Neuroimmune crosstalk and evolving pharmacotherapies in neurodegenerative diseases

Neurodegeneration is characterized by gradual onset and limited availability of specific biomarkers. Apart from various aetiologies such as infection, trauma, genetic mutation, the interaction between the immune system and CNS is widely associated with neuronal damage in neurodegenerative diseases. The immune system plays a distinct role in disease progression and cellular homeostasis. It induces cellular and humoral responses, and enables tissue repair, cellular healing and clearance of cellular detritus. Aberrant and chronic activation of the immune system can damage healthy neurons. The pro‐inflammatory mediators secreted by chief innate immune components, the complement system, microglia and inflammasome can augment cytotoxicity. Furthermore, these inflammatory mediators accelerate microglial activation resulting in progressive neuronal loss. Various animal studies have been carried out to unravel the complex pathology and ascertain biomarkers for these harmful diseases, but have had limited success. The present review will provide a thorough understanding of microglial activation, complement system and inflammasome generation, which lead the healthy brain towards neurodegeneration. In addition to this, possible targets of immune components to confer a strategic treatment regime for the alleviation of neuronal damage are also summarized.


INTRODUCTION
Neuroinflammation is a hallmark of the vicious cycle of neurodegeneration. 1 It is responsible for the development and gradual progression of neurodegenerative conditions such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), huntington disease (HD) and multiple sclerosis (MS). It is also an obvious consequence of cerebral ischaemia. 2 These proteinopathies show ramification of organelles and synaptic impairment of nervous system. 2 An epidemiological study carried out in 2016 revealed that about 276 million people were suffering from neurological disorders worldwide, and among them, every year, the extent of fatality was about 9 million people. 3,4 As dementia is one of the outcomes of neurological disorders, the estimated count of a global dementia patient is around 35 million, and if compelling measures are not identified, it will rise threefold by 2050. 5 Neurodegenerative diseases affect both the immune system and the central nervous system. 6 The immune system can also be constructive as it triggers repair of the damaged tissues, neurotrophic factor production and remyelination in response to toxins, trauma and injury, thus maintaining the functional integrity of the brain. During neurodegeneration, T cells, microglia, astrocytes, oligodendrocytes, inflammasomes and the complement system can induce neuroinflammation. 7,8 Microglia and inflammasomes act as a host defence mechanism against infections and dysfunctional neurons. 9,10 However, aggregation of amyloid-b, a-synuclein, superoxide dismutase (SOD), mutant huntingtin (mHtt), etc. can cause aberrant activation of microglia, complement system and inflammasomes, which promotes the release of inflammatory cytokines leading to neuroinflammation. 11,12 The interplay between these two systems is of interest, albeit the mechanism is yet to be explored. 13 The present review focuses on the involvement of the immune system in regulating neurodegeneration and the available therapeutic approaches. Further, the detailed mechanism of immune component activation and how they contribute towards developing the pathology has been dealt with an elaborative summary of various therapeutic approaches of targeting immune components in neurodegenerative diseases.

COMPLEMENT SYSTEM
The complement system is an element of the body's innate immunity whose components are primarily synthesized in the liver and then enter into systemic circulation for activation. 14,15 These components cannot cross the blood-brain barrier; hence, neurons and glial cells synthesize them locally. 16 The complement system consists of more than 30 fluid phases to destroy pathogens and foreign cells. 17 The three pathways of complement system activation, that is classical (CP), lectin (LP) and alternative (AP), are mainly involved in protection against microbial infections, the connection of innate and adaptive immunity, and clearance of the debris of immune products. 18,19 Complement system in CNS The complement system helps in maintaining homeostasis in the brain by acting against infections and by removal of waste products. 20 The classical pathway of the complement system starts with component C1q, which binds with the fragment crystallizable region (Fc region) of antibody. 21 Microglia is the primary source of C1q, which stimulates C1r and cleaves C1s to activate C1. 21,22 Activated C1 binds with C4 and cleaves it into C4a and C4b. C4b attaches itself to the surface of the pathogen and activates C2a and C2b. C2a and C4b together form C3 convertase (C4b2a and C3bBb). 23 The mannan-binding lectin (MBL) pathway attaches to the carbohydrates of the pathogen and activates MBL-associated serine proteases (MASPs). MASP-2 splits C4 and C2 to form C3 convertase. 24 The other complement system pathway, that is the alternative pathway, is an independent antibody pathway responsible for distinguishing between self and non-self. Hydrolysis of native C3 leads to activation of C3 (H 2 O), which associates with factor B (serine protease, which stimulates B cell to instigate inflammatory responses) and gives rise to C3 convertase. 25 Properdin can directly activate and amplify AP and is also accepted as a recognition factor for AP. 26 C3 convertase in all three pathways then splits into C3a and C3b. C3b attaches to C3 convertase to give rise to C5 convertase, which then activates membrane attack complex (MAC), resulting in phagocytosis. 27 However, this activation of the complement system is highly specific. 28 and at times can be detrimental to neurons. 29 These have self-renewal properties and are responsible for eliciting immunological responses in the CNS. 30 As per different neurological disorders, it undergoes proliferation and configurational changes to become reactive, which is known as microgliosis. 31 These reactive microglia, on the one hand, stimulate the release of neurotrophic factors and guide stem cells towards injury. 32 On the other hand, chronic activation of microglia secrets pro-inflammatory mediators such as tumour necrosis factor (TNF-a), interleukin-1b (IL-1b) and reactive oxygen species (ROS), hydrogen peroxide (H 2 O 2 ), hydroxyl radicals (OH À ) and superoxide anions (O 2À ), which are detrimental for neurons. 33,34,35 Once microglia are activated by Toll-like receptor (TLR), they stimulate the adaptor protein MyD88, which promotes autophosphorylation of IL-1Rassociated kinase (IRAK) and releases pro-inflammatory cytokines. 36 Furthermore, it promotes the generation of ROS, leading to neuroinflammation. 37 In addition to this, microgliosis also involved in the synaptic loss in diseases such as AD and PD, which correlates with the gradual progression of dementia. 38 It has been observed that M1 microglia releases pro-inflammatory proteins such as IL-6, TNF-a and IL-1b at 24 hours post-stroke, which cause secondary injuries a few days afterwards. Unlike this, M2 microglia is anti-inflammatory and reported to be increased and proliferated in the core region at the same time. Modulation of TLRs or IFN-c causes activation of pro-inflammatory M1 phenotype, whereas anti-inflammatory M2 phenotype is activated by regulatory factors such as IL-4, IL-10, IL-13 and TGF-b 39 ( Figure 1).

INFLAMMASOME-MEDIATED INFLAMMATION
The inflammasome is a complex protein that is responsible for the inflammation-mediated response. The three chief components of inflammasomes are NOD-like receptors (NLRs), apoptosis-associated spec-like protein (ASC) and pro-caspase-1. 40 NLRs, a tripartite structure, include N-terminal pyrin domain (PYD) or caspase recruitment domain (CARD), NACHT or NOD (nucleotide-binding oligomerization domain), and C-terminal leucine-rich repeats (LRRs). 40 Neurodegenerative diseases activate the NOD-like receptor (NLR) pyrin domain containing 1 and 3 (NLRP1 and NLRP3) inflammasome, ASC and pro-caspase-1 via either MAPK or NF-jB pathway. NLRP3 contributes to inflammation and injury in CNS and in neurodegenerative diseases such as AD, PD, MS and ALS, whereas both NLRP1 and NLRP3 play a role in the pathogenesis of ischaemic stroke. 41 Upon activation, the interaction of NLRP3 protein with ASC followed by the interaction of ASC with the CARD domain via PYD recruits pro-caspase-1 and forms the NLRP3-ASC-procaspase-1 complex (NLRP3 inflammasome). 42 It leads to the conversion of inactive pro-inflammatory cytokines (pro-IL-1b, pro-IL-18) into active inflammatory cytokines (IL-1b, IL-18) and activation of caspase-1 that ultimately cause neuroinflammation and pyroptosis. 43 Pyroptosis is mediated by producing gasdermin-D (GSDMD), which is responsible for producing pores on the plasma membrane. 44 Damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) such as energy depletion, ion flux (Ca 2+ influx, Na + influx, K + efflux, and Cl À efflux), cathepsin release, oxidized mitochondrial DNA release, PKR (protein kinase R) activation and ROS could activate inflammasome signalling 45,46 (Figure 2).
The immune response is a harmonized interplay of potential threat recognition by the innate immune system initially and response by the adaptive immune system afterwards. 13,47 Studies support that pattern recognition receptor (PRR) not only is involved in recognition of DAMPs and PAMPs, but it also informs and influences the adaptive immune response via TLRs. 48 PRRs include (nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin containing domain) NLRP-1, NLRP-3 NLRC-4 and absent in melanoma 2(AIM2). 49,50 Microglia express these PRRs whose increased expression causes inflammasome signalling activation. 51

Complement system: Associated pathways of activation
Alzheimer's disease (AD) is a neurodegenerative tauopathy that manifests as dementia and affects more than 6% of people older than 65 years. 18,52 AD is characterized by the formation of neurofibrillary tangles consisting of hyperphosphorylated tau and deposition of insoluble amyloid-beta (Ab) plaque. In genomic studies, it has been identified that single nucleotide polymorphisms (SNPs) of genes encoding complement protein complement receptor 1 (CR1) and clusterin (CLU) are associated with the risk of late-onset AD. 53,54 CR1, a transmembrane protein that increases the phagocytosis of C3b, C4b and C1q, opsonized particles. 55 Interaction between CLU and Ab prevents proteolytic degradation of oligomers leading to plaque formation. 56 The co-localization of the classical pathway proteins (C1q, C3 and C4) with neurofibrillary tangles, amyloid fibrils and Ab deposits, particularly in the hippocampus, temporal cortex and amygdala, has been observed in Immunohistochemistry (IHC) study of post-mortem AD brains. 55 The terminal pathway marker of the complement system, terminal C5b-9 complement complex (TCC), is predominantly present in the cortex of the AD brain, which is associated with the formation of neurofibrillary tangles and aggregated Ab. 18,57 Dystrophic neurites in Ab plaque and neurofibrillary tangles in neurons of the frontal cortex and hippocampus of AD brain express C5aR1 and C5L2 receptors predominantly. C5a fragment, generated by complement activation, is a chemotactic factor for glia. 58 C5a-CD88 (C5aR1) interaction has a detrimental effect in AD either by direct impact on a neuron or by indirectly activating the microglia, whereas the anti-inflammatory effect of activated C5L2 receptor is reported. 58 Accumulation of toxic Ab leads to activation of the complement system by secretion of C1q from neurons and the production of C3 by astrocytes. 14 C1q binds to Ab plaque and C1q receptor (C1qR) on microglia, thus leads to synaptic pruning and initiates phagocytosis. 59 CD14, CD36, CD47, a6b1 integrins, TLRs and RAGE receptors of microglia get activated by soluble Ab oligomer and Ab fibrils, which in turn instigate phagocytosis. 59 Binding of Ab oligomers to astrocytic TLR4 or CD36 receptors leads to the production of inflammatory cytokines or chemokines by activating NF-jB signalling. 60 Furthermore, this activated signalling cascade along with microglial post-phagocytic processes (lysosomal injury, acidification of cytosol, etc.) contributes to the activation of NLRP-3 inflammasome. 61 C3 is further cleaved into C3a and C3b that ultimately produce MAC. C3b along with C3a contributes to the activation of DNAX-activating protein of 12 kD (DAP12), which stabilizes the triggering receptor expressed on myeloid cells 2 (TREM-2) to modulate microglial function leading to neuroinflammation. 61 In the case of Parkinson's disease, the presence of Lewy bodies and melanized neurons in the substantia nigra (SN) is responsible for the activation of the classical complement system. 62 These depositions show positive staining in IHC for early complement activation C3b, C4d and C7 and late activation of C9. 63 Later on, positive staining in IHC of microglia-derived C1q and C5a levels was also found in SN. 64 Earlier studies have been reported that C1q also helps in removal of aggregated neuromelanin in the SN. 65 Lewy bodies and oligodendrocytes show MAC activation, which leads to phagocytosis in the SN in PD. 66 Microglial NADPH oxidase (Nox2) is stimulated by CR3, which suggests the role of the complement system in neuroinflammation-mediated loss of  dopaminergic neurons. 67 Increased neuroinflammation in PD is marked by elevated C3 and factor H (FH) along with reduced Ab42 in cerebrospinal fluid (CSF), which helps to correlate with cognitive and motor dysfunction. 68 In contrast to this study, Rozemuller et al 69 found negative double staining for a-synuclein (a-syn) and components of complement, suggesting that reactive microglia and the complement system are not involved in the formation of Lewy body in the cortex of PD patients.

Implication of microglia
In AD, the structure of Ab oligomer is sensed by the microglia as DAMP and they are morphologically altered to the 'reactive' or 'primed' microglia state. 13 Activation of microglia has both degenerative and protective effects in AD. 13 On a negative aspect, activation of microglia can produce pro-inflammatory cytokines such as IL-1b, IL-6 and TNF-a that act on the cholinergic neurons and induce apoptosis. 70 Conversely, it can also produce a proteolytic enzymes such as insulin-degrading enzyme (IDE), matrix metalloproteinases (MMPs) and neprilysin to degrade Ab plaque to provide neuroprotection. Phagocytic function of microglia is inhibited by a mutation in the TREM-2 and CD33 protein 71 . TREM is a protein of immunoglobulin (Ig) superfamily, which is expressed in macrophages, microglia and dendritic cells of the brain, whereas CD33 is a cell-surface protein of the sialic acidbinding Ig-like lectin (SIGLEC) family, which majorly expressed in myeloid and lymphoid cells. 72 TREM-2 is involved in cell survival, phagocytosis, proliferation and production of inflammatory cytokines. 73 It has been observed in various studies that the risk of AD gets increased up to threefold to fourfold due to arginine to histidine mutation (R47H mutation) of TREM-2, which results in functional loss of the protein. 74 Other mutant variants of TREM-2 such as D87N, T96K and R62H have also been associated with AD generation. 75,76 Progression of PD is the result of the infiltration of lymphocytes from the periphery to CNS, excessive activation of microglial cells and astrogliosis. 77 Researchers have observed early and prolonged activation of microglia . Inflammasome-mediated neurodegeneration. PAMPs or DAMPs activate TLR and mediate non-transcriptional priming of the inflammasome. TNF activates the TNF receptor and increases the expression of NLRP3, ASC and pro-caspase-1 via NF-jB signalling. It also leads to the priming of inflammasome, which ultimately contributes to inflammasome assembly. It will further activate pro-caspase-1 into caspase-1, which converts the pro-inflammatory cytokines (pro-IL-1b, pro-IL-18) into inflammatory cytokines (IL-1b, IL-18). P2X7 receptor and mitochondrial stress also activate the inflammasome assembly. It will ultimately lead to pyroptosis and, thus, neurodegeneration (PAMPspathogen-associated molecular pattern, DAMPsdamage-associated molecular pattern, TNFtumour necrosis factor, NLRP3nucleotide-binding domain, leucine-rich repeat-containing receptor (NLR) family pyrin domain containing 3, ASCapoptosis-associated speck-like protein containing CARD, NF-jBnuclear factor kappa-light-chain- within the territory of damaged neuronal death using positron emission tomography (PET) scanning. 78 Reactive microglia phagocyte aggregated a-syn and led to stimulation of Nox2 and generation of ROS to elevate dopaminergic neurodegeneration in SN. 79 Aggregation of a-syn enables the upregulation of leucine-rich repeat kinase (LRRK2) along with activation of iNOS and CD68 through macroglia and the invading peripheral cells. 80 Activated microglia facilitate the release of proinflammatory cytokines such as interleukin-1b (IL-1b), IL-23, IL-12, epidermal growth factor (EGF) and TNF-a and chemokines such as CCL5, CCL2 and CXCL10 in the striatum, CSF and peripheral blood 81,82,83 ( Figure 3). Interferon-gamma and TNF-a are also stimulated in the SN. 84 Increased levels of TNF-a in serum lead to four times of exacerbation in cognitive decline, which gives an idea about the role of inflammatory markers in cognitive function. 85 The release of IL-1b impairs the spatial navigational learning and cognitive functions in female C57BL/6 mice. 86 These cytokines then trigger iNOS and COX-2, which results in the generation of ROS. 87,88 A massive release of ROS leads to oxidative stress, which is responsible for aggravating neurodegeneration. 89 Inflammasome activation AD is the most studied disease for understanding inflammasome signalling. 90,91 Excessive generation of Ab leads to its oligomerization and subsequently senile plaque formation. These Ab oligomeric or fibrillar form can act as DAMPs to activate inflammasome signalling. 90 In experiments with AD patients, it has been observed that activation of NLRP3 inflammasome is an early-stage event rather than a late-stage event. 92 Another clinical study on AD has shown the release of IL-18 and IL-1b and coexpression of ASC, NLRP3 and caspase-1 in the monocyte. 93 Fibrillar Ab stimulates microglia to release IL-1b based on the interaction of NLRP3 and ASC, whereas soluble Ab peptides activate NLRP3 inflammasome majorly through CD36. 93 NLRP3 inflammasome activation in response to Ab can also be regulated by autophagy-related protein 7 (ATG7). Deficiency of cellular ATG7 causes increased cleavage of caspase 1, the formation of speck by ASC, and the release of IL-1b in microglia and BV2 cell (alternative model for primary microglial culture). Moreover, microglia, which are deficient in ATG7, cause a more remarkable loss of neuronal dendrites suggesting the activation of microglial inflammasome to limit neuronal destruction. 94 Following uptake of Ab in the astrocytes, IL-1b is produced through inflammasome signalling. 95 According to Liu et al, 90 palmitate increases NLRC4 inflammasome activation in astrocytes of rat and causes maturation of IL-1b. In an IL-1 and MyD88 knockout rat model, microglia were not activated. This suggests that Ab activates MYD88, which results in activation of NLRP3 inflammasome and ultimately cleavage of caspase 1.
In PD, aggregation of a-syn stimulates TLR, which further contributes to the assembly of NLRP3 inflammasome and conversion of its downstream marker pro-IL-1b to IL-1b. 96 Fyn kinase enables the transfer of a-syn to microglia via PKC-delta-dependent NF-jB-p65 nuclear translocation. This transfer further contributes to mitochondrial ROS generation and an increase in the level of cathepsin B, which therefore activates NLRP3 inflammasome 97,98 . Apart from oxidative stress, mitochondrial DNA disruption can stimulate pro-inflammatory mediators to activate NLRP3 inflammasome. 99 Lee et al 100 found that microglial NLRP3 inflammasome activation in mouse model of MPTP-induced PD is essential for the motor deficit and dopaminergic neuronal loss. Elevated levels of IL-1b were also found in CSF and blood of the PD patients. 101 In addition to this, increased NLRP3, caspase-1 and IL-1b were detected in transgenic a-syn-A53T overexpressed mouse model of PD. 12 The serine-threonine kinase cyclin-dependent kinase 5 (CDK-5) is responsible for phosphorylating NLRP, ASC and POP1 ASC-2, which ultimately leads to inflammasome activation. 102 Immunotherapeutics for age-related neurodegenerative diseases Hitherto, FDA-approved drugs for symptomatic treatment targeting Ab and Tau tangle are, donepezil, galantamine, rivastigmine and AADvac1 (a vaccine developed by Axon Neuroscience (NCT02579252), currently in clinical trial phase II) are in practice. 103 Now, targeting immune cells such as microglia and macrophages for modulation of neuroinflammation is of great interest. Ab plaque burden can also be reduced by injections of anti-Ab monoclonal antibodies (mAbs) into the systemic circulation. 104,105,103,106 Tyrosine kinase inhibitors, which are currently in phase III clinical trial, can inhibit mast cell differentiation and degranulation, exhibiting beneficial effects as an adjunct with conventional therapy (NCT01872598). Endocannabinoids inhibit the neuronal microglial activation by promoting the receptor interaction of CD200. 107

Vaccination
In April 2020, Elan/Wyeth's group is planning to launch a dose-escalation, multiple-dose, randomized and doubleblind phase II clinical trial for the AN1792 vaccine (NCT00021723) against AD. It comprises QS21 and preaggregated Ab1-42 as an adjuvant. 108  targets the small Ab fragment (Ab 1-6), which is a B-cell epitope. 109 It showed marked improvement of cognitive behaviour in phase I trials, but the result of phase II clinical trial is yet to be disclosed (NCT02565511). Janssen and Pfizer conducted a phase II clinical trial on ACC-001 (NCT01284387). It also targets Ab(1-6) fragment, which is coupled to a carrier protein and the adjuvant QS-21, a surface-active saponin. 110 Passive immunization using monoclonal antibody is the recent trend seen in immunotherapy, in which administration of exogenous anti-Ab monoclonal antibody is performed without the chance of Th1-mediated antibody production. 111 Janssen/Pfizer conducted phase II clinical trial on bapineuzumab AAB-001 (NCT00606476) and bapineuzumab AAB-003, 112 while Eli Lilly conducted phase III clinical trial on solanezumab (NCT02760602). 111 However, these drugs failed to show any significant clinical improvement in phase III trial. The limitation of passive immunization is the unavailability of appropriate antigen targets, repeated injections in chronic diseases, haemorrhagic risk, blood-brain barrier (BBB) penetration, high costs and generation of the immune response against injected antibodies.
In PD, immunotherapeutics such as peroxisome-proliferated receptor gamma (PPAR-c) agonists such as fenofibrate help to reduce inflammation and oxidative stress, and simultaneously elevate the dopamine level. 113 NSAIDs such as aspirin, an irreversible COX-1 inhibitor, decrease inflammation and oxidative stress by elevation of lipoxin. 114 Statins inhibit TNF-a, IL-1b and IL-6 and lead to a decline in inflammation. 115 In an experiment by Gordon et al., it has been observed that daily oral administration of MCC950 (20 mg/kg) can inhibit NLRP3 inflammasome-mediated caspase-1 activation in striatum. 101 Han et al. 116 revealed that kaempferol inhibits the formation of the NLRP3 inflammasome by activating autophagy in microglia, which further reduced the NLRP3 protein expression. Bushen-Yizhi formula suppresses NLRP3 inflammasome activation in the SN-and MPP + -stimulated BV-2 microglia cell lines. 117 PMX-205, the antagonist of C5a, protects against dopaminergic neurodegeneration. Anti-a-synuclein immunotherapy not only can be a promising approach, but it acts by inhibiting the movement of extracellular a-synuclein to other neurons. 118 Other vaccinations such as 9E4 (humanized mouse monoclonal antibody) and AFF-1 (short peptides-AFFITOPEs) minimize the aggregation of calpain cleaved a-synuclein and oligomers of a-synuclein, respectively. 13 There are many limitations regarding the development of immunotherapy, one of them is the direct administration of a-synuclein antibody to the brain, which hinders asynuclein homeostasis. Moreover, these clinical trials are costly and take a longer duration to develop new approaches for synucleinopathy 119 (Table 1).

Complement system
Experimental autoimmune encephalomyelitis (EAE) is the best model for studying the role of the complement system in MS. 120 Binding of C3d to CR3 receptor mediates phagocytosis of myelin by microglia. 121 Further, it leads to the activation of TNF-a and the generation of NO, which culminate in demyelination. 122 At 2 h post-formation, terminal complex C5b-9 in lytic dose in macrophages, neurons and oligodendrocyte progenitor cells induces demyelination. 123 In contrast, the sublytic dose of C5b-9 can prevent oligodendrocyte apoptosis via phosphatidylinositol 3-kinase/Akt pathway (PI3K/Akt). 124 Watkins et al. 125 revealed clinical data of 22 MS patients, which confirm the upregulation of C1q protein of the classical complement system and fragment Bb of the alternative complement pathway in the grey matter of cortex. To validate the role of complement system in MS, transgenic mice deficient in C3 or factor B were used in an antibody-independent model of EAE, which showed less severity of disease. 126 A marked decrease in the infiltration of T cells and macrophages, along with a reduced expression of P-selectin, was detected in PVG/C6-deficient rats. 127 Therefore, it can be observed that the complement system has a dualistic role. Hence, its modulation instead of complete inhibition can improve the quality of life in MS patients. 27 In the case of ALS, the upregulation of various components of the complement system, such as C1q, C3, is reported. 128 Activation of its components such as MBL, MASP-1, C3, C4a and C5a leads to the destruction of BBB integrity. 128 One of the critical elements is C5a peptide, which binds to the C5aR receptor of neurons and glia. 129 It also links to another G protein-coupled receptor C5a-like receptor 2 (C5L2), which leads to the activation of mitogen-activated protein kinase (MAPK) and protein kinase B (PKB/Akt) pathways. 130 C5a is responsible for the entry of macrophages in the skeletal muscles of the hSOD1 G93A mouse model, contributing to muscle denervation in ALS. 131 SOD1 G93A C5aR1 knockout mouse model has shown to increase the survival rate, which indicates that C5aR1 activation exacerbates the deleterious effect of ALS. 132 Another mouse model, TDP-43 Q331K , shows similar activation of complement C5a in the lumbar region of the spinal cord and tibialis anterior muscle. 133 This indicates that C5a can be a potential therapeutic target for ALS. Microarray analysis and lasercapture microdissection in the transgenic animal model revealed that the levels of C1q and C4 were elevated throughout the progression of the disease. 134 Moreover, ª 2020 John Wiley & Sons Ltd, Immunology, 162, 160-178 mRNA and protein of classical components C1q and C4 in addition to terminal complements C3 and C5b-9 were elevated in 16 ALS patients. 135 Farber et al. found that activated C1q is responsible for converting resting microglia into reactive microglia via Ca 2+ signalling, which further stimulates pro-inflammatory markers such as TNF-a and IL-6, which are detrimental for neurons. 136 Thus, modulating complement system activation can be a promising approach for neuroprotection in ALS (Figure 4).

Microglia activation in ALS
Microglia play the role of a double-edged sword. In rNLS8 mouse model, human TAR DNA binding protein 43 (hTDP-43) pathology-related ALS microglia exhibit a protective role by reducing inflammatory markers. Inhibitors of microgliosis such as PLZ3397, CSF1R and c-kit failed to recover motor function in rNLS8 mice. 137 Moreover, after microglial activation, the M2 phenotype secretes neuroprotective factors such as BDNF, while M1 releases inflammatory cytokines to detrimental effect. 138 Fractalkine, a chemo-attractant bound to surface receptor CX3CR1 of microglia, which helps them in intercellular signalling of impaired neurons. 139 It acts as neuroprotective by stimulating PI3K/Akt, which leads to activation Bcl-x L (antiapoptotic protein) and quelling of BAD (proapoptotic protein). 140 The SOD1 G93A transgenic mouse model is extensively used for studying human ALS. 141 In the SOD1 G93A mouse model, reactive microglia and astrocytes cause induction of nerve growth factor (NGF) expression correlated with p75 neurotrophin factor (p75NTR) expression in the area of degenerating motor neurons, which lead to apoptosis. 142,143 Reactive microglia causes upregulation of lymphocyte function-associated molecule-1 (LFA-1), leucocyte common antigen (LCA), complement receptor CR3 and CR4, immunoglobulin receptor FccR1 and MHC-II complex. 144 These reactive microglia generate the superoxide anions (O 2À ) to promote oxidative stress, which worsens the ALS condition. 33 In addition to this, reactive microglia secretes pro-inflammatory markers such as IL-1, IL-6 and TNF-a. IL-1 further recruits IL-6, colony-stimulating factor (CSF), IL-8 and IFN-a/b. 145 Apart from these inflammatory markers, there is a threefold elevation in the expression of COX-2 protein and mRNA in astrocytes, microglia, neurons and in the spinal cord of the transgenic mouse model during the progression of disease. 146

Microglia and T-cell interaction in MS
The induction of the immunological response against CNS antigen in MS is still paradoxical. 147 Activated microglia contribute to oxidative stress following oligodendrocytes, and neuronal and axonal injury-associated demyelination. 148 Two contrary hypotheses are involved in describing the functions of the immune system in lesions of MS. First, the pathology starts in the periphery by stimulation of innate and adaptive immunity and progress towards the CNS. 149 The second hypothesis states that it begins from CNS and then shifts to the periphery. 150 Activated microglia helps in the infiltration of T cells responsible for adaptive immunity such as Th1, Th2 and Th17. Th1 releases pro-inflammatory markers (IFN-c and TNF-a), Th-2 secretes anti-inflammatory markers (IL-4, IL-6, and IL-13), and Th17 releases IL-17 and granulocyte-macrophage colony-stimulating factor (GM-CSF). 151 Both MHC-I and MHC-II are expressed by activated microglia, which later stimulate CD8 + T cells and CD4 + T cells, respectively. 152 CD4 + release antibodies against myelin sheath and oligodendrocyte antigens result in demyelination. 153 In contrast, stimulated CD4 + cells can modulate T cells into iT reg (regulatory T cells), which suppress the immune system by inhibiting the antigenpresenting cell (APC) and induce neurotrophic factors such as BDNF and GDNF, but impaired activation of iT reg leads to autoimmune disease. 154,155,156 Inflammasome activation Dysregulation of NLRP3 inflammasome can be detrimental and eventually end up with degeneration of neurons. 157 In the SOD1 G93A mouse model, increased expression of TLR-4 and NF-jB in the diseased brain suggests that NLRP3 inflammasome activation culminates in pyroptosis. 158 Scientists have also observed an increased expression and co-localization of NLRP3 inflammasome in the spinal cord of SOD-1 G93A mouse model. 159 Co-localization and activation of NLRP3 and upregulation of IL-18 in the serum of ALS patients were reported by Kadhim et al., which then further activates caspase-1. 160 Increased expression of the NLRP3 inflammasome, along with aggregation of p62 and damaged mitochondria, is indicative of diminished autophagy in ALS. 161 According to Meissner et al., in the SOD-1 G93A mouse model, activation of microglial IL-1b and caspase-1 occurs without activation of NLRP3. 162 In contrast, protein aggregates such as TDP-43 Q331K , TDP-43 A315T , TDP-43 WT , and SOD-1 G93A showed microglial NLRP3dependent inflammasome activation in TDP-43 Q331K and a SOD-1 G93A mouse model of ALS, respectively, by using Nlrp3 À/À knockout and NLRP3 inhibitor MCC950. 163 In MS, elevated levels of IL-1b and caspase-1 in peripheral blood mononuclear cells (PBMC) indicate the contribution of NLRP3 inflammasome in experimental mouse model. 164 NLRP3 inflammasome stimulates the migration of Th cells and APC in CNS to increase the expression of chemotactic proteins such as osteopontin, CXCR6 and CCR2. 164 Co-localization and activation of NLRP3 through IL-1b aid in demyelination, while stimulation of NLCR4 helps in neuroprotection. 165 Moreover, NF-jB facilitates pro-IL-1b transcription, while expression of pro-IL-18 is constituted, which increases after cellular activation. 166 Progression of relapse-onset MS was marked by the upregulation of IL-1b in CSF. 167 In addition to this, MS patients showed a significant increase in serum and CSF levels of IL-18 levels. 168 IL-18 is responsible for the stimulation of CD8 + mucosal-associated invariant T (MAIT) cells along with activation of integrin, which enables the infiltration of CD8 + T in CNS. 169 Increased rare genetic variants in NLRP1/3 and CASP1 lead to its inactivation through autophagy, mitophagy and type 1 interferons. 170 Immunotherapeutics for modulating the immune system in ALS and MS To date, US FDA-approved drug, riluzole, can increase life expectancy for about 2-3 months. 171 The mechanism of action is to suppress the glutamate release at synaptic cleft. 171 Recently in May 2017, edaravone becomes the second FDA-approved drug. It shows functional recovery by preventing the nitration of a tyrosine residue in CSF. 172 Inflammatory markers such as TNF-a and FasL expression are suppressed by COX-2 inhibitors, thalidomide and its congener lenalidomide in the SOD-1 G93A mouse model. 173 Another COX-2 inhibitor cyclosporin has been proven effective in the transgenic mouse model, and currently, it is in clinical trial phase IV (NCT01795872). 174,175 A clinical trial of glatiramer acetate at a dose of 40 mg/day failed due to inappropriate regime to modulate ALS. 176 Anakinra is an antagonist of IL-1, which then further inhibits activation of IL-6 and other pro-inflammatory markers in ALS. 177 PMX205, the antagonist of C5aR, shows extended survival and slow progression of disease. 133 Cyclo(His-pro) helps in the reduction of ROS generation and NF-jB and therefore inhibits ª 2020 John Wiley & Sons Ltd, Immunology, 162, 160-178 NLRP3 inflammasome in ALS. 178 Two vaccines, tgG-DSE2lim and tgG-DSE5b, were tested in hSOD1 G37R mouse, which resulted in late-onset and increased life expectancy. 179 NEDA (no evidence of disease activity) is a broadly accepted approach for early treatment of MS that starts with injectables followed by oral doses and infusions. 180 Injectable disease-modifying therapies include interferonbs (IFN-bs), glatiramer acetate (GA) and natalizumab (NTZ). 181 Immunomodulating drugs such as mitoxantrone intercalate with DNA and halt humoral immunity and T-cell count. 182 Infusion of rituximab and ocrelizumab that are anti-CD-20 antibodies is approved for relapsing-remitting multiple sclerosis (RRMS) and primary progressive multiple sclerosis (PPMS). 183 Sphingosine 1 phosphate (S1P) modulator, fingolimod, is the first oral approved drug that is phosphorylated by sphingosine kinase to form fingolimod phosphate, impeding with circulating immune cells. 184 Other orally approved drugs are teriflunomide and dimethyl fumarate for RRMS. 185,186 The drugs that are in clinical trials are laquinimod, ponesimod, siponimod and ceralifimod. 183 A combination of two antibodies trastuzumab and pertuzumab can be used for regulating complement system. 187 Intranasal administration of genetically engineered CAR/FoxP3 T reg cells can quell the EAE model of MS. 188 As myriads of treatment are emerging, these therapies are expected to improve the livelihood of MS patients (Figure 5).

Complement system
HD is an inherited, dominant and autosomal neurodegenerative disease that is triggered by an expansion of three-base-pair (CAG) repeat in exon 1 of the HTT gene. 189 This expansion repeats translate into a polyglutamine tract at the N-terminus of the protein. 190,191 Experimental data suggest that in HD, microglia activate the classical pathway of the complement system via pathological peptide-like mutant huntingtin (mHtt). Mutation in the gene of chromosome 4p 16.3 leads to the generation of mHtt. 192,191 In situ hybridization study reveals that the striatum, neurons, astrocytes and myelin sheath of the HD brain are strongly positive for C1q, C1r, C3, C4, C9-neoepitope and iC3b-neoepitope deposition. 190,193 In the early disease stages, negative outcome for staining of complement component suggests that early neuronal damage precedes local synthesis and activation of complement components. Microarray analysis reports of HD brain tissue indicate that in the caudate nucleus and motor cortex area, complement components C3, C4A and C4B get highly expressed. 194 Researchers have worked on various animal models of HD. Among them, the R6/2 transgenic mouse model was proven to be a more realistic model despite having the limitations to

Microglial activation
Marked microgliosis has been found in the post-mortem human HD brains. 193 It was reported that medium spiny GABA neurons (MSNs) of the striatum are most vulnerable to the neurotoxicity mediated by mHtt. 196 mHtt mostly accumulates in nerve terminals and neuronal dendrites rather than in soma and other cells, leading to microglial activation. 196 Activated and proliferated microglia contributes to neurodegeneration by upregulating inflammatory cytokines. 197,198 There are several pathways through which microglial activation occurs in HD, which are as follows:

NF-jB pathway
Microglia mostly express TL-2 receptors, which are accountable for IL-10 and IL-6 secretions. 199,200 It also expresses TLR-3 and TLR-4 receptors that are linked with the production of pro-inflammatory cytokines such as IL-6, IL-10, IL-12, TNF-a and CXCL-10. 200 A group of inhibitory proteins, IjBs, sequestered NF-jB in the cytoplasm. 199 When IjB kinase (IKK) phosphorylates IjBs, it leads to the dissociation of NF-jB from IjBs and translocation of NF-jB to the nucleus. 201 IKKa, IKKb and IKKc Components of the complement system, microglia and astrocytes reside near the blood-brain barrier and help in homeostasis and protection of CNS. It promotes neurogenesis by releasing BDNF, neurotrophin-5, etc. It also helps in removing debris and suppressing inflammation and therefore leads to neuroprotection. (B) On tissue injury, activation of C4a, C5a and C3 of the complement system is responsible for the entry of microglia and astrocytes to promote its conversion from resting to its activated form. Reactive microglia and astrocytes release pro-inflammatory markers such as TNF-a, IL-1b and iNOS. These markers further lead to the generation of ROS and culminate in oxidative stress. In addition to this, reactive microglia causes upregulation of LFA-1, LCA, complement receptor CR3 and CR4, immunoglobulin receptor FccR1 and MHC-II complex. Chronic activation of C3a and C5a promotes entry of peripheral immune cells, which secretes chemokines and cytokines on endothelial cells and disrupts the integrity of BBB. It also causes induction of NGF correlated with p75NTR in an area of degenerating motor neurons, which leads to apoptosis (BDNFbrain-derived neurotrophic factor, TNF-atumour necrosis factor, ILinterleukin, iNOSinducible nitric oxide synthase, ROSreactive oxygen species, LFA-1lymphocyte function-associated molecule-1, LCAleucocyte common antigen, NGFnerve growth factor, p75NTR-p75neurotrophin factor, FccR1fragment crystallizable gamma receptor 1, BBBblood-brain barrier, MHCmajor histocompatibility complex) ( are the subunits of IKK that is activated by soluble mHtt, ultimately leading to NF-jB pathway activation. 202 Trager et al. reported that the interaction of mHtt with IKK causes transcriptional changes in the NF-jB signalling with an induction in the level of IL-6, IL-8, IL-1b and TNF-a. siRNA lowers the mHtt level and causes attenuation of the NF-jB pathway with concurrent downregulation of pro-inflammatory cytokine levels in HD. 203 Kynurenine pathway Studies on the R6/2 HD mouse model have shown that peripheral macrophages exacerbate the HD pathogenic condition through the Kynurenine mono-oxygenase pathway (KP). 204 KP is the major route of metabolism of Ltryptophan in mammals and the principal pathway for the formation of nicotinamide adenine dinucleotide (NAD + ). 205 Levels of two neurotoxic metabolites of the kynurenine pathway such as 3-hydroxykynurenine (3-HK) and quinolinic acid (QUIN) are increased in neocortex and striatum in the early stage of HD. 204 These QUIN and 3-HK, in addition to astrocytic deficiency and altered glutamate reuptake, have a role in N-methyl D-aspartate (NMDA) receptor overstimulation (excitotoxicity), free radical formation, lipid peroxidation and decrease in dopamine release. 206,207 This will ultimately contribute to neurodegeneration and cognitive dysfunction.

Cannabinoid pathway
Cannabinoid receptors are GPCR and mainly consist of CB1 and CB2 types of receptor. 208 CB2 plays a role in cell survival, differentiation and proliferation by adenylyl cyclase, cyclic AMP-protein kinase A (PKA), extracellular signal-regulated kinase 1 (ERK1) and ERK2, p38 mitogen-activated protein kinase and JUN N-terminal kinase (JNK) pathways. 209 CB 1 receptors are mostly present in striatal MSNs in R6/2 HD mouse model. 210 It plays a neuroprotective role by increasing the expression of brain-derived neurotrophic factor (BDNF). 211 In the R6/2 mouse model, the genetic knockdown of CB2 receptors shows microglial activation along with exacerbation of behavioural abnormalities and reduction in life span. 212

Inflammasome activation
The mHtt aggregates in the caudate and putamen area and acts as DAMPs to cause neostriatal atrophy. 213 Although recent studies have observed that in the absence of any stimuli, mHtt overexpression in the primary murein microglia induces inflammatory gene expression, 214 studies on the R6/2 mouse model suggest that caspase-1 and caspase-3 get activated and further aggravate neurodegeneration. 215 Another research shows that inhibition of ATP-sensitive P2 purinergic receptor (P2X7) leads to the alleviation of HD through the suppression of NLRP3 inflammasome signalling. 216 An increase in expression, as well as activity of the major myeloid transcription factors such as CCAAT/enhancer-binding protein alpha/beta (C/ EBPa/b) and Pu.1, may cause the production of pro-inflammatory cytokines, whereas the aggregation of mHtt triggers activation of inflammasome, but the mechanism to activate Pu.1 and C/EBP activity in microglia is yet to be explored. 217,214 Immunotherapeutics in HD Immunotherapy is a promising area for the treatment of HD. Recently, Vaccinex -'a Rochester'a New Yorkbased biotechnology company, is conducting a doubleblinded clinical trial on antibody VX-15. 218 It inhibits Semaphorin 4D, which is a transmembrane protein causing neuroinflammation and neurodegeneration. 219 Apart from this, an antisense oligonucleotide (ASO) therapy is also in phase I clinical trial. 220 The limitation of ASO therapy is that it can be administered through lumbar puncture only. 220

INVOLVEMENT OF THE IMMUNE SYSTEM IN EXACERBATION OF ISCHAEMIC STROKE
Complement system The complement system plays a significant role in the pathogenesis of ischaemic stroke. 221,222 After an ischaemic attack, the levels of C3a, C4d and soluble C5b-9 in plasma are increased instantly, while the elevation of C5a occurs within 7-14 days. 223,222 The role of C1q, initiator of the classical pathway in the pathogenesis of ischaemic stroke, is quite complex. Reports suggest that the accumulation of C1q occurs at 3 to 6 h post-ischaemic stroke. 224 After 24 h of ischaemic insult, biosynthesis and functional activity of C1q by microglia get drastically increased. 224 However, C1q-deficient transgenic mice do not show any protective role in ischaemic stroke. 223 Conversely, neonatal C1q-deficient mice show less functional deficit. 225 This discrepancy between neonatal and adult C1q is mainly due to the differences in the presence of C1q in the CNS of adult and neonatal mice. 226 MBL, the initiator of the lectin pathway, also exacerbates ischaemic stroke pathology in transgenic mice and exhibits less cerebral infarct with better functional outcome poststroke. 227 Compared with the above two pathways, the alternative pathway has also been reported in ischaemic stroke pathology. 221,222,223 Factor B-deficient mice, when treated with CR2-fH, an alternative pathway inhibitor, show better improvement 24 h post-reperfusion. 228

Microglial responses after ischaemic stroke
Microglial activation shows characteristic temporal and spatial patterns in the peri-infarct zone. 230 In the case of focal cerebral ischaemia, microgliosis is prominent in the core region, accumulation region and marginal regions. 230 Within the cortical region where blood flow is normal, ramified microglia are majorly observed, whereas microglia with few stout and short processes are observed in the marginal zone. Finally, the hypertrophic cell body containing amoeboid microglia is found in the accumulation region and in the core region. 231 The distribution pattern signifies the transformation of morphology microglia that reflects the changes of its function and also a different pathological state of the tissue. 51 After global transient ischaemia, the hippocampal CA1 region has an extensive microglial expansion. 232 Immediately after an ischaemic episode, the release of IFN-c shifts the microglial polarization towards pro-inflammatory M1 phenotype, whereas at a later stage, CXCL-16 and IL-4 lead to anti-inflammatory M2 polarization. 233,234 In the early stage, activation of pro-inflammatory M1 microglia leads to the production of cytokine, TNF-a, IL-1, ROS, RNS and protease, while at the reparative phase, anti-inflammatory M2 microglia helps in attenuation of inflammation by producing IL-10 and TGF-b. 235,39 Prolonged overexpression of microglia further leads to peripheral immunosuppression that may predispose to bacterial infection, pneumonia and urinary tract infection (UTI). 236 Hence, microglial activation following ischaemic stroke leads to exacerbation of pathology, whereas at the late stage, microglia contribute to neuronal protection. 51

Role of inflammasome signalling
The activation of inflammasome signalling is involved in atherogenesis, a major risk factor for stroke. 237,238 Macrophages are transformed into foam cells, and these can activate NLRP3 inflammasome. Atherogenesis causes the formation of plaque that leads to lysosomal rupturing of foam cells. 238 It further causes the release of cathepsin, various proenzyme and ROS, which ultimately activate NLRP1 and NLRP3. 239 TLR-4 and TLR-12 identify minimally oxidized LDL and FFA, and increase myeloid differentiation primary response protein MyD88 following activation of TIR-domain-containing adaptor-inducing interferon-b (TRIF) to induce the expression of NF-jB. 240 Further, an increase in the expression of NLRP-3, ASC and pro-caspase-1 contributes to inflammasome assembly. 241,242 Inflammatory cytokines such as IL-1b and IL-18 activation also induce neutrophil, macrophage and lymphocyte infiltration, followed by their activation. 243 Release of MMPs 1, 2, 3, 9 and 12 is triggered by the inflammatory cytokines. 244 These MMPs play an important role in extracellular matrix remodelling and also trigger plaque stability. 245 This cyclic mechanism activates NLRP3 and aggravates atherosclerosis, which therefore contributes to the development and exacerbation of stroke pathology.

Post-stroke activation of inflammasome signalling
After ischaemic stroke, damaged cells release DAMPs, which leads to activation of PRRs such as TLR-2, TLR-4, IL-1b and RAGE, which may further activate NLRP1 and NLRP3 inflammasome via MAPK and NF-jB pathways. 45,246 . In the core area, depletion of ATP leads to activation of NLRP1 by conversion of ATP-bound inactive inflammasome to ADP-bound active inflammasome. 247 Increased expression of NLRP3 or NLRP1, along with ASC and pro-caspase-1, leads to inflammasome assembly, and thus maturation of pro-inflammatory cytokines into inflammatory cytokines that further leads to neuronal cell death via pyroptosis. 248

Immunotherapy in ischaemic stroke
Recent studies reported that b-hydroxybutyrate and MCC950 could inhibit the NLRP3 inflammasome by reducing the secretion of pro-inflammatory cytokine (IL-1b) by macrophage. 244,249,250 Treg cells are suppressors of neuroinflammation. 251 Forkhead box transcription factor (FOXP3) is a marker of Treg cells, and FoxP3+ Treg cells are reported to attenuate inflammation. 251 Trichostatin A activates FoxP3 and reduces the proliferation of Th1 cell, which suppress the production of IFN-c by promoting the Treg cells. 252

MICROGLIAL MODULATION VIA GUT MICROFLORA AFFECTING CNS DISORDERS
As discussed previously, microglial activation leads to generation of NO. Aberrant NO generation has been linked with various neurodegenerative diseases, specifically AD and PD. 253 Studies have reported that gut commensal bacteria produce short-chain fatty acids (SCFA) to a certain extent, which are particularly responsible for microglial maturation and functional regulation. 254 Disruption of gut microflora has been observed to contribute to different CNS disorders via microglial dysfunction. 254,255 Hence, targeting the link between microglia and host gut microflora is a promising therapeutic approach. The Kallyope company is conducting a study for investigating the communication pathways of the gut/ brain axis for developing novel therapies for various CNS disorders. However, this study is still in its infancy, which requires further research to determine how probiotics can be utilized to improve microglial function and finally be beneficial in CNS disorders. 256 CHALLENGES TO TREAT NEURODEGENERATIVE DISORDERS VIA IMMUNE SYSTEM MANIPULATION Manipulation of immune system as a novel therapeutic approach for neurodegenerative disorders possesses few challenges. Studies have shown that there are major gaps in understanding the disease mechanism, which often restricts bedside translation of promising in vivo results. 257 In vivo studies for neurodegenerative diseases are extensively based on genetic models, which reflect the familial forms of the disease. Hence, development of effective therapies is limited with the use of such models. 257 Along with this, age-related factors and effect of lifestyle are not considered extensively in preclinical studies. As discussed in the previous section, host gut microflora can affect both brain function and immune response. 257 It has been recently found that heterogeneity among individuals can also be a result of latent CNS infections causing neurodegenerative disease such as AD. 258 This appears consistent with ageing-related IFN-1 expression at the choroid plexus. 259,260 Therefore, common immunological factors contributing to different neurodegenerative diseases can be modified by systemic immunomodulation and microglial phenotype modulation.

CONCLUSION
This review summarizes the neuroimmune crosstalk with fundamental mechanisms and potential therapeutic approaches. As discussed in the article, the innate immune reaction can be detrimental to neurons and oligodendrocytes, and at the same time, it can also be beneficial for recovery. This response depends upon the sources of cytokines and on their cognate receptor's nature. The production of innate immune protein, as well as the presence of adaptive immune cells in the brain environment, is an important feature of neurodegeneration. Dysregulation of components of the immune systems such as microglia, T cells, complement system and inflammasome signalling plays a vital role in degenerating neurons, but the genetic background, gender and environment also contribute towards progression of the pathology. Singlet therapeutic approaches, in addition to the present choices of therapy, can suffice to reduce neuroinflammation and toxicity, thus preventing the induction and exacerbation of