Although often referred as neuronal ion channels, ASICs are expressed in numerous types of immune cells (Huang et al., 2010; Tong et al., 2011; Kong et al., 2013; Yu et al., 2015) and they are potentiated by molecules associated with neuroinflammation such as arachidonic acid, histamine, lactate, and nitric oxide (Immke and McCleskey, 2001; Cadiou et al., 2007; Smith et al., 2007; Rash, 2017; Shteinikov et al., 2017). Acidity can provoke different responses in immune cells depending on the source of the acidosis, as exemplified in Table 2 for macrophages. Therefore, whether or how inflammation is triggered by acidity depends on the source of the lowered pH. As proton-coupled monocarboxylate transporters (MCTs) shuttle lactate in a 1:1 ratio with H⁺ (Hertz and Dienel, 2005), altering lactate levels will have secondary effects on the H⁺ concentration.

Neutrophils exposed to acidic pH undergo various functional changes, including significantly impaired migration, defective chemotaxis, reduced speed of apoptosis, increased CD18 expression, and increased phagocytosis but with decreased bactericide action (Rotstein et al., 1988; Trevani et al., 1999; Cao et al., 2015). The combination of acidosis-induced decreases in migration, chemotaxis and apoptosis together with increased phagocytic activity might suggest that once neutrophils enter a region of low pH, they become trapped there with enhanced lifespan and inflammatory activity. CD18 is a cell-surface molecule expressed by neutrophils that promotes cell adhesion, a process that allows neutrophils to aid tissue repair in the periphery. Altered expression of CD18 is believed to facilitate neutrophil breach into the CNS (Serrano et al., 1996; Neri et al., 2018). The acidic blood pH after trauma (as low as 6.6) likely induces altered CD18 expression on peripheral neutrophils which then undergo facilitated extravasation into the CNS, where they accumulate in acidic regions (Mitra et al., 2012). Furthermore, in patients with SCI, neutrophils exhibit a higher “respiratory burst” in the blood (Kanyilmaz et al., 2013). The myeloperoxidase (MPO) produced by neutrophils enhances conversion of H₂O₂ to HOCl, and thus contributes to the secondary damage after SCI (Kubota et al., 2012). Neutrophils in the blood of SCI patients in rehabilitation have decreased ability to phagocytose Escherichia coli and Staphylococcus aureus (Campagnolo et al., 1997; Kanyilmaz et al., 2013). These data suggest that, at least for neutrophils, typical immune responses are altered in the presence of acidity and this may explain why neutrophils can have a detrimental impact in the CNS after neurotrauma.

Vermeulen et al. (2004) and Martínez et al. (2007) found that murine and human DCs mature upon exposure to acidity. Acidosis modifies the inflammatory profile of monocytes and macrophages, and is proposed to induce an inflammatory state in the latter (Riemann et al., 2016). Microglial motility decreases after exposure to acidic pH (Faff and Nolte, 2000). Rat macrophages exposed to acidic environments (pH below 7.4) upregulate nitric oxide synthase (NOS), and thus increase production of NO (Bellocq et al., 1998). Excess amounts of NO are detrimental in neurodegenerative pathologies such as HD and after striatal lesions, causing DNA damage and oxidative stress to cells (Schulz et al., 1996; Browne and Beal, 2006; Calabrese et al., 2007). These responses are discussed further below when considering the role of ASICs.

Decreased extracellular pH, therefore, can modify the function of immune cells by altering motility, impacting phagocytosis, aiding CNS invasion, and increasing the production of potentially neurodamaging agents such as NO. Although the function of neutrophils is modified by acidity, there are, to date, no studies on whether they express ASICs. Thus, the role of ASICs in acidosis-induced responses of neutrophils is currently unknown.

The key immune cell receptors involved in the innate immune response are complement and cytokine/chemokine receptors and pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). The PRRs recognize both PAMPs and also DAMPs which are produced by dying or damaged cells and trigger sterile inflammatory processes. As part of the adaptive immune response, the TCRs and antibody-binding receptors are involved in cascades that facilitate the immune cell response. Immune cells also possess ion channels that are important for their function, including the voltage-gated potassium channel Kv1.3, the calcium-activated potassium channel KCa3.1, calcium release-activated calcium channels (CRAC), and transient receptor potential (TRP) channel M7 (Cadiou et al., 2007; Chandy and Norton, 2017; Chiang et al., 2017; Nadolni and Zierler, 2018; Clemens and Lowell, 2019). The function of these ion channels in immune cells and their contribution to pathology have been reviewed (Feske et al., 2015; Vaeth and Feske, 2018) but neither of these reviews discuss the presence or role of ASICs in immune cells.

Microglia are the main immune cell type in the CNS. Although considered CNS-resident macrophages, microglia branch off earlier in development from yolk sac precursors (Ginhoux et al., 2010; Schulz et al., 2012; Kierdorf et al., 2013). In non-pathological conditions, microglia are involved in clearing tissue debris and surveying the environment (Nimmerjahn et al., 2005). During neuropathology, microglia can enter an activated state and release enzymes such as NADPH oxidase, which causes neuronal damage. Microglia can additionally modify myelination, affecting developmental myelination as well as beneficial re-myelination processes (Popovich et al., 2002; Butovsky et al., 2006). The divergent context-specific roles of microglia in CNS pathologies is an emergent field and has been recently reviewed (Norris and Kipnis, 2019; Butler et al., 2021).

It has been known for some time that the severity of disease progression in MS patients is linked to microglial activation (Heppner et al., 2005; Jack et al., 2005; Geladaris et al., 2021), and it seems that microglial phagocytosis of stressed neurons contributes to neurodegeneration in AD, PD, ischemic stroke, CNS viral infections, and aging (Butler et al., 2021). Rat microglia have been shown to express ASIC1, ASIC2a and ASIC3. In cultured primary rat microglia stimulated with lipopolysaccharide (LPS), a known immune activator, ASIC1 and ASIC2a expression increased both at the surface and intracellularly (Yu et al., 2015) and ASIC-specific inward currents could be recorded using whole-cell patch-clamp electrophysiology. Both PcTx1 and amiloride reduced the amplitude of these currents and reduced the expression of inflammatory cytokines (Yu et al., 2015). Treatment of cultured primary microglia with amiloride also decreased the extent of phagocytosis under acidic conditions (Vergo et al., 2011). A decrease in phagocytosis was also seen in microglia cultured from ASIC1^–/– mice, with no change upon the addition of amiloride, indicating that the effect of amiloride is ASIC1-mediated.

These data collectively show that ASICs, and ASIC1a specifically, are present in microglia and contribute to their function in acidic microenvironments. In turn, this indicates that microglial ASIC1a contributes to microglia function, affecting release of inflammatory cytokines and phagocytosis, potentially impacting the immune response in the CNS during MS and many other CNS pathologies.

Astrocytes fulfill a variety of roles in the CNS, such as regulating the formation and maintenance of the BBB (Abbott et al., 2006). After brain injury and disease, astrocytes can become reactive, participating in repair cascades and gene upregulation, and forming an astroglial scar (Zamanian et al., 2012; Anderson et al., 2016). Reactive astrocytes are key players in CNS disease, an area reviewed recently by Dossi et al. (2018). Stimulation of astrocytes by cytokines such as interferon gamma (IFN−γ) leads to expression of the antigen-presenting major histocompatibility complex II (MHC II) on the cell surface alongside other costimulatory molecules such as intercellular adhesion molecule 1 (ICAM-1) (Wong et al., 1984; Lee et al., 2000). MHC II expression occurs in MS plaques and may contribute to the inflammatory cascade through antigen presentation to T cells (Zeinstra et al., 2000). Ponath et al. (2018) recently reviewed the differing roles of astrocytes in MS.

ASIC1, ASIC2a, ASIC3 are expressed in the nucleus of rat astrocytes, and astrocytes express an ASIC-like current that is blocked by amiloride (Huang et al., 2010; Yu et al., 2015). Yang et al. (2016) noted increased ASIC1a expression in the membrane and cytoplasm of reactive astrocytes from the hippocampi of deceased TLE patients, findings which were replicated in a mouse model of chronic epileptogenesis. To explore normal astrocytic function, the group used primary cultures from wild-type mice. At 24 h after stimulation with LPS, astrocytes upregulated ASIC1a and exhibited increased Ca²⁺ influx upon exposure to pH 6.0, which was significantly reduced by PcTx1.

Yang et al. (2016) showed that seizure frequency in TLE mice is reduced by knockdown of astrocytic ASIC1a, with restoration of the ASIC1 gene increasing the frequency of spontaneous seizures. Thus, in contrast to what is believed regarding epilepsy and the beneficial function of ASIC1a (likely via neuronal expression), these data suggest that activation of astrocytic ASIC1a causes pathological Ca²⁺ influx and contributes to the pathogenesis of TLE.

Oligodendrocytes deliver critical trophic support to neurons, producing the proteins and lipids that comprise the myelin sheath that wrap around and insulate axons, and they engage in significant cross-talk with microglia during the process of myelination (Peferoen et al., 2014). Oligodendrocytes secrete cytokines such as C-C motif chemokine ligand 2 (CCL-2) and IL-8 in response to neuroborreliosis, the neurological manifestation of Lyme’s disease (Ramesh et al., 2012). IL-8 and CCL-2 are chemotaxic for a number of peripheral immune cells, such as neutrophils and T cells respectively (Turner et al., 2014).

Oligodendrocytes are especially sensitive to ischemic insults (Bradl and Lassmann, 2010), and resident ASICs may contribute to ischemia-induced injury of these cells. ASIC1 is upregulated in oligodendrocytes in chronic brain lesions of patients with MS, and use of amiloride in patients provided neuroprotection at primary stages of the disease (Arun et al., 2013). ASIC1a, ASIC2a/b (non-selective primers were used, and thus ASIC2a and ASIC2b could not be distinguished), and ASIC4 mRNA were found in oligodendrocytes, and ASIC-specific inward currents recorded from oligodendrocyte lineage cells (OLCs) were inhibited by PcTx1 (Feldman et al., 2008). Cultured mouse oligodendrocytes express ASIC1 (ASIC1a and ASIC1b were not separable) and they were protected from acidosis-induced damage by PcTx1, as were oligodendrocytes derived from ASIC1 KO mice (Vergo et al., 2011). Thus, the combined data suggest that ASIC1 activation contributes to oligodendrocyte injury during and after periods of tissue acidosis.

Macrophages are key players in inflammation and autoimmune disease. They produce NO under acidic conditions and promote demyelination in the CNS (Bellocq et al., 1998; Yamasaki et al., 2014; Li and Barres, 2018). RT-PCR and Western blots were used to demonstrate expression of ASIC1 and ASIC3 in the cytoplasm of macrophages (Kong et al., 2013). When exposed to acidic conditions (pH 6.5), bone marrow-derived macrophages (BMMs) increase their rate of phagocytosis (Kong et al., 2013). Extracellular acidosis also causes an upregulation of key cell surface markers related to BMM maturation (e.g., CD80, CD86 and MHC II). These effects were blocked by application of amiloride before exposure to tissue acidosis. Interestingly, expression of IL-10—thought to be a beneficial anti-inflammatory cytokine—was increased in macrophages after acid exposure.

Thus, the limited available data suggest that ASICs can modulate the macrophage response to acidic microenvironments. CNS macrophages/monocytes are believed to be a primary contributor to MS demyelination, and ASICs may contribute to their response in this pathology (Yamasaki et al., 2014).

T cells play a major role in coordinating and executing multiple functions of the adaptive immune response. Autoreactive T cells that have lost their ability to differentiate self (i.e., host cells and antigens) from non-self (i.e., foreign/pathogenic cells and antigens) are major players in autoimmune diseases such as MS. In the first study that identified a role for ASIC1a in the neuropathology of MS, ASIC1b, ASIC3 and ASIC4 were identified at the mRNA level in mouse T cells, with the presence of ASIC1 protein confirmed in these immune cells via Western blot (Friese et al., 2007). In this study, no obvious function for ASIC1 in T cells was observed based on responses of wild-type and ASIC1 knockout T cells in terms of proliferation and cytokine secretion. Thus, the role of ASICs in T cells remains to be determined, but they do not seem to play a role in these cells in the context of MS, at least not in the mouse EAE model.

Mature DCs are professional antigen presenting cells that constitute a key part of the inflammatory cascade, forming a major link between the innate and adaptive immune systems. The expression of MHC II and co-stimulatory molecules such as CD86 on their cell surface allows them to present antigens to T cells leading to T cell activation and proliferation (Reis e Sousa, 2006; Dudek et al., 2013).

When mature DCs are exposed to acidic conditions (pH 6.5) for a moderate time period (∼4 h), the expression of their specific cell-surface markers is increased (Vermeulen et al., 2004). Human DCs exposed to acidosis increase production of proinflammatory IL-12 and the authors suggest that this triggers a bias toward a proinflammatory Th1 response (Martínez et al., 2007). The acidosis-induced increase in cell-surface marker expression was replicated using cultured DCs derived from mouse bone marrow, a response that was blocked by amiloride (Tong et al., 2011). Using non-specific antibodies for ASIC subtypes (i.e., not distinguishing between splice isoforms), Tong et al. (2011) found that mouse DCs express ASIC protein in the cytoplasm (ASIC1 and ASIC2), on the plasma membrane (ASIC2), in the endoplasmic reticulum and perinuclear regions (ASIC1), and in the mitochondria (ASIC3). Functional surface expression of ASICs was confirmed using patch-clamp electrophysiology experiments and the observation of amiloride-sensitive, acidosis-induced inward currents with a pH₅₀ of ∼6.0. Acidosis (pH 6.5) increased the antigen-presenting ability of DCs as assessed by increased ability to stimulate T cell proliferation, and this effect was blocked by amiloride (Tong et al., 2011).

These results strongly suggest that ASIC activation on DCs enhances expression of cell-surface proteins involved in antigen presentation and subsequent T cell activation and proliferation. As T cells can be highly destructive to healthy tissue in chronic inflammatory conditions, acidosis-enhanced communication between these two CNS-invading peripheral immune cells may exacerbate CNS damage in disease (Korn and Kallies, 2017).

Data from the human binary protein interactome (Luck et al., 2020) indicate an interaction between ASIC1a and the killer cell immunoglobulin-like receptor 3DL3, a key regulator of NK cell function. Reduced levels of NK cells have also been implicated in depression (in which immune responses tend to be impaired). Brain acidity is altered in several mental health conditions (Coryell et al., 2009; Suzuki et al., 2017), but it is not yet clear whether this impacts on NK levels or function or whether ASICs have any role in these changes.

Axonal degeneration plays a major role in MS, with associated inflammation causing dysfunctional activity of mitochondria (Waxman, 2006). Using the EAE model of MS, it was found that mice in which the ASIC1 gene was genetically inactivated or the channel was inhibited with amiloride exhibited marked axonal preservation (Friese et al., 2007). Consistent with the hypothesis that ASIC1a is activated during MS, increased levels of lactate are found in brain lesions of MS patients (Bitsch et al., 1999). ASICs are also expressed in microglia, astrocytes and oligodendrocytes, major players in MS that engage in crosstalk (Domingues et al., 2016). While microglia are the primary phagocytotic cells in the CNS, astrocytes also possess the ability to phagocytose neuronal debris, axonal mitochondria, and pathological protein aggregates (Lee and Chung, 2021). In aging mice, microglia appear to accumulate myelin debris at the same time as myelin degeneration (Hill et al., 2018). Indeed, Popovich et al. (2002) demonstrated that direct activation of “CNS macrophages” (covering both invading peripheral macrophages and CNS resident microglia) results in axonal damage and demyelination. This conclusion has gained strong support over the past 20 years and activated microglia in particular seem to represent promising targets in MS (Geladaris et al., 2021). As described above, ASICs are functionally expressed in both macrophages and microglia, and contribute to their activation during periods of acidosis. Thus, the protection afforded by ASIC1a inhibition in the EAE mouse model of MS is possibly due to decreased macrophage/microglial activation as well as direct neuroprotection.

In a PD model, knockout of ASIC1a made no difference to the number of dopaminergic neurons, the key subset of neurons that are lost in this pathology and lead to motor impairment (Komnig et al., 2016). A significant build-up of lactate has been observed across all brain regions in animal models of HD, and in the frontal cortex of HD patients (Harms et al., 1997; Dautry et al., 1999; Tsang et al., 2006). Administration of an amiloride derivative in a HD model caused a reduction in polyQ aggregation, one potential effector in this pathology, both in vivo and in vitro (Wong et al., 2008). Although the authors state that this indicates a role for ASIC1a in HD, their use of a non-specific ASIC inhibitor means that one cannot rule out the potential involvement of other channels/transporters in this neurodegenerative disease.

Epileptic seizures are associated with increased acidity in the brain, and upon CO₂ administration the resultant hypercapnia allows for seizure termination through lowering of brain pH (Lennox, 1928; Wang and Sonnenschein, 1955; Miller, 2011). Overexpression of ASIC1a in mice appears to limit the duration and progression of chemoconvulsant-induced seizures, but not the number, and knockout of the gene has the converse effect (Ziemann et al., 2008). Furthermore, the beneficial effect of CO₂ inhalation requires ASIC1a to interrupt induced seizures. This may be due to activation of ASICs causing generation of action potentials in inhibitory interneurons, although ASICs are also expressed in excitatory pyramidal neurons (Cho and Askwith, 2008; Ziemann et al., 2008; Weng et al., 2010). Another study found that amiloride suppressed pilocarpine-induced seizures, but the use of this pan-ASIC inhibitor makes it difficult to relate this outcome to specific involvement of ASIC1a (Liang et al., 2015). Consistent with ASIC1a playing a role in seizure termination, hippocampal levels of ASIC1a are elevated in patients with temporal lobe epilepsy (TLE) and in epileptic mice (Yang et al., 2016). It has been suggested that in a neurotypical system, seizures should be self-limiting due to the ASIC1a response to increased acidity at seizure onset; in support of this hypothesis, a single nucleotide polymorphism (SNP) in the ASIC1 gene is associated with TLE (Lv et al., 2011; Wemmie et al., 2013).

Patients with schizophrenia exhibit lower pH and increased lactate in the cerebellum (Halim et al., 2008), whereas patients with bipolar disorder show decreased pH in the dorsolateral prefrontal cortex and increased lactate in gray matter regions of the brain (Dager et al., 2004; Sun et al., 2006). The frontal cortex and brain homogenate of rodent models replicated these findings with both increased lactate and reduced pH observed (Halim et al., 2008; Hagihara et al., 2017). ASIC1a is abundantly expressed in the amygdala, a brain region that has been described as a chemosensor that promotes fear behavior after detection of CO₂ and/or acidosis (Wemmie et al., 2003; Coryell et al., 2007; Ziemann et al., 2009). Activation or inhibition/disruption of ASIC1a and altered expression of the ASIC1 gene all modify fear-related behaviors. Coryell et al. (2007) found that loss of the ASIC1 gene affected neuronal activity in the amygdala after exposure to fear-inducing odors. Not only do ASIC1 KO mice exhibit reduced fear, overexpression of the channel increases fear responses (Wemmie et al., 2002, 2004). In humans, two SNPs in the ASIC1 gene are associated with anxiety, linking with amygdala structure and function and also risk of panic disorder (Smoller et al., 2014). Lastly, Coryell and colleagues used a mouse model to examine the link between ASIC1a and depression. They found that pharmacological inhibition and or genetic ablation of ASIC1a reduced depression like-symptoms, while restoring the gene to the amygdala returned responses back to baseline (Coryell et al., 2009).

As described earlier, vascular disruption due to trauma can result in acidification of the brain, and the resultant drop in pH is often sufficient to activate neuronal ASIC1a (Xiong et al., 2004; Li et al., 2010; Chassagnon et al., 2017). It was originally envisaged that neuronal ASIC1a might exacerbate ischemia-induced brain injury by contributing to excitotoxicity by virtue of its ability to mediate flux of Ca²⁺ into neurons in addition to Na⁺ (Xiong et al., 2004; Yermolaieva et al., 2004). However, recent studies suggests that, at least in the brain, activation of ASIC1a also leads to recruitment and activation of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), a key mediator of necroptosis (Wang et al., 2015, 2020). Remarkably, this activation of RIPK1 occurs independently of ion flow through the channel (Wang et al., 2015).

How does ASIC1a contribute to ischemic injury when it is subject to rapid steady-state desensitisation (SSD) when exposed to sustained acidic pH in vitro (Gründer and Chen, 2010), and is distinguished from other ASICs by a reduced responsiveness to successive acid stimulations, a phenomenon known as tachyphylaxis (Chen and Grunder, 2007)? One might assume that these properties would limit the persistence of ASIC1a currents during sustained periods of tissue acidosis in vivo. However, a variety of ensuing biochemical events act to persistently activate ASIC1a during a sustained drop in tissue pH. First, ASIC1a currents are potentiated by several ischemia-related factors, including: (i) extracellular lactate which increases from a basal level of 1–2 mM to 12–20 mM during ischemia (Allen and Attwell, 2002; Gonzalez-Inchauspe et al., 2020); (ii) membrane stretch resulting from the rise in extracellular [K⁺] (Allen and Attwell, 2002); and (iii) CaMKII phosphorylation of ASIC1a (Gao et al., 2005). Second, during ischemic stroke, the increase in extracellular spermine prolongs ASIC1a currents and enhances recovery from SSD (Duan et al., 2011). Third, the increase in intracellular Ca²⁺ inhibits tachyphylaxis. Fourth, arachidonic acid, which is elevated during stroke due to activation of phospholipase A₂ (PLA₂), potentiates ASIC1a currents and enhances the sustained component of the current (Allen and Attwell, 2002). Finally, and perhaps most importantly, the train of cell-death signaling set off by the initial activation of ASIC1a continues regardless of the subsequent state of the channel. This includes activation of necroptosis (Wang et al., 2015; Redd et al., 2021), apoptosis (Song et al., 2019), and the stimulation of cell executioners such as calcium-activated proteases, endonucleases, and PLA₂ due to the increased levels of intracellular Ca²⁺ (Yermolaieva et al., 2004; Xiong et al., 2007).

Thus, despite its susceptibility to desensitization (which has largely been studied under control conditions in in vitro assays), activation of ASIC1a during sustained tissue acidosis is a major contributor to ischemic injury, as evidenced by the fact that genetic ablation or specific pharmacological inhibition of ASIC1a greatly reduces the tissue damage caused by ischemic stroke (Xiong et al., 2004; McCarthy et al., 2015; Chassagnon et al., 2017). Genetic knockout or knockdown of ASIC1a, as well as pharmacological inhibition of the channel, also provide neuroprotective effects post-SCI (Hu et al., 2011; Koehn et al., 2016). However, it should be noted that these studies have focused exclusively on the role of ASIC1a in CNS neurons. Despite the critical role of immune cells in damage progression after CNS trauma, very few studies have explored whether the resident population of ASIC1a, or other ASICs, in immune cells contributes to ischemic jury of the CNS.