Uncontrolled Neuroinflammation in Aging and Neurodegenerative Diseases

Neuroinflammation generally serves to protect and restore homeostasis to the brain following an insult. This is achieved by endogenous innate immune cells of the brain known as microglia, that detect distress and injury signals through pattern recognition receptors (PRRs) resulting in their activation and recruitment to the insult. Upon activation, microglia serve as housekeepers to clear the insult (e.g., oxidative burst for microbial infections, phagocytosis for cellular debris, environmental exposures and ‘prion-like’ protein aggregates, and glial scar formation for stroke and head trauma) and subsequently self-regulate their own inactivation by releasing pro-resolution factors to quench the inflammation once the distress signals are no longer detected. Factors such as the severity and distribution of the insult, genetic and environmental predisposition to alter the inflammatory responses and age dictate the extent and duration of the staged neuroinflammatory response. Deregulated immune resolution can occur when a severe enough insult is coupled with preexisting susceptibilities that limit an appropriate response to resolve the insult, resulting in pathological chronic neuroinflammation that contributes to collateral neurodegeneration near ‘dysfunctional microglia’ persistently undergoing oxidative bursts [71].

Aging microglia share many similar features to pathological microglia found in brain injuries and diseases—displaying amoeboid morphologies with enlarged somas and thick, shortened processes that lose their arbor-like complexity [72, 73] with severe alterations in their abilities to host an adequate response to insults. Yet, detailed evaluations of aged microglia have characterized them as senescent or dystrophic rather than pathologically activated due to their greater density of cytoplasmic vacuoles and inclusions, deramified dendritic arbors with fragmented processes, enlarged peri-nuclear cytoplasms with beading suggesting cytorrhexis, membrane blebbing, and excessive accumulation of ferratin and neuromelanin [74, 75]. As microglia age, one notable feature is that they accumulate undegradable lipofuscin, polymeric chains of cross-linked proteins that form intracellularly during oxidative stress [76] or accumulated from phagocytized dysfunctional neurons within their lysosomes. Together, the morphological changes observed in dystrophic microglia impair the dynamic nature of microglial processes utilized in immune surveillance [77] and synaptic remodeling [78-80] and thus likely influence their function.

Functionally, aged microglia display delayed response times, slower rates of motility, low phagocytic activity, exaggerated inflammatory responses and greater proliferative capacity to insults compared to younger microglia [81-91]. This was verified in vivo by examining how aged microglia respond to exogenous ATP to simulate cell lysis or laser injury using two-photon imaging. Prior to injury, aged microglia display slower process dynamics, which were maintained even after insult. Aged microglia responded to the injuries with delayed ramification and motility and lingered at the site of insult far longer than microglial responding to the same insult in younger animals [82]. Age also impaired the rate and degree of microglial phagocytosis of p-amyloid in a model of Alzeihmer’s disease [92, 93] and of myelin in an experimental autoimmune encephalomyelitis model of multiple sclerosis [94]. These findings suggest that the functional changes in aging microglia afflict both the ability to detect and respond to the initial immunoactivating stimuli, but also may alter their ability to resolve insults.

Gene expression levels of the PRRs TLR1, TLR2, TLR4, TLR5, TLR7, and CD14—used to detect insults—are up-regulated with age and support the exaggerated inflammatory responses observed in aged microglia [95, 96]. Paradoxically, the expression levels of microglial P2 purinergic receptors [82, 97], integrins CD11b and CD11c [88], scavenger receptors CD68 [84, 98] and RAGE [88], TREM2 [99] and FcyRs [100] involved in ATP-mediated chemotaxis, adhesion and phagocytosis of opsonised debris and pathological protein aggregates are up-regulated, rather than down-regulated, with aging. Age-related hypometabolism could partly explain this contradiction between gene expression and altered function in microglia, since even though the microglial machinery for motility and phagocytosis are present, they are highly energy-intensive processes [101]. Another explanation is that repeated insults generate large amounts of debris, many of which are undigestable with age, that overwhelm microglia and render them dysfunctional over time [102]. However, the expression of degrading enzymes such as IDE, neprilysin, and matrix metalloprotease 9 (MMP9) [92] and the rate of authophagy in aging microglia [101] are consistently decreased with the functional impairment of phagocytosis by aged microglia.

Aged microglia express increased levels of effector molecules associated with their activated states, including elevated basal levels of the pro-inflammatory cytokines IL-1p, IL-6 and TNF-a [84, 103-109] coupled with a minor decrease in the expression of anti-inflammatory cytokines IL-10 [104, 110, 111]. Likewise, healthy elderly individuals show increased basal levels of inflammation as detected on positron emission tomography (PET) using [11C]-PK11195 [112]. This imbalance between inflammatory and anti-inflammatory factors potentiate age-related neuroinflammatory responses [113-116], more importantly this study suggests that many aged microglia displaying graded yet chronic states of ‘semi-activation’ or ‘para-inflammation’ within the brain due to the aging process [117]. In support of this theory, many studies found aged brains to be leakier to environmental factors circulating in the blood and more abundant in persistent inflammagens within the parenchyma, thus capable of being chronically stimulated into a maladaptive activated phenotype [118-120].

Aged microglia in ‘semi-activated’ states have reduced activation thresholds, a phenomenon known as priming, resulting in exaggerated inflammatory responses to additional insults [105]. In situ hybridization for MHC II, a marker of primed microglia, showed ~2 % of adult microglia in mice were MHC II positive, whereas ~25 % of aged microglia were MHC II positive in the absent of an insult [121]. Priming was confirmed in aged microglia to generate larger, more sustained immune responses to insults resulting in more collateral degeneration and dysfunction compared to similar lesions on younger animals. Insults ranging from infectious agents and their cellular components [105, 107, 121-127]; hemorrhagic stroke [128, 129], physiological stress [130, 131], and mechanical- or toxicant-induced neurological injury [132-134] all significant potentiated the release of IL-1p, IL-6, and TNF-a in aged rodent brains. Suppressing inflammation with minocycline in aged mice prior to LPS stimulation attenuated the priming-induced amplification of pro-inflammatory factors released by microglia [135].

Though the age-related shift of microglia into primed states can explain the altered sensitization and reactivity to immune challenges, the theory of replicative senescence suggests that since quiescent microglia are rarely thought to replicate compared to activated microglia [136, 137], it is only microglia in chronic states of activation that may reach their lifetime replication limits (as defined by telomere attrition with each replication; [138]). To accommodate for the age-related loss in microglial turnover, the resident brain macrophage population can be steadily replaced with infiltrating monocytes that adapt to the brain environment yet express slightly altered phenotypes that could be perceived as priming [139]. Though monocyte-derived microglia-like macrophages can be identified as CD11b+ CD45high cells through flow cytometry, it is nearly impossible to differentiate these two distinct populations through conventional histological methods due to the similarity in expression of markers, thus more work needs to be done to verify this theory.

The pathway by which IL-1p is released in its active form from microglia has recently been determined to require two environmental cues [140]—a priming signal usually initiated that activate the NF-kB pathway to transcribe immature Pro-IL-1p and a second signal to activate NLRP3 inflammasome (e.g., ATP through P2X7/ Pannexin, Cathepsin B released from phagolysosome rupture from indigestible lipid crystals and misfolded proteins and direct stimulation by cytosolic viral vectors) to activate caspase-1 to catalyze Pro-IL-1p into its active form [141]. Though inflammasome activation within microglia has been associated with cognitive impairment and dementia [142, 143], recent findings support that aging is associated with increases in the phosphorylation of NF-kB signaling and the increased expression of inflammasome assembly genes and activation of caspase 1 [144]. Evolutionary, the two-step activation process required to generate active IL-1p in the brain likely served as an additional checkpoint to regulate neuroinflammation from becoming pathogenic. Yet, since aging shifts microglia into primed, dystrophic state associated with basal levels of NF-kB signaling (produced by distress signals or ROS from dysfunctional mitochondria), only the second signal is required for NLRP3 inflammasome formation and signaling in aging brains [145]. Since a large population of aging microglia release autocrine pro-inflammatory mediators such as ATP and TNF-a and store a-synuclein aggregates and p-amyloid fibrils that are implicated in lysosomal damage that release cathepsins, the secondary signals to form the inflammasome may also be present in aged microglia [146, 147]. Interestingly, 95 % of the isolated of primed microglia (MHC II positive) showed co-expression of IL-1p once stimulated with LPS, compared to 31 % of the non-primed microglia [121]. Since the release of IL-1p requires inflammasome formation, this finding suggesting that the population of primed aged microglia have sufficient secondary signals to induce inflammasome assembly.

Several soluble and membrane bound ligands interacting with microglial receptors may modulate the activation state of microglia [148]. For instance, astrocytes and neurons are thought to possess immunosuppressive functions within the brain to regulate microglia and infiltrating leukocytes [149]—limiting the collateral damage they may cause during inflammation on the brain’s network of non-regenerating, post-mitotic neurons that are highly vulnerable to lipid peroxidation. Age-related modifications (e.g., loss of immunosuppressive factor expression on astrocytes and neurons, loss of expression of their respective receptors on microglia) are also thought partake in the exaggerated inflammatory responses observed in primed, aged microglia. Though astrocytes secrete S100p, TGFp and neurotrophins that suppress microglial activation [150], their expression in aged astrocyte seems to be either unaltered or overexpressed compared to younger astrocytes—and thus are negligible with regards to aging-related shifts in modulating activation. Neurons, on the other hand, gradually lose many of the secreted and cell-to-cell contact factors that suppress microglial activation with aging (see Table 1; [110, 151-173]). Though activated adult microglia can undergo immunosuppression through M1/M2 polarization in the presence of IL-4 or IL-10, one nuance of activated aged microglia is that they remain in the M1 activation state upon treatment with either of these anti-inflammatory factors—suggesting that aging impairs the microglial response to IL-4 and IL-10 regardless of the expression of levels of either the ligand or receptor.

Aged microglia have enhanced leukocyte transmigration into the brain, most likely a result of age-related increased IL-1p- and TNF-a-mediated upregulation of endothelial cell adhesion molecules [174] and secretion of the chemokines monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein-1 (MIP)-1a by reactive astrocytes [175, 176]. In support of this, aged microglia express more MHC class II complexes that bind to T cell receptor (TCR) on naive CD4+ T cells and costimulatory molecule CD80 and CD86 that bind to T-cell CD28 [177]. Though T cells are rarely found in the brain parenchyma of young adults [178], observation in mice indicate that lymphocyte extravasation in brain increases after 12 months of age and accumulate near activated microglia and astrocytes that secrete IL-1p and TNF-a [179]. Likewise, dendritic cells, which are rarely found within the brain parenchyma in young adults, accumulate within the brain in an age-dependent manner [179]. The adaptive immune cells are active participants in amplification of the brain immune response that has been observed with advanced age.

Neuroinflammation serves as a primary pathological hallmark of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease and multiple sclerosis. Despite their different etiologies and specific nuclei of degeneration, many of the immunologically-active endogenous factors released by aged, degenerating neurons that maintain chronic neuoinflammation are shared among many diseases. Our group has previously reported how chronically activated microglia may participate in exacerbating neurodegeneration through collateral damage. For instance, anti-inflammatory interventions that inhibit the formation of superoxide by NOX2 inhibition [169, 180-191] and through antioxidant natural compounds [192-195] have been shown to be effective at preventing dopaminergic degenerations by feducing inflammation and oxidative stress in in vitro models of Parkinson’s disease.

Table 1 A list of immunosupressent neuronal ligands and their respective receptors on microglia that have altered expression with aging

Neuronal ligand

Microglial receptor

Function

Age-related changes

Sources

Cell adhesion and contact inhibition

CD22

CD45

Suppresses microglial activation and proliferation

Gradual loss of receptor

[151-153]

CD47

SIRPoc (CD 172a), ECM glycoprotein thrombospondin

Suppresses microglial activation and phagocytosis

Gradual loss of ligand

[154, 155]

CD200 (OX-2)

CD200R

Suppresses microglial activation

Gradual loss of ligand

[110]

NCAM;

Polysialylated

NCAM

NCAM, Singlec-11

Suppresses microglial activation

Gradual loss of ligands and Singlec-11

[156-159]

FasL (CD95L)

Fas (CD95)

Suppresses microglial activation while promoting apoptosis

Gradual loss of ligand

[160, 161]

Cytokines and chemokines

IL10

IL10R

Promote Ml/М2 Polorization

Gradual loss of ligand

[111]

CX3CL1

CX3CR1

Suppresses microglial activation and chemotaxis

Gradual loss of ligand and receptor

[162, 163]

Transmitters and peptides

NE

alA, a2A, (31, (32

Suppresses microglial activation and chemotaxis

Gradual loss of ligand

[164, 165]

VIP, PACAP

VPACl, VPAC2

Suppresses microglial activation

Gradual loss of ligand and VPAC2

[166-169]

Neurotrophins

NGF

p75, NTR, TrkA

Suppresses microglial activation

Cortical decrease of ligand

[170, 171]

BDNF

p75, NTR, TrkA

Suppresses microglial activation

Gradual loss of ligand

[170, 171]

Neurotrophin-3

llp75, NTR, TrkB, TrkC

Suppresses microglial activation

Cortical decrease of ligand, systematic decline in pathological models

[170, 171]

 
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