et al. 1993; Jurga et al. 2020). Similar to IBA1 and CD163, cells expressing CD68 are present in human cochlea; however, CD68 was not present fully in the cytoplasm, as seen with anti-IBA and anti-CD183 antibodies. In fact, CD68 can be difficult to identify because of its punctate pattern with immunostaining (Fig. 1 (e)). These results support previous work by Holness and Simmons (1993) in which immunoreactivity for CD68 was often found in cytoplasmic granules and that CD68 localizes mainly to lysosomes and endosomes of activated macrophages. MHCII, another activated state marker, mobilizes immune cells in pathological conditions (Lebedeva et al. 2005). Interestingly, many IBA1-expressing cells also express MHCII in the SV and spiral ganglion of human temporal bones (Fig. 1 (d, d″)) (Liu et al. 2019b).

The studies that found abundant cells expressing well-established macrophage/microglia markers (i.e., IBA1, CD68, CD163, and MHCII) in multiple cochlear locations were conducted mostly in human temporal bones with no known otologic pathology. These data suggest that the human ear does not have a completely “immune-privileged” site, and that macrophages may regulate cochlear physiology and pathophysiology in humans.

AGE-RELATED CHANGES IN MACROPHAGES/MICROGLIA OF MOUSE AND HUMAN COCHLEA

Macrophage/microglia maintains and restores tissue homeostasis and integrity by surveying the microenvironment and recognizing debris or pathogens (Aloisi 2001). Macrophages/microglia are phenotypically heterogeneous in other nervous systems (Olah et al. 2011; Tan et al. 2020). For example, two cellular forms of microglia were found in brain tissues (Davis et al. 1994). The activated state and function of microglia could be determined by their distinct morphology, gene expression pattern, and the production of pro-inflammatory or anti-inflammatory cytokines (Stence et al. 2001; Leone et al. 2006). Under physiological conditions, “resting” (or “ramified”) microglia are in a surveillance mode that allows them to sense the “well-being” of surrounding cells. In this mode, microglia are uniformly distributed with a highly branched morphology (e.g., multiple long branching processes and a small cellular body). Ramified macrophages/microglia perform limited phagocytosis and produce few immune molecules. With pathological conditions such as injury, activated microglia/macrophages have an amoeboid shape with no or only a few extended cellular processes. In this shape, macrophages can move freely as scavenger cells that phagocytose dying cells or cellular debris. These activated cells often signal cytokine activation that rapidly stimulates neighboring immune cells, which secrete tumor necrosis factor-α and increase inflammation (Aloisi 2001). In general, activated macrophages/microglia remove cellular debris during development and in adult tissue. Although ramified macrophages were originally considered resting macrophages and a precursor to activated macrophages, we now know that ramified macrophages also maintain extracellular fluid, transmitter inactivation, and synaptic pruning (Stevens et al. 2007).

Macrophages interact with sensory hair cells, non-sensory cells, and neural cells in the inner ear of several animal models of noise-induced hearing loss (see reviews by Hirose et al. 2017; Jiang et al. 2017; Kalinec et al. 2017; Wood and Zuo 2017; Watson et al. 2017; Hu et al. 2018; Frye et al. 2019; Warchol 2019). In human cochleae, macrophages have distinct morphology in different regions of cochlear specimens from donors who range from 52- to 88-year-old and have no known pathology other than the aging process (O’Malley et al. 2016). To better understand how macrophages and cochlear inflammation affect age-related degeneration of cochlear structures and declines in auditory function, we need to characterize changes in activation states and the phenotypic heterogeneity of macrophages in aged cochleae.

Unlike acute cochlear injury, such as noise-induced hearing loss, cochlear cell degeneration with aging is a chronic change. For example, a significant physiological loss of sensory hair cells, non-sensory cells, or neural cells in the AN may not be identified for months or even years. To understand how macrophages affect cochlear immunity in response to the slow degeneration of sensory hair cells, Frye et al. (2017) examined changes in the distribution, number, and morphology of macrophages in different age groups of C57BL/6 J mice, a model of age-related loss of sensory hair cells. As these mice aged, Frye et al. (2017) uncovered dynamic changes in macrophage number and morphology. Macrophages with an amoeboid shape spatially correlated with the degeneration of sensory hair cells, demonstrating that macrophage activation occurs at the sensory epithelium site where death of hair cells begins. These findings suggest that macrophages change their active state in response to the cochlear environment that is pathologically changed by sensory hair cell loss. They also suggest that the initial macrophage-derived inflammatory response may contribute directly to degeneration of sensory hair cells.

Macrophages may also be involved in degeneration of the SV in the cochlear lateral wall. In this region, the integrity of the strial microvasculature maintains the blood-labyrinth barrier and ensures proper function of cells within the SV, where the endocochlear potential (EP) is generated (Gratton et al. 1996; Shi 2016). With aging, the strial microvasculature shows pathological changes that include fewer capillaries, dilated capillaries, merged capillaries, thickened basement membrane, and altered transport of charged macromolecules (Gratton et al. 1997; Thomopoulos et al. 1997; Saitoh et al. 1995; Ohlemiller et al. 2008) (Fig. 2a). Age-related changes to macrophages and strial blood-labyrinth barrier occurred around the non-sensory cells in the cochlear lateral wall, suggesting that macrophage activation may contribute to strial microvasculature degeneration (Neng et al. 2015). Macrophages within the SV, also known as perivascular-resident macrophage-like melanocytes (Zhang et al. 2012), are a key part of the blood-labyrinth barrier and may play an important role in maintaining the ionic, osmotic, or metabolic balance needed for EP generation, cochlear structural integrity, and auditory function. As shown in Fig. 2a (left panel), IBA1⁺ macrophages directly interact with endothelial cells in the SV of young adult mice. These macrophages appear “ramified-like,” with multiple cellular processes physically intermingled with CD31⁺ endothelial cells of the strial capillary network, suggesting the involvement of macrophages in regulating blood-labyrinth barrier integrity. This hypothesis is supported by an elegant in vitro study by Neng et al. (2015) showing the presence of perivascular-resident macrophage-like melanocytes is necessary for endothelial cell tube formation and stability.

Fig. 2.

Age-related morphological changes in macrophages in the stria vascularis of mouse and human cochleae. The schematic (the top panel) shows two forms of macrophages (Davis et al. 1994). (a) Strial microvasculature (CD31+ endothelial cells, red) and GFP+ macrophages (green) in young adult and aged CX3CR1-GFP mice show macrophage pathology and their abnormal spatial relationships to the microvasculature. Images were taken from whole-mount preparations of the stria vascularis (SV). (b) Morphometric analysis of strial macrophages reveals significantly reduced volume and area in aged (n = 45) versus young-adult (n = 28) CX3CR1-GFP mice; *p < 0.01 (Student’s unpaired t-test). (c) Aged-related morphological alterations of macrophages in the spiral ligament (SpL) from tissues of CX3CR1-GFP mice. The macrophages were randomly selected from the whole-mount preparations of the spiral ligaments from young-adult and aged animals. (d) Strial macrophages from sections of human temporal bone show age-related changes like those seen in aged mice in (a). Age of donor is indicated in white at the top. Scale bars in (µm) = 20. Images in d were modified from Noble et al (2019)

To study age-related phenotypic changes in macrophages, we examined macrophages in the cochlear lateral wall of aged CX3CR1-GFP mice (Fig. 2a, b. c), which express EGFP under control of the endogenous Cx3cr1 locus. This mouse model has been used to examine pathological changes in macrophages in the peripheral auditory system (Hirose et al. 2005; Kaur et al. 2015). In contrast to the ramified shape in young adult mice, aged macrophages reduce their process number and cellular volume, and they appear less ramified (Fig. 2a, b). In addition, interactions (contacts) between macrophages and the microvasculature were disrupted in the SV of aged mice. In the SpL of these aged mice, macrophages were clearly transformed to an active state with an amoeboid shape (Fig. 2c). These findings aligned with a previous study showing that aged strial macrophages stain positive for the lectin griffonia simplicifolia (IB4), a marker of macrophage activation (Neng et al. 2015). This study also found significantly fewer macrophages in the SV of aged mice, which correlated with loss of capillary density. These observations suggest that the aging process significantly changes the state and phenotype of macrophages. They also suggest that deficiency (and/or dysfunction) of macrophages may disrupt the integrity of the blood-labyrinth barrier, resulting in enhanced cochlear inflammatory responses, cochlear cell degeneration, and hearing loss.

The ability to compare results from animal models and human temporal bones offers an unparalleled opportunity to address both basic science and translational questions about how cochlear macrophages and the innate immune system affect human presbyacusis. However, limited availability of human temporal bones that were properly prepared for cellular and molecular assays has hindered the progress of our investigations into the role of macrophages and the inflammatory response in the aged human cochlea. In a recent study, we compared 12 human temporal bones obtained from donors aged from 20 to more than 89 years. In these bones, we identified age-dependent changes in macrophage morphology in both the cochlear lateral wall and AN (Noble et al. 2019). We found that macrophages of both the SV and SpL have fewer processes and increased cytoplasmic volume around the nucleus in older donors than younger donors, suggesting that aged macrophages transform to a more active state (Fig. 2d). Although we did not see significant age-related changes in macrophage numbers in the cochlear lateral wall, we did see more macrophages in the AN of the middle turns of human temporal bones from older donors. We also saw more physical interactions between macrophages and myelinating glial cells and neuronal components in the ANs of the older donors (unpublished observation). These findings revealed that with aging, macrophage activation increases in the AN and cochlear lateral wall of the human inner ear, suggesting that activation of cochlear macrophages and chronic inflammation influence presbyacusis.

Understanding how inflammation and macrophage activation contribute to sensorineural hearing loss has become an important area of research (see reviews Hu et al. 2017; Kalinec et al. 2017; Watson et al. 2017; Wood and Zuo 2017). Our current knowledge of the cochlear immune response is largely centered on how macrophages are involved in cochlear development, and on their role in hearing loss after cochlear injury (Rai et al. 2020; Fredelius et al. 1990; Bhave et al. 1998; Sekiya et al. 2001; Hirose et al. 2005; Frye et al. 2017; Wood and Zuo 2017; Kishimoto et al. 2019; Kaur et al. 2015, 2018; Brown et al. 2017; Panganiban et al. 2018; Noble et al. 2019; Warchol 2019). To better understand the causes of pathophysiological changes in presbyacusis, we need more studies on the molecular mechanisms that mediate macrophage activation and abnormal interaction between macrophages and cochlear cells. Although many studies have uncovered molecular mechanisms that regulate microglia/macrophage dysfunction in multiple neurodegenerative diseases, few have deciphered the molecular mechanisms of macrophage dysregulation in aged cochleae. Here we will discuss two regulatory systems of inflammation and macrophage activity: complement dysregulation and the related NLRP3 inflammasome.

COMPLEMENT SIGNALING AS A REGULATOR OF MACROPHAGE FUNCTION

The complement system is a key component of the innate immune system that contributes to rapidly clearing dead or degenerating cells and pathogens by mounting a non-specific defense response to injury or other pathological conditions. Dysregulation of the complement cascade plays a vital role in age-related neurodegenerative disorders (Schnabolk et al. 2014; Armento et al. 2021). The complement system comprises more than 40 soluble and membrane-bound proteins. This system can be activated via three distinct routes: the classical pathway, the lectin pathway, and the alternative pathway (Fig. 3). All of these pathways converge on the cleavage of complement component 3 (C3), the most abundant complement protein (Merle et al. 2015; Sarma and Ward 2011). When cleaved, C3b fragments covalently bind receptors on the surface of foreign bodies and apoptotic cells, signaling phagocytes to engulf opsonized targets. Also, when C3b complexes generate, a C5 convertase begins to form, which triggers the membrane attack complex (MAC) that causes transmembrane pores to form on the cell surface, leading to cell lysis (Ramaglia et al. 2008; Sarma and Ward 2011). This rapid complement cascade helps phagocytic cells (such as macrophages) of the innate immune system recognize and eliminate pathogens.

Fig. 3.

Schematic of the three pathways of the complement system that converge on C3 to activate macrophages. The classical and lectin pathways involve cleavage of C4 and C2 to make C3 convertase. Activation of C3 induces production of the cleavage products C3a and C5a, which promote inflammation. The alternative pathway can spontaneously act on C3b through Factor B (FB) in a feedback loop. Activation of C3 and the terminal pathway ultimately leads to formation of the membrane attack complex (MAC) and cell lysis. Binding of C3a and C5a with their specific receptors on macrophages triggers release of pro-inflammatory signaling molecules, leading to macrophage activation

Three Complement Pathways

The classical pathway is triggered by cleavage of C1 into three molecules: C1q, C1s, and C1r. This cleavage prompts cleavage of C4 and C2 to form the C3 convertase, C4bC2a, and stimulates generation of the MAC complex (Sarma and Ward 2011; Fig. 3). Similarly, the lectin pathway is triggered when mannose-binding lectin (MBL) binds the surface of foreign cells, causing a conformational change that generates MBL-associated protein 2. Like C1q, MBL-associated protein 2 (MASP2) cleaves C4 and C2 to form C4bC2a, prompting C3 cleavage and activating the MAC pathway (Sarma and Ward 2011). Unlike the classical and lectin pathways, the alternative pathway does not require pathogens or immune complexes. In this pathway, complement is continuously activated to assist with the rapid and acute response to pathogens (Merle et al. 2015; Morgan and Harris 2015). When the alternative pathway is activated, C3 is spontaneously hydrolyzed and a C3bB complex forms with complement Factor B. This process occurs spontaneously and at a low continuous rate, which has been defined as “tick-over” (Thurman and Holers 2006). C3bB is then cleaved by complement Factor D to form a C3bBb convertase, activating the MAC pathway (Merle et al. 2015; Sarma and Ward 2011).

Synthesis of Complement Proteins and Macrophage Activation

Multiple cell types can synthesize complement proteins. Hepatocytes in the liver generate most of the complement molecules. Tissue resident and migratory immune cells, including macrophages, produce initiator complement components (Heeger et al. 2005; Peake et al. 1999; Strainic et al. 2008). In nervous tissue, microglia/macrophages, neurons, and glial cells express surface-bound and soluble complement proteins (de Jonge et al. 2004; Lian et al. 2016; Schafer et al. 2012). In the peripheral auditory system, the developing AN secretes high levels of classical complement components, demonstrating that complement signaling may play a key role in nerve development during hearing onset (Brown et al. 2017).

The target cell types for the bioactive complement subunits are immune cells, which express a variety of complement receptors and regulators on their cell surface (e.g., receptors C3aR and C5aR of macrophages) (Dustin 2016; Fig. 3). As such, the complement system is considered the primary effector of soluble proteins in the innate immune system (Fearon and Locksley 1996). Given the phagocytic nature typically attributed to macrophages, complement molecules assist with phagocytosis by targeting cells and debris for engulfment, a process called opsonization. For example, after light-induced retinal degeneration, deletion of the C5aR receptor results in reduced macrophage recruitment and complement component expression (Song et al. 2017). Deletion of the C3 convertase regulator receptor (Crry) induces microglial cell priming, and AD-mouse models deficient in Crry have enhanced amyloid beta plaque and accumulation of degenerating neurons (Ramaglia et al. 2012; Wyss-Coray et al. 2002). Furthermore, deficiency of complement regulator Cd59a leads to enhanced expression of complement components with age in the retinal pigment epithelium choroid (Herrmann et al. 2015). Thus, dysregulation of complement gene expression or protein function contributes to impaired activity of resident macrophages.

Complement Dysregulation in Age-related Neurodegeneration

Dysregulation of the complement cascade plays a vital role in age-related neurodegenerative disorders (Schnabolk et al. 2014; Armento et al. 2021). During aging processes, dysregulation of complement signaling may significantly contribute to cochlear pathology. A common aspect of aging includes recurrent insults (e.g., sterile inflammation may develop from repeated sound exposures over one’s lifetime). Exposure to lipopolysaccharides is a common model of inflammatory response in rodents. Long-term systemic exposure to lipopolysaccharides activates the classical complement signaling pathway and enhances the inflammatory microenvironment in the brain (Bodea et al. 2014). This persistent inflamed microenvironment precedes neural cell death and is rescued in C3-deficient mice (Kuehn et al. 2008). During age-related macular degeneration in these mice, localized inflammation, excessive leakiness, and neovascularization were dampened (Tan et al. 2015). In hypo-ischemic injury, immune cell activity and resulting neural cell loss are linked to the signaling of C1q and C3 (Ten et al. 2005). Although, C1qa-deficient mice did not show differences in developmental synaptogenesis nor auditory function (Calton et al. 2014), complement molecules (including C1s and Cfi) were enriched in the cochlea following noise injury (1 day after 120 dB sound pressure level and 2-h 1–7 kHz broadband noise exposure) (Patel et al. 2013). Consequently, cochlear aging may be due to dysregulated complement activation (e.g., upregulation of C3 and C1q; unpublished observations). This dysregulated complement-related inflammatory signaling would contribute to aberrant macrophage activity, resulting in damage to cochlear tissues (e.g., cochlear lateral wall and AN of aged cochlea) and declines in auditory function.

POTENTIAL ROLES OF NLRP3 INFLAMMASOME IN COCHLEAR CELL DEGENERATION

Inflammasomes are intracellular molecular complexes that are expressed in myeloid-derived cells, such as macrophages and microglia (Sarkar et al. 2009; Qu et al. 2007) (Fig. 4). Inflammasomes are triggered by infection or other cellular stress, resulting in maturation of pro-inflammatory cytokines that further activate innate immune defenses (Schroder and Tschopp 2010; Guarda et al. 2011).

Fig. 4.

Schematic of NLRP3 inflammasome activation associated with macrophages in presbyacusis. During aging, many pathological factors (e.g., TRL4) can evoke expression of Gal-3. Gal-3 then activates NFκB through a positive-feedback loop. Also, the presence of extracellular DAMPs and PAMPs triggers lysosomal damage as shown by Gal-3 aggregation on ruptured lysosomes. Furthermore, Gal3 prevents clearance of ruptured lysosomes and enhances generation of NLRP3 inflammasomes. Furthermore, NLRP3 inflammasomes can be activated by K⁺ efflux via binding the P2X7 receptor. K⁺ efflux leads to mitochondrial damage and ROS production, critical elements of inflammasome assembly. Activation of NLRP3 inflammasomes leads to maturation of IL-1β and IL-18, and activation of caspase 1. This pathway results in chronic neuroinflammation that may contribute to the degeneration of cochlear cells and presbyacusis

Inflammasomes use pattern recognition receptors (PRRs), which are expressed on macrophages, to detect microbial patterns and other danger signals. PRRs can be further delineated by their extracellular or intracellular location (Jo et al. 2016; Schroder and Tschopp 2010). Extracellular PRRs are represented by C-type lectins and Toll-like receptors (TLRs), which can detect pathogen-associated molecular patterns (PAMPs) (Jo et al. 2016). Intracellularly, PRRs can detect DNA and RNA. The signal cascade that occurs because of a PAMP or danger-associated molecular patterns (DAMP) binding a PRR differs depending on the unique combination of PRR and PAMP or DAMP (Schroder and Tschopp 2010). A specific subset of intracellular PRRs, known as nucleotide-binding oligomerization domain-like receptor (NLRs), can detect PAMPs similar to the PRRs discussed above. They can also detect endogenous signals that indicate DAMPs (Schroder and Tschopp 2010). A specific NLR, NLRP3, is involved with the NLRP3 inflammasome, which leads to the development of interleukin-1β (IL-1β) and interleukin-18 (IL-18) (Jo et al. 2016).

The NLRP3 inflammasome was discovered when researchers found a link between inflammatory conditions and mutations in the genes responsible for the NLRP3 inflammasome (Hoffman et al. 2001; Aganna et al. 2002). More specifically, genes for the NLRP3 inflammasomes are expressed within the cochlea (Nakanishi et al. 2017). Few studies have examined inflammasome-related pathways in cochlea, but several have studied the NLRP3 inflammasome in age-related chronic inflammatory conditions, such as age-related macular degeneration (Gao et al. 2015), cardiovascular disease (North and Sinclair 2012), and Alzheimer’s disease (Halle et al. 2008). Several DAMPs that activate the NLRP3 inflammasome are elevated with age (Cordero et al. 2018). Studies have proposed that the NLRP3 inflammasome pathway could promote caspase-1 and IL-1β to activate macrophages and microglia, thereby contributing to chronic sterile inflammation throughout the body with aging (Youm et al. 2013). These studies suggest that in age-related hearing loss, the NLRP3 inflammasome pathway contributes to macrophage activation and abnormal macrophage-glia interactions in cochlea (Noble et al. 2019).

NLRP3 Inflammasome Priming

The first step in activating the NLRP3 inflammasome is upregulation of NLRP3, also known as priming (Jo et al. 2016; Fig. 4). Several candidate pathways contribute to inflammasome priming in both aged and noise-exposed macrophages of cochlea, and they all converge on increased expression of nuclear factor kappa B (NFκB), which ultimately induces expression of NLRP3 (Schroder and Tschopp 2010). The NFκB pathway can be induced by interacting with TLR4. Indeed, TLR4 and NFκB downstream signals were upregulated after traumatic cochlear noise exposure (Zhang et al. 2019). Amyloid plaques, the hallmark of Alzheimer’s disease, also interact with TLR4, possibly contributing to inflammasome priming (Song et al. 2011). Further, the pro-inflammatory cytokine TNF was linked to inflammasome priming, which is significantly upregulated in aging people (de Gonzalo-Calvo et al. 2010; Bauernfeind et al. 2016). When macrophages derived from murine bone marrow were incubated with TNF for 2 h, NLRP3 expression increased, whereas IL-1β expression did not change. This result shows that the inflammasome was primed but not yet activated (Bauernfeind et al. 2016), and that the primed inflammasome was activated after being primed with TNF but not without TNF (Bauernfeind et al. 2016).

NLRP3 Inflammasome Activation

Once primed, NLRP3 can be activated by PAMPs or DAMPs. The NLRP3 inflammasome responds to a variety of DAMPs, many of which are found in aging tissues (Cordero et al. 2018). These DAMPs include ATP (Sadatomi et al. 2017), amyloid-β fibrils (Halle et al. 2008; Abderrazak et al. 2015), and reactive oxygen species (ROS) (Abderrazak et al. 2015). In some tissues, such as blood monocytes, stimulation of the NFκB pathway primes and activates the NLRP3 inflammasome. However, in macrophages, the NFκB pathway only primes; a second signal is still needed for activation (Netea et al. 2009).

An array of molecules can activate the NLRP3 inflammasome, suggesting that these molecules may not directly interact with NLRP3, but rather rely on common triggers (Yang et al. 2019). One common trigger is the fluctuation of varying ions, such as the efflux of K⁺ and Cl⁻ (Muñoz-Planillo et al. 2013; Green et al. 2018), influx of Na⁺(Gianfrancesco et al. 2019), and Ca⁺ variations (Murakami et al. 2012). Extracellular ATP causes efflux of K⁺ by binding the P2X7 receptor, a ligand-gated ion channel (Próchnicki et al. 2016). Also, adding extracellular ATP led to increased P2X7 receptors in cochlea and reduced EP (Muñoz et al. 1995; Housley et al. 1999).

Another trigger is the production of ROS (Zhou et al. 2011). We do not fully understand how ROS activates the inflammasome, but a newly discovered serine-threonine kinase, NEK7, interacts with the NLRP3 inflammasome and regulates ROS signaling (Shi et al. 2016). Also, the anti-inflammatory interleukin-10 prevented activation of NADPH oxidase in microglia, which reduced synthesis of NLRP3 and its downstream effectors (Gao et al. 2020). Also, traumatic noise exposure induces production of ROS in cochlea (Shi et al. 2003; Yamashita et al. 2005; Henderson et al. 2006). ROS levels are also higher in aging neural tissue, implicating the inflammasome in age-related hearing loss (Lee et al. 2000). Moreover, in the senescence-accelerated mouse prone 8 model, which shows premature hearing loss and cochlear neural degeneration, lipid peroxidation products were significantly higher at 9 months of age. This finding indicates increased oxidative stress (Menardo et al. 2012), which could contribute to NLRP3 inflammasome activation and, ultimately, chronic inflammation.

Lysosomal destabilization influences neurodegenerative diseases and may activate the NLRP3 inflammasome (Hoffman et al. 2001; Hafner-Bratkovič et al. 2012). Lysosomal destabilization occurs during the normal aging process, which could link increased activation of macrophages to aging cochlea (Shi et al. 2003; Yamashita et al. 2005; Henderson et al. 2006). A molecule that may affect this process is galectin-3 (Gal-3). This β-galactoside-binding protein, originally named Mac-2, was discovered on thioglycolate-elicited macrophages (Ho and Springer 1982; Dumic et al. 2006). Gal-3 contains a C-terminal domain that recognizes carbohydrates (common in all galectins), and a unique N-terminal domain that supports the formation of oligomers (Dumic et al. 2006). Plasma levels of Gal-3 are elevated in several neurodegenerative diseases, such as Huntington’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis (Wang et al. 2015; Yan et al. 2016; Siew et al. 2019). Gal-3 expression is also correlated with aging. Indeed, immunohistochemistry of brain tissues from aged monkeys showed greater levels of Gal-3-positive microglia than that of young controls (Shobin et al. 2017). Also, single-cell RNA sequencing of microglia revealed upregulated Lgals3, the gene coding for Gal-3, in aged microglia (Hammond et al. 2019). In a mouse model of Huntington’s disease, Gal-3 puncta were seen with lysosomal-associated membrane proteins (Siew et al. 2019), and these Gal-3 puncta indicate lysosomal destabilization (Aits et al. 2015). Gal-3 also acts as a sensor by attaching to the normally luminal β-galactosidases of lysosomes when they become exposed after loss of structural integrity (Siew et al. 2019). Based on co-immunoprecipitation experiments, Gal-3 interacts with the adapter apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) of the NLRP3 inflammasome to aids in its activation (Simovic Markovic et al. 2016; Chen et al. 2018). Gal-3 also induces and is promoted by the NFκB pathway in a positive feedback loop, which could aid in priming the NLRP3 inflammasome (Zhao et al. 2017; Siew et al. 2019; Fig. 4). In Huntington’s disease, diabetic retinopathy, and Alzheimer’s disease, Gal-3 knockout markedly reduced inflammation (O’Malley et al. 2016). Multiple studies have linked Gal-3 with microglia and neurodegenerative disorders, but few have linked the molecular changes of the cochlea and hearing loss. Although no reports describe protein expression of Gal-3 in cochlea, a study that immunostained aged human cochlea for CD68, a marker of the lysosomal/endosomal-associated membrane glycoprotein, found punctate staining patterns that are often associated with Gal-3 (Salminen et al. 2012). Activated macrophages and lysosomal punctate staining patterns in cochlea suggest that Gal-3 may affect presbyacusis, similar to other neurodegenerative disorders. Thus, Gal-3 is a promising target for future research on the connection between activated macrophages and lysosomal destabilization in the cochlear pathophysiological conditions (Salminen et al. 2012; Noble et al. 2019).

Downstream Effects of the NLRP3 Inflammasome

When the NLRP3 inflammasome is primed and activated, the complex recruits ASC, which then recruits and activates pro-caspase-1 to form a protein complex (Bergsbaken et al. 2009). Pro-caspase-1 then undergoes cleavage and activation, which completes the maturation of pro-inflammatory cytokines IL-1β and IL-18 (Youm et al. 2013). The activated caspase-1 then initiates pyroptosis, a cell death cascade that starts by forming plasma-membrane pores that lead to the loss of ion homeostasis. This process ultimately culminates in cell lysis and failure to contain the pro-inflammatory intracellular contents (Wang et al. 2019; Fig. 4). Pyroptosis is one way that the NLRP3 inflammasome contributes to neural degeneration (Lee et al. 2019). Neuron cell death is triggered by IL-1β production, which increases recruitment of additional microglia and intensifies their neurotoxicity capabilities (Qiao et al. 2017). Also, caspase-1 can induce the caspase-7/PARP1/AIF pathway, contributing to neuron cell death in Parkinson’s disease (Jha et al. 2010). In Parkinson cell lines, overexpression of caspase-7 diminished the anti-apoptotic effects of caspase-1 inhibitors (Hoffman et al. 2001).

As we have already described, the NLRP3 inflammasome could be activated in multiple ways that result in cochlear inflammation. Other factors that could link the NLRP3 inflammasome to cochlear inflammation include gain-of-function mutations on cochlear macrophages. In one study, researchers performed genetic mapping on patients with auto-inflammatory diseases known as cryopyrin-associated periodic syndromes. In these patients, they discovered a gain-of-function mutation in the NLRP3 inflammasome that leads to activation of the inflammasome without an activation signal (Nakanishi et al. 2017; Nakanishi et al. 2018). This aberrant activation in cochlear macrophages leads to increased IL-1β production and sensorineural hearing loss (Goldbach-Mansky et al. 2006; Nakanishi, et al. 2017). Interestingly, other studies found that blocking IL-1β could improve or completely reverse hearing loss, implicating IL-1β as a major cause of hearing loss (Goldbach-Mansky et al. 2006; Nakanishi et al. 2017).

CONCLUSIONS

Traditionally, the environment of the human inner ear was considered immune-privileged. With abundant evidence, we now know that this paradigm is outdated and invalid. We have gained an understanding of how macrophages migrate and persist during development, and are present with other immune cells. However, we still lack the knowledge of how precisely macrophages influence cochlear maturation and age-related structural and functional maintenance and degeneration.

Based on previous observations, activated cochlear macrophages have an intricate relationship with resident cells in both mouse and human ears. This relationship suggests that dysregulation of immune cells influences pathophysiological changes in many cochlear cell types in aged cochlea, particularly in the cochlear lateral wall and AN. These changes are similar to the inflammaging contributions of macrophages and microglia seen in other age-related neurodegenerative disorders, such as age-related macular degeneration, Alzheimer’s disease, and Parkinson’s disease (Denver and McClean 2018; Yang et al. 2019; Jha et al. 2010).

Complement signaling has a clear effect on age-related and neurodegenerative disorders in mouse models (Lian et al. 2016; Qin et al. 2007; Tan et al. 2015; Bodea et al. 2014; Scholl et al. 2008). Complement molecules were associated with synapse loss, neural cell death, and inflammatory microenvironments in models of Alzheimer’s disease, age-related macular degeneration, and multiple sclerosis (Qin et al 2007; Tatomir et al. 2017; Armento et al. 2021). Similar characteristics were identified in aged human cochlea, suggesting that dysregulation of the complement system contributes to inflammaging of the cochlea and promotion of presbyacusis.

Comparably, the NLRP3 inflammasomes affects macrophage and microglia activation in neurodegenerative disorders (Jha et al 2010; Lee et al 2019; Bauernfeind et al 2009; Bauernfeind et al 2016). The presence of NLRP3 inflammasomes in the cochlea (Nakanishi et al. 2017) suggests that macrophage-derived inflammasomes may have a role in exacerbating damage in the cochlea during presbyacusis. Additional studies of immune cells in aged cochlea may reveal potential therapeutic strategies for targeting macrophages/microglia to treat presbyacusis. Furthermore, targeting inflammatory signaling, such as complement pathways or NLRP3 inflammasome priming and activation, in aged cochlea may uncover new therapeutics for treating presbyacusis and other forms of sensorineural hearing loss.

Funding

This work was supported by grants from the National Institutes of Health including P50DC000422 (H.L.), SFARI Pilot Award #649452 (H.L.), and K18 DC018517 (H.L.).

Declarations

Competing interests

The authors declare no competing interests.