**4. Involvement of neuroimmunity in motoneuron degeneration**

#### **4.1. Pathological phenotype of the immune system in ALS**

In addition to astrocytes and microglia, immune cells may also play synergistic and critical roles in ALS neuroinflammation and disease progression. Presence of a systemic immune activation is suggested by abnormalities observed in the blood and CSF of ALS patients such as increased numbers of circulating lymphocytes (CD4+ helper T cells, CD8+ cytotoxic T lymphocytes (CTL) and natural killer (NK) cells), increased expression of MHC class II molecules on monocytes as well as higher levels of inflammatory chemokines and cytokines (regulated on activation normal T cell expressed and secreted (RANTES), monocyte chemo‐ tactic protein (MCP-1), IL-12, IL-15, IL-17 and IL-23)[142-146]. Further, *post-mortem* studies of brain and spinal cord from ALS patients show that the activation and proliferation of microglia is associated with an infiltration of activated macrophages, mast cells and T lymphocytes which are found in close proximity to degenerating tissues [147-149]. An in-depth autopsy of six ALS patients reveals an enrichment of T-cell receptor Vβ2 positive T cells in the spinal cord and CSF, suggesting an antigen-driven T cell selection [150]. Finally, ALS patients with a more rapidly progressing pathology show decreased numbers of regulatory T lymphocytes (Tregs), suggesting that the numbers of Tregs are inversely correlated with disease progression [144, 151]. Tregs secrete anti-inflammatory cytokines such as IL-4, IL-10 and transforming growth factor beta (TGF-β) as well as the neurotrophic growth factors glial-derived neurotrophic factor (GDNF) and BDNF. Tregs are also able to dampen a Th1 pro-inflammatory response and

sitivity to normally innocuous sensory stimulations. Spasticity develops following spinal cord injury and is also regarded as an ALS clinical symptom [138]. The main mechanisms hypothe‐ sized to be responsible for spasticity are increased motoneuron excitability and increased synaptic inputs in response to muscle stretch due to reduced inhibitory mechanisms. Recently, it has been demonstrated that, following spinal cord injury, increased levels of BDNF mediated spasticity, due to post-transcriptional down regulation of KCC2 [139]. Together, these studies suggest that reactive microglia in ALS may exert an aberrant effect on the electrical activity of motoneurons and highlight the importance of furthering our understanding of this functional

Lastly, a hypothetical scenario relates to the defect in astrocytic glutamate transporter and the neurotoxic accumulation of the excitatory amino acid that we have mentioned above. It has been demonstrated that TNFα promotes the release of glutamate by activated microglia through the cystine/glutamate exchanger (Xc)[140]. Though the implication of the Xc system in ALS has not yet been investigated, it is intriguing that the Aβ peptide induces a neurotoxic phenotype in microglia through the Xc-mediated release of glutamate Therefore, system Xc represents a potential mechanism of microglia-mediated excitotoxicity that warrants further

The potential non-cell-autonomous mechanisms involving microglial cells in the selective

In addition to astrocytes and microglia, immune cells may also play synergistic and critical roles in ALS neuroinflammation and disease progression. Presence of a systemic immune activation is suggested by abnormalities observed in the blood and CSF of ALS patients such as increased numbers of circulating lymphocytes (CD4+ helper T cells, CD8+ cytotoxic T lymphocytes (CTL) and natural killer (NK) cells), increased expression of MHC class II molecules on monocytes as well as higher levels of inflammatory chemokines and cytokines (regulated on activation normal T cell expressed and secreted (RANTES), monocyte chemo‐ tactic protein (MCP-1), IL-12, IL-15, IL-17 and IL-23)[142-146]. Further, *post-mortem* studies of brain and spinal cord from ALS patients show that the activation and proliferation of microglia is associated with an infiltration of activated macrophages, mast cells and T lymphocytes which are found in close proximity to degenerating tissues [147-149]. An in-depth autopsy of six ALS patients reveals an enrichment of T-cell receptor Vβ2 positive T cells in the spinal cord and CSF, suggesting an antigen-driven T cell selection [150]. Finally, ALS patients with a more rapidly progressing pathology show decreased numbers of regulatory T lymphocytes (Tregs), suggesting that the numbers of Tregs are inversely correlated with disease progression [144, 151]. Tregs secrete anti-inflammatory cytokines such as IL-4, IL-10 and transforming growth factor beta (TGF-β) as well as the neurotrophic growth factors glial-derived neurotrophic factor (GDNF) and BDNF. Tregs are also able to dampen a Th1 pro-inflammatory response and

**4. Involvement of neuroimmunity in motoneuron degeneration**

degeneration of motoneurons in ALS are illustrated in Figure 2.

**4.1. Pathological phenotype of the immune system in ALS**

interaction.

108 Current Advances in Amyotrophic Lateral Sclerosis

study [141].

**Figure 2.** Proposed mechanisms by which microglial activation and inflammation contribute to the neurodegenera‐ tive process in ALS. Microglia can influence astrocytes and immune cells as well as directly impact motoneuron viability via several mechanisms.

attenuate toxic microglial responses. Contribution of the innate immune system is also suggested by the presence of immunoglobulins and complement deposition as well as a significant increase of NK cells in the blood of ALS patients [87, 144, 152]. While these investigations of ALS samples and tissues do not assess the contributory role of the immune system to the disease pathogenesis, they do highlight its active presence.

In support of what is observed in humans, ALS rodent models also display a particular immunological phenotype. Indeed, *SOD1G93A* mice demonstrate that the inflammatory cell subtypes are phenotypicaly and functionally different depending upon the disease stage [96]. During the initial stages, infiltrating CD4+ T cells are almost mainly Th2 (IL-4+) while as the disease progresses there is a skew toward Th1 (IFNγ+) cells and CD8+ T cells (both IL-17A positive and negative)[106, 153]. Alteration in inflammatory cell subtypes is associated with, and maybe driven by, differences in Tregs. Interestingly, early symptomat‐ ic *SOD1G93A* mice have increased numbers of Tregs and a decreased proliferation of effectors T lymphocytes (Teffs), whereas decreased numbers of Tregs and increased proliferation of Teffs is found in end-stage animals [151, 154]. The innate immune system is also affected in ALS rodents, displayed by the substantial increase of NK and NKT in the spinal cord of *SOD1G93A* mice [155, 156].

Whether neuroinflammation is a cause or a consequence of motoneuron death is still debated. It is interesting to note that inflammation is not limited to the CNS but systemic with increased levels of plasma LPS associated with increased numbers of activated circulating monocytes and T lymphocytes that correlate with disease evolution [142, 157]. A thymic dysfunction is also observed in parallel to the neurodegenerative process in mutant SOD1 mice and ALS patient [158]. In the CNS of ALS patients, TAR DNA-binding protein 43 (TDP-43) increased and interacts with nuclear factor kappa B (NF-κB) in glial and neuronal cells. LPS-activation of NF-κB in microglial cells expressing the TDP-43 mutant is associated with the production of pro-inflammatory cytokines, including TNFα, IL-1β, IL-6 and IFNγ [159]. The central role of inflammation and NF-κB in ALS was recently confirmed by the description in familial ALS of mutations in the gene encoding optineurin, a negative regulator of TNF-induced NF-κB activation [160].

role in ALS and targeting the Tregs/M2 signaling pathway may be an attractive therapeutic

The Neuroinflammation in the Physiopathology of Amyotrophic Lateral Sclerosis

http://dx.doi.org/10.5772/56489

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T lymphocytes could mediate motoneuron damage either directly through cell-cell contact, secretion of cytokines or indirectly through activation of microglia and macrophages [165]. As mentioned above, the effect of the immune system varies during disease progression from a protective role at early stages to a neurotoxic activity when disease accelerates [151]. Neuro‐ protective activity has been associated with a Tregs/M2 response and expression of trophic and anti-inflammatory factors such as BDNF, GDNF and IL-4 whereas neurotoxic effects are associated with an M1/Th1/CTL pro-inflammatory immune response [106]. Accordingly, mutated SOD1 Teffs proliferate to a greater extend and produce more IFNγ (Th1-driven) during the rapidly progressing phase than Teffs isolated during slowly progressing phase [154]. Different death pathways can be induced by Th1/CTL lymphocytes and promote motoneuron loss in ALS. For instance, activation of Fas (CD95) has been demonstrated to trigger a motoneuron-restricted death pathway. Motoneurons expressing ALS-linked SOD1 mutants showed an increased susceptibility to Fas-mediated death through activation of an amplification loop [166-168]. Accordingly, mutant *SOD1* mice with homozygous FasL mutation present a reduced loss of motoneurons and a prolonged life expectancy [169]. Likewise, the RNA interference-mediated silencing of Fas following intrathecal delivery of Fas-specific small interfering RNA improves motor function and survival in ALS mice [170]. While it remains unclear if T lymphocytes contribute to Fas-induced motoneuron degenera‐ tion, these studies suggest the possibility of their direct participation in the degenerative

T lymphocytes could also amplify the neuroinflammation in ALS via glial cells. Upon activa‐ tion, microglia cells increase membrane expression of MHC class II molecules, becoming efficient antigen presenting cells able to actively drive T cell activation and differentiation. In turn, cytokines secreted by T cells modulate microglia phenotype and function. For instance, TNFα and IFNγ, two major pro-inflammatory cytokines produced by Th1 lymphocytes induce and activate M1 microglial cells and cause neurotoxicity toward motoneurons. Experimental studies in ALS mice demonstrated that inflammatory cell subtypes were phenotypicaly and functionally different depending upon the disease stage [96]. At initial stages, microglia exhibits anti-inflammatory M2 phenotype (Ym1+, CD163+) and infiltrating T cells are almost exclusively CD4+ while end-stage disease is associated with a skew of microglia toward a pro-

inflammatory M1 phenotype (Nox2+) and T lymphocytes are mainly Th1 cells [106].

The neurotoxic effect of NK cells is suggested by the neuroprotective effect of the immuno‐ modulation of NK cells, which increases lifespan of ALS mice and is accompanied by a reduced astrocytosis. While the pathological modalities of NK cells in ALS remain elusive, several hypothetical mechanisms can be raised. Indeed, activated NK (and to a lesser extent CD8+ T cells) inhibit neurite outgrowth of cerebellar neurons in a cell contact-dependent manner *in vitro* [171]. In sensory neurons, IL-2-activated NK cells have a killing activity that requires cellular contact and perforin [172]. Further, the production of IFNγ by activated NK cells might

strategy for this neurodegenerative disease.

process.

**4.3. The neurotoxic function of the immune response in ALS**

Altogether, the information from pre-clinical models and ALS patients suggest that systemic immune activation (innate and adaptive) might play a key role in ALS pathogenesis and may represent an interesting target for the development of novel treatments. However, a better understanding of the specific roles played by the different subtypes of immune cells is of utmost necessity. Indeed, accumulative evidence suggests that inflammatory cells mediate both protective and deleterious effects on motoneuron survival and that these functions vary during disease progression.

#### **4.2. The protective function of the immune response in ALS**

Protective immunity, a homeostatic phenomenon important in the repair of damaged tissues, results from both the clearance of debris and the effects of cytokines and growth factors delivered by inflammatory T-cells to the site of injury [161, 162]. The neuroprotective ability of immune cells is also evident in ALS. Indeed, when *SOD1G93A* mice are bred with mice lacking functional T cells or CD4+ T cells, microglia skew towards an M1 inflammatory phenotype and disease progression accelerates, suggesting that CD4+ T cells provide neuroprotection by suppressing the cytotoxic activation of microglia. Accordingly, reconstitution of T cells following bone marrow transplantation of *SOD1G93A* mice lacking functional T and B cells prolonged their survival and suppressed the activation of M1 microglia [163]. Further analysis shows that the increased numbers of CD4+/CD25+/Foxp3+ Tregs during early symptomatic stages secrete IL-4, thus promoting the M2 protective microglia while inhibiting the neurotoxic Th1 response and IFNγ secretion. As described above, these neuroprotective Tregs are decreased as the disease progression accelerates. Co-culture experiments show that Tregs suppress the expression of cytotoxic factors Nox2 and iNOS from *SOD1G93A* microglia through IL-4. Tregs also inhibit the proliferation of *SOD1G93A* Teffs via the combined secretion of IL-4, IL-10 and TGF-β[154]. The neuroprotective properties of Tregs are also reinforced by their ability to secrete GDNF and BDNF, thus attenuating toxic microglial responses [164]. Impor‐ tantly, the passive transfer of endogenous Tregs into *SOD1G93A* mice lengthens disease duration and prolongs survival, suggesting that Tregs is likely the neuroprotective subpopulation among CD4+ T lymphocytes. Therefore, a subtype of immune cells appear to have a beneficial role in ALS and targeting the Tregs/M2 signaling pathway may be an attractive therapeutic strategy for this neurodegenerative disease.

#### **4.3. The neurotoxic function of the immune response in ALS**

Whether neuroinflammation is a cause or a consequence of motoneuron death is still debated. It is interesting to note that inflammation is not limited to the CNS but systemic with increased levels of plasma LPS associated with increased numbers of activated circulating monocytes and T lymphocytes that correlate with disease evolution [142, 157]. A thymic dysfunction is also observed in parallel to the neurodegenerative process in mutant SOD1 mice and ALS patient [158]. In the CNS of ALS patients, TAR DNA-binding protein 43 (TDP-43) increased and interacts with nuclear factor kappa B (NF-κB) in glial and neuronal cells. LPS-activation of NF-κB in microglial cells expressing the TDP-43 mutant is associated with the production of pro-inflammatory cytokines, including TNFα, IL-1β, IL-6 and IFNγ [159]. The central role of inflammation and NF-κB in ALS was recently confirmed by the description in familial ALS of mutations in the gene encoding optineurin, a negative regulator of TNF-induced NF-κB

Altogether, the information from pre-clinical models and ALS patients suggest that systemic immune activation (innate and adaptive) might play a key role in ALS pathogenesis and may represent an interesting target for the development of novel treatments. However, a better understanding of the specific roles played by the different subtypes of immune cells is of utmost necessity. Indeed, accumulative evidence suggests that inflammatory cells mediate both protective and deleterious effects on motoneuron survival and that these functions vary

Protective immunity, a homeostatic phenomenon important in the repair of damaged tissues, results from both the clearance of debris and the effects of cytokines and growth factors delivered by inflammatory T-cells to the site of injury [161, 162]. The neuroprotective ability of immune cells is also evident in ALS. Indeed, when *SOD1G93A* mice are bred with mice lacking functional T cells or CD4+ T cells, microglia skew towards an M1 inflammatory phenotype and disease progression accelerates, suggesting that CD4+ T cells provide neuroprotection by suppressing the cytotoxic activation of microglia. Accordingly, reconstitution of T cells following bone marrow transplantation of *SOD1G93A* mice lacking functional T and B cells prolonged their survival and suppressed the activation of M1 microglia [163]. Further analysis shows that the increased numbers of CD4+/CD25+/Foxp3+ Tregs during early symptomatic stages secrete IL-4, thus promoting the M2 protective microglia while inhibiting the neurotoxic Th1 response and IFNγ secretion. As described above, these neuroprotective Tregs are decreased as the disease progression accelerates. Co-culture experiments show that Tregs suppress the expression of cytotoxic factors Nox2 and iNOS from *SOD1G93A* microglia through IL-4. Tregs also inhibit the proliferation of *SOD1G93A* Teffs via the combined secretion of IL-4, IL-10 and TGF-β[154]. The neuroprotective properties of Tregs are also reinforced by their ability to secrete GDNF and BDNF, thus attenuating toxic microglial responses [164]. Impor‐ tantly, the passive transfer of endogenous Tregs into *SOD1G93A* mice lengthens disease duration and prolongs survival, suggesting that Tregs is likely the neuroprotective subpopulation among CD4+ T lymphocytes. Therefore, a subtype of immune cells appear to have a beneficial

activation [160].

during disease progression.

110 Current Advances in Amyotrophic Lateral Sclerosis

**4.2. The protective function of the immune response in ALS**

T lymphocytes could mediate motoneuron damage either directly through cell-cell contact, secretion of cytokines or indirectly through activation of microglia and macrophages [165]. As mentioned above, the effect of the immune system varies during disease progression from a protective role at early stages to a neurotoxic activity when disease accelerates [151]. Neuro‐ protective activity has been associated with a Tregs/M2 response and expression of trophic and anti-inflammatory factors such as BDNF, GDNF and IL-4 whereas neurotoxic effects are associated with an M1/Th1/CTL pro-inflammatory immune response [106]. Accordingly, mutated SOD1 Teffs proliferate to a greater extend and produce more IFNγ (Th1-driven) during the rapidly progressing phase than Teffs isolated during slowly progressing phase [154]. Different death pathways can be induced by Th1/CTL lymphocytes and promote motoneuron loss in ALS. For instance, activation of Fas (CD95) has been demonstrated to trigger a motoneuron-restricted death pathway. Motoneurons expressing ALS-linked SOD1 mutants showed an increased susceptibility to Fas-mediated death through activation of an amplification loop [166-168]. Accordingly, mutant *SOD1* mice with homozygous FasL mutation present a reduced loss of motoneurons and a prolonged life expectancy [169]. Likewise, the RNA interference-mediated silencing of Fas following intrathecal delivery of Fas-specific small interfering RNA improves motor function and survival in ALS mice [170]. While it remains unclear if T lymphocytes contribute to Fas-induced motoneuron degenera‐ tion, these studies suggest the possibility of their direct participation in the degenerative process.

T lymphocytes could also amplify the neuroinflammation in ALS via glial cells. Upon activa‐ tion, microglia cells increase membrane expression of MHC class II molecules, becoming efficient antigen presenting cells able to actively drive T cell activation and differentiation. In turn, cytokines secreted by T cells modulate microglia phenotype and function. For instance, TNFα and IFNγ, two major pro-inflammatory cytokines produced by Th1 lymphocytes induce and activate M1 microglial cells and cause neurotoxicity toward motoneurons. Experimental studies in ALS mice demonstrated that inflammatory cell subtypes were phenotypicaly and functionally different depending upon the disease stage [96]. At initial stages, microglia exhibits anti-inflammatory M2 phenotype (Ym1+, CD163+) and infiltrating T cells are almost exclusively CD4+ while end-stage disease is associated with a skew of microglia toward a proinflammatory M1 phenotype (Nox2+) and T lymphocytes are mainly Th1 cells [106].

The neurotoxic effect of NK cells is suggested by the neuroprotective effect of the immuno‐ modulation of NK cells, which increases lifespan of ALS mice and is accompanied by a reduced astrocytosis. While the pathological modalities of NK cells in ALS remain elusive, several hypothetical mechanisms can be raised. Indeed, activated NK (and to a lesser extent CD8+ T cells) inhibit neurite outgrowth of cerebellar neurons in a cell contact-dependent manner *in vitro* [171]. In sensory neurons, IL-2-activated NK cells have a killing activity that requires cellular contact and perforin [172]. Further, the production of IFNγ by activated NK cells might directly trigger motoneuron death through the LIGHT/LT-βR pathway or potentiate a cytotoxic Th1/CTL response via the combined action of other NK-related cytokines such as IL-17 or IL-22 [173]. Of note, NK cells also produce IL-4 upon activation, which as described earlier, mediates a neuroprotective effect. Therefore, NK cells represent an appealing branch of the immunopathology that could be considered as a therapeutic target for ALS.

In addition to the adaptive immune system, several studies suggest that humoral immunity and immunoglobulins could also contribute to the disease. Autoantibodies to voltage-gated Ca2+ or K+ channels have been described in ALS patients, which induce specific motoneuron alterations both *in vitro* and *in vivo* after passive transfer in mice [174-178]. Accordingly, C5a and other complement activation products released after activation of the classical comple‐ ment pathway by antibodies are elevated in the CSF and spinal cord of ALS mice and patients and specific inhibition of C5a receptor ameliorates disease in *SOD1G93A* mice [179, 180]. Additionally, abnormal levels of anti-Fas antibodies, able to induce neuronal apoptosis *in vitro*, have been detected in the serum of patients with ALS [181, 182]. Thus, both the innate and adaptive immune system appear to have deleterious consequences on the survival and maintenance of motoneurons in ALS.

Figure 3 illustrates the potential mechanisms implicating different populations of immune cells in ALS pathogenesis.
