The immune system and the central nervous system (CNS) cross-talk, and this interaction are pivotal to the onset and the progression of central neurodegenerative diseases (i.e., Alzheimer’s disease and amyotrophic lateral sclerosis), as well as in classic autoimmune-inflammatory disorders [i.e., multiple sclerosis (MS)]. Although inflammation probably does not represent an initiating factor, new evidence suggests that pro-inflammatory molecules contribute to the derangement of chemical synapses favoring disease progression (1). In fact, a number of endogenous molecules (i.e., cytokines and chemokines) released by the immunocompetent cells as well as by activated astrocytes control chemical transmission at active synapses, affecting the main functions of these cells including transmitter release and second messenger production. These endogenous molecules are main transducers of the pathological “glial to neuron” cross-talk (1–5).

The endogenous level of CCL5 is very low, almost undetectable, in cerebral spinal fluid (CSF) of healthy individuals, but it increases dramatically when human immunodeficiency virus 1 (HIV-1) infection occurs (31–35), at the onset and during the progression of MS (9, 36–40). Increased central and peripheral CCL5 levels are also detected in mice suffering from the experimental autoimmune encephalomyelitis (EAE), an animal model reproducing most of the spinal cord pathological features of MS in humans [(41) and references therein]. Two are the mechanisms determining the dramatic increase of CCL5 bioavailability in CNS. First, the permeabilization of the blood–brain barrier that occurs in inflammation favors the entry of CCL5 from the periphery into the brain. Second, the concomitant massive local production of CCL5 from astrocytes and, to a lesser extent, from microglial cells triggered by pro-inflammatory citokines.

Microglia cells coordinate brain innate immunity, rapidly expand their population, and then migrate chemotactically to sustain inflammation and cells death. Microglia exist in the “resting” and the “activated” forms. In the resting state, that is an active surveying state, microglia cells in close proximity to neurons and astrocytes participate to the central network by releasing regulatory agents and by controlling homeostasis. In the “active” state, microglia releases pro-inflammatory effectors including TNF-alpha and IL-1beta, which diffuse to neighboring astrocytes, influencing their functions (42). These factors are the principal inducers of chemokine overproductions from astrocytes. Microglia filopodia make dynamic contact with astrocytes and neurons and have a pivotal role in controlling synaptic plasticity (43–45). The intimate contact of microglia with chemical synapses also makes the neuronal control of microglial functions possible. Actually, microglia cells are endowed with receptors for transmitters (46) the activation of which controls their activation state. Excitatory transmission favors the shift from the “resting” to the “activated” state of microglial cells, while inhibitory transmission reduces activation of microglial cells to the “resting” condition (47).

Astrocytes are innate immune sentinels that ensheath cerebral blood vessels controlling the entry of peripheral cells into the CNS parenchyma during infection. Astrocytoma cells as well as cultured astrocytes produce CCL5 upon incubation with the microglial effectors TNF-alpha or IL-1 beta, through an inflammatory-like response mediated by the p55 receptor-dependent signaling (42, 48). IFN-gamma from microglial cells, inactive on its own (42), seriously amplifies the effect of either TNF-alpha or IL-1 beta, worsening the pro-inflammatory signaling.

The expression and the release of CCL5 from astrocytes is tightly controlled by several receptor subtypes, including opioid receptors (49, 50), group III metabotropic glutamate receptors (2), alpha/beta noradrenergic receptors (51–53), and sphingosine-1-phosphate receptor (S1PR) subtype 1 (54), as well as by endogenous neurotoxin such as quinolinic acid acting at NMDA receptors (55). These receptors represent targets of therapeutics for inflammatory autoimmune disease typified by overexpression of CCL5.

Differently, data supporting the active production and release of the endogenous chemokine from neurons are lacking, suggesting that these cells do not release the chemokine.

Once released in the synaptic cleft, CCL5 regulates the function of glial cells (microglia and astrocytes) themselves through autocrine processes.

The autocrine control of microglia functions is permitted by the existence of CCR5 receptors, and, to a less extent, of CCR1 and CCR3 receptors in these cells, which also permit the chemotactic movements of microglial cells during inflammation (56). CCR5s are also the receptors functional to HIV-1-induced pathogenetic mechanisms (57–59). As to the astrocytes, the existence of CCR1, CCR3, and CCR5 proteins in these cells has been a matter of discussion, and, despite some initial discrepancies (60), it was definitively demonstrated that adult astrocytes possess CCR1, CCR3, and CCR5 (61–63). CCR1, CCR3, and CCR5 proteins are expressed in cultured fetal astrocytes and in adult astrocytes from mammalian brains [(29), but see for a concise review Ref. (60)], as well as in astrocytic processes [gliosomes (64, 65)] isolated from the cortex and the spinal cord of adult mice (Table 1) (66). In particular, in physiological conditions, the expression of CCR5 in astrocytes is low but rapidly increases to abnormal pathological levels upon stimulation with TNF-alpha and by IL-1beta released by neighboring microglial cells, which causes persistent adaptation leading to a significant increase in the expression of most of the CCR proteins (55). This cascade of events occurs during inflammation, so that the overexpression of the receptors targeted by CCL5 is an event intimately linked to pathological conditions.

In cultured astrocytes from both fetal simian and human brains, a significant increase in internal Ca²⁺ ions mobilization was observed upon exposure to CCL5 (29). Since intraterminal Ca²⁺ ions’ mobilization is a prerequisite to transmitter exocytosis also in these cells [(77) and references therein] the CCL5-mediated control of cytosolic Ca²⁺ bioavailability might suggest that the chemokine could favor/modulate glutamate exocytosis from astrocytes. Despite the expectation, however, the spontaneous and the depolarization-evoked release of [³H]d-aspartate ([³H]d-Asp) from gliosomal particles isolated from human brain tissue was not modified by CCL5 (29), compatible with the idea that activation of these receptors cannot modify the glutamate outflow of the excitatory aminoacid. Similarly, exposure of human and mouse glial particles [we refer to as gliosomes (64)] to CCL5 in the absence or in the presence of a concomitant depolarizing stimulus (i.e., 20 mM KCl-enriched solution, KCl substituting for an equimolar amount of NaCl) did not significantly modify the outflow of glutamate. This is compatible with the idea that the amount of Ca²⁺ ions mobilized in gliosomal cytosol by CCL5 acting at its own receptors was insufficient to prime vesicle docking and fusion with astrocyte membranes (66, 71). So far, data concerning the impact of CCL5 on glutamate uptake in microglia and astrocytes are not available and further investigations are needed to address these aspects.

The serum level of CCL5 was found to be significantly increased in patients suffering from MS (89–91) as well as in EAE animals (15, 41, 92). The highest levels of the chemokine were detected in the peripheral blood mononuclear cells (PBMCs) of MS patients suffering from the secondary progressive form of the disease, while lower level was observed in the PBMCs from patients with the relapsing–remitting form of MS (93). CCL5 levels are also increased in the CNS of MS patients as well as of EAE mice (9, 13, 22, 88, 93–100). The huge elevation of the CCL5 levels in CNS mainly depends on the increased peripheral production of the chemokine, but it also reflects the local overexpression of the chemokine in astrocytes activated by IL-1beta, TNF-alpha, and IFN-gamma (55), from neighboring microglia cells. All these observations are predictive of the role of CCL5 in the onset and progression of disease in MS patients. Accordingly, a CCL5 polymorphism [the CCL5-403 G/A single nucleotide polymorphism (22, 99)] is associated to a higher risk of susceptibility to the onset of the disease, while modified CCL5 ligands are efficacious in controlling the symptoms and the neurodegenerative processes in EAE mice (15).

CCR1, CCR3, and CCR5 exist in different cell types, including T lymphocytes, monocytes/macrophages, and immature dendritic cells, but also exist in neurons and astrocytes (101–104). In particular, CCR1 and CCR3 are expressed by circulating T cells as well as in monocytes, which are occasionally found in perivascular infiltrates in the brain of MS patients. Differently, CCR5-positive T cells and macrophages are concentrated in the active demyelinating lesions in CNS of MS patients (13, 22). To note, the expression of CCRs in CNS correlates with disease severity (105–107) as proved by the observation that clinical symptoms are reduced in CCR1, CCR3, or CCR5 knockout (k.o.) EAE animals, which also suggest redundancy in the chemokine system (7, 8, 99, 108).

Glutamate is the major excitatory neurotransmitter in CNS, where it mediates important physiological functions (i.e., synaptic plasticity, learning, and memory), but also triggers excitotoxic degenerative processes. Glutamate concentration in the synaptic cleft is finely tuned by several cellular mechanisms including active re-uptake and release from nerve terminals as well as presynaptic mechanisms of control mediated by auto- and/or heteroreceptors (65, 82, 109–116). Glutamate bioavailability is also affected by neighboring astrocytes [i.e., the cells that take up and release the aminoacid (77)] as well as by altered glutamate metabolism. l-Glutamate signaling, however, is not restricted to neuron/astrocyte compartments since glutamate receptors (GluR3-containing AMPA receptors and mGluR1/5 receptors) exist also in immune cells, including cells of the T lineage (117–119). Therefore, besides its role in controlling chemical transmission and excitotoxicity, glutamate may represent a chemo-attractant driving force for the recruitment and the migration of leukocytes and T cells into CNS site where glutamate release occurs (119).

Increased glutamate levels are found in the cerebrospinal fluid of MS patients (119–121) possibly because of the down-regulation of glutamate-metabolizing enzymes (glutamate dehydrogenase and glutamine synthase) and up-regulation of glutamate-producing enzyme glutaminase (122). In 2003, Sarchielli et al. (119) compared the levels of aspartate and glutamate in the CSF of patients suffering from different forms of MS and of controls healthy individuals. The authors find a significant increase of the glutamate levels in patients suffering from the relapsing–remitting form of MS. Interestingly, the glutamate levels were significantly higher in individuals suffering from the relapsing–remitting form of MS with active central lesions during the stable phase than in patients suffering from a similar form of disease, but without lesions. Inasmuch, high levels of glutamate were also detected in patients suffering from the secondary progressive form of MS.

Impaired glutamate bioavailability was also observed in EAE animals. However, depending on the animal model used and the brain region under study, opposite modifications of glutamate release efficiency were observed, consistent with the view that, in demyelinating disorders, impaired glutamate transmission at active synapses is a complex event. Increased glutamate release was detected in the spinal cord of EAE rats (123, 124) as well as in striatal and spinal cord nerve terminals of EAE mice (72, 88, 124–126), while reduced glutamate release was observed in cortical and hippocampal nerve endings of both mice and rats suffering from EAE disease (72, 88, 100, 126, 127). As to glutamate receptors, both metabotropic and ionotropic glutamate receptors (namely mGlu1/5 and mGlu4, mGlu2/3 receptors, and NMDA and AMPA receptors) control glutamate release (83, 109, 113, 115, 116, 128, 129). The expression and the function of these receptors were found to be altered in EAE mice when compared with controls (109, 128–136), suggesting that they represent suitable targets of drugs for the cure of MS symptoms Besides receptors, also glutamate transporter expression is modified in EAE rats (124), determining increased glutamate bioavailability (137) and consequent neurotoxicity.

Generalized ongoing subclinical axonal degeneration in lesioned and non-lesioned white matter, as well as in gray matter, is detectable in CNS of MS patients and seems to occur independently from inflammation or demyelination, representing an early cause of CNS damage in MS (138). Interestingly, besides the spinal cord, neurodegeneration also takes place in other brain regions, such as the cortex and the hippocampus, and could be responsible of cognitive and affective dysfunctions (121, 139–141) that represent common and early manifestations of MS (142, 143).

The thesis that central chemokines and classic transmitters functionally cross-talk in CNS has several implications and add new aspects of interest to the role of chemokines in the synaptic derangements that typify neuro-inflammatory central disease.

As already stated, an abnormal overproduction of CCL5 in the spinal cord, and to a lesser extent in the cortex, of EAE mice was evidentiated in immunocytochemistry analysis and confirmed in tissue homogenate (88, 100, 126) and in blood (88). Concomitantly, changes in glutamate exocytosis from nerve endings isolated from the cortex and the spinal cord of EAE mice were observed. Quite interestingly, the modifications of glutamate exocytosis observed in cortical and spinal cord synaptosomes from EAE mice recall the presynaptic modulation elicited by CCL5 in nerve terminals in healthy mice. Actually, glutamate exocytosis was reduced in cortical nerve endings from EAE mice [i.e., in this CNS region, CCL5 inhibits glutamate exocytosis in control animals (66, 71)], but it was drastically increased in spinal cord terminals [where a positive role of CCL5 on glutamate release was observed in healthy animals (66)].

In both CNS regions, the altered glutamate exocytosis was paralleled by impaired second messenger production. Again, the alterations in cAMP ad IP₃ productions observed in both cortical and spinal cord synaptosomal subpopulations from EAE mice were reminiscent of the modulatory presynaptic effects exerted by CCL5 on the corresponding enzymatic pathways in nerve terminals from control, non-immunized, mice. In fact, endogenous cAMP was drastically reduced in cortical synaptosomes but not in the spinal cord, where IP₃ production, the second messenger accounting for the CCL5-mediated presynaptic facilitation of glutamate exocytosis was significantly augmented (Figure 1) (66, 88).

The changes in second messenger productions and release efficiency could be explained by assuming that the prolonged in vivo CCR activation elicited by the high CCL5 could have triggered adaptive intraterminal changes in nerve terminals (144–146), which are retained in “ex vivo, in vitro” synaptosomal preparations and can emerge in “in vitro” functional studies as changes in glutamate release efficiency and second messenger production. As a matter of fact, the abnormal expression of the chemokine could have reverberated on the CCR repertoire presynaptically located on glutamatergic nerve terminals, leading to adaptation of the CCRs heteromers controlling glutamate exocytosis. These adaptations might lead to changes in the receptor expression and/or associated signaling that might account for the profound changes in glutamate exocytosis in nerve terminals from EAE mice. The CCR composition of the presynaptic chemokinergic oligomers involved in the CCL5-mediated control of glutamate exocytosis in both cortical and spinal cord nerve endings of EAE mice, however, was conserved when compared to control mice, indirectly suggesting that adaptation in CCR subunits assembly were not involved in the EAE-induced changes to the CCL5-mediated control of glutamate exocytosis described above (72).

As to the second messengers, the strict correlation linking CCL5 levels, glutamate release efficiency and IP3, and cAMP accumulation was confirmed by the observation that administration of drugs able to reduce the overexpression of CCL5 in CNS [i.e., the antidepressant desipramine (DMI)] (126) restored both presynaptic functions (i.e., transmitter exocytosis as well as second messenger production) at glutamatergic nerve endings in the cortex of mice suffering from EAE. The beneficial effects exerted by DMI were mediated by the change in noradrenaline bioavailability, due to blockade of the noradrenaline transporters. Actually, the transient increase in the endogenous amine in the synaptic cleft elicited by DMI activates the α and β receptors expressed in astrocytes in the near proximity of the noradrenergic nerve terminals, the activation of which hampers the central endogenous production and release of pro-inflammatory chemokines, including CCL5 (53, 126, 147) from these cells. Interestingly, the peripheral production of the chemokine (which at that stage of disease is already augmented) was unaffected (126).

To conclude, hampering the central overproduction of CCL5 leads to a marked amelioration of the presynaptic defects in terms of release of glutamate and second messenger production. Altogether, these observations clearly support a strict correlation between the increased production of CCL5 in the CNS and the onset of synaptic glutamatergic alteration in EAE mice, also strengthening the pathological role of the “glial to neuron” “CCL5–glutamate” interaction.

In 2016, Centonze et al. (40) demonstrated that the endogenous concentration of CCL5 in the cerebrospinal fluid of MS patients suffering from the active form of the disease was largely increased when compared to healthy individuals and to patients at the inactive stage of disease. Inasmuch, the authors showed a significant correlation between the endogenous level of CCL5 and the amount of IL-1 beta, used as a marker of gravity of the disease. CSF levels of RANTES were associated with enhanced cortical excitability in the cortex, as suggested by results from studies in which cortical excitability and plasticity was monitored with transcranial magnetic stimulation in MS patients. In these experiments, the authors highlighted a high correlation between the increased intracortical facilitation and the endogenous amount of CCL5. Differently, no correlation emerged when studying the relation linking CCL5 and a long-term potentiation-like synaptic plasticity measured through theta burst stimulation in the same patients. Despite the contrasting observation, the authors concluded that CCL5 couples inflammation and synaptic excitability in MS brains.

As already stated in this review, CCL5 production is increased in MS patients suffering from both the remitting and the non-remitting form of the disease (15, 41, 89–92). Interestingly, most of the therapeutics currently in use for the cure of MS reduces significantly the overexpression of CCL5.

It is the case of interferon-beta-1b (IFN-beta-1b); administration of this drug prevents CCL5 overproduction in the sera, in the peripheral blood and in the adherent mononuclear cell supernatants during both the relapse and the remission stage of the pathology. These observations are compatible with the idea that CCL5 might be involved in determining the molecular events accounting for the action of IFN-beta-1b in MS patients (38).

Similarly, glatiramer acetate, an approved drug for the treatment of MS, was reported to reduce the TNF-alpha-induced CCL5 mRNA overexpression in human U-251 MG astrocytes. This effect was attributed to the inhibition of mRNA transcription and led to the conclusion that glatiramer acetate may exert its therapeutic effect in MS also by inhibiting pro-inflammatoy signaling (148).

Activation of cannabinoid receptors, which represents a therapeutic approach useful to control the progression of central neuroinflammation in EAE mice and MS patients, also reduces the endogenous availability of the chemokine CCL5 being concomitantly beneficial to the progression of the demyelinating disorder (41, 149).

Laquinimod is a novel orally administered drug for the treatment of relapsing–remitting MS. The molecular events accounting for its therapeutic effects are far from to be elucidated. Monocytes obtained from laquinimod-treated patients tended to secrete lower levels of the pro-inflammatory chemokines CCL2 or CCL5 (150).

Another orally active disease-modifying drug is Fingolimod. Fingolimod is a pro-drug, rapidly metabolized to its active form, the fingolimod-phosphate (fingolimod-P). By acting at the S1PRs in microglia cells, in circulating T cells, and in the spleen, fingolimod-P prevents the egress of lymphocytes and exerts central anti-inflammatory effects favoring remyelination (151). Recent data demonstrated that in vivo oral (the drug dissolved in the drinking water) administration of this drug largely ameliorated the clinical symptoms in EAE mice. The treatment was beneficial to the inflammation and demyelination in the spinal cord of EAE mice, also significantly reducing the endogenous content of CCL5 in this CNS region (100).

Quite interestingly, the abovementioned therapeutics were also described to ameliorate glutamatergic synaptic transmission (152–155) further supporting the strict connection linking CCL5 overexpression and glutamatergic synaptic derangements.

The scope of this manuscript is to review the literature concerning the physio-pathological role of CCL5 in controlling glutamate transmission in the CNS of healthy mammals, as well as of individuals and animals suffering from demyelinating disease, in order to highlight the main role of CCL5 as a modulator of the neuroimmune cross-talk in the “quad partite” synapse in CNS (Figure 2).

These effects, together with the well-known chemo-attractant role of the chemokine toward glial cells, suggest that CCL5 exerts a dual role in the CNS of individuals suffering from MS. On one hand, the chemokine impairs the chemical transmission at asymmetric synapses in selected region of the CNS. On the other hand, it worsens the course of disease progression by favoring the recruitment of pro-inflammatory glial cells in the site of the lesion.

When considering its role as modulator of glutamate transmission, the chemokine preferentially emerges as a key effector of the “astrocytes to neurons” signaling in CNS. Actually, the chemokine released from astrocyte and microglia is an efficient paracrine modulator of glutamate release at synaptic boutons of glutamatergic neurons in both healthy and demyelinating conditions, while its autocrine role of modulator of glutamate overflow from astrocytes is less evidnet (Table 1).

Therapeutic approaches aimed at containing the overexpression of the chemokine might represent therefore a useful approach to the cure of MS.

The author confirms being the sole contributor of this work and approved it for publication.

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.