We present a novel hypothesis for the pathogenesis of PD in that the adaptive immune system can modulate microglial inflammatory responses and as such affect the homeostatic environment of the brain (). In this way opposing immune regulatory responses as those affected by Treg or Teff could lead to divergent outcomes in the tempo and progression of disease. This is based on activation of microglial responses by aggregated and oxidized proteins, particularly α-syn. In early stages of disease where limited N-α-syn accumulates in the extracellular brain, the regulatory functions of the immune system predominate and microglial-induced inflammatory responses are controlled. This may occur by limited antigenic stimulae or by alterations in the microglial phenotypes (36
). We posit that prior to onset of symptomatic disease (asymptomatic), adaptive immune responses, in which Treg predominate, are operative on microglia to attenuate microglial activation and neuroinflammatory responses. As a result reactive oxygen species (ROS) generation and the degenerative activities that occur subsequent to DA neuronal damage and release of α-syn from LB are controlled. At this stage of disease microglia are actively phagocytic and produce a broad spectrum of regulatory factors that principally maintain central nervous system (CNS) homeostasis. Therefore, aggregation and nitration of α-syn that accumulates in the extravascular space is limited. Such events preclude the development of potent adaptive neurodegenerative immune responses and the widespread, often adverse affects of oxidized and misfolded proteins. Regrettably, there are as yet no diagnostic biomarkers to confirm PD when few dopaminergic neurons are affected by disease. Although it is not clear if Treg is activated more in the early stage and Teff in the later stages of PD our data would support both ideas amongst another notion and that is Treg is active in early stage disease but is dysfunctional in the later stages. On balance, bioimaging data obtained from PD patients and animal models of human disease indicate that levels of activated mid-brain microglia correlate with diminished levels of dopamine transporters in the putamen and that microglial activation is increased with disease or lesion progression (37
). During overt disease the presence of N-α-syn engages both innate and adaptive immune responses leading to losses in homeostatic function coupled to robust inflammatory and neurotoxic responses. During active disease, regulation of adaptive immunity breaks down and significantly influences control of the neural homeostatic environment (21
). Moreover, changes in T cell populations in PD suggest that peripheral adaptive immune alterations and immune-related inflammation are important in disease pathogenesis (40
). However, analysis of T cell subsets in PD and aged populations has yielded conflicting results with regards to CD4+CD25+ Treg numbers and function (40
). Profound oxidative-associated damage and death of nigral DA neurons lead to increased release of α-syn with subsequent oxidation and misfolding. With increased exposure to N-α-syn, microglia become activated yielding a phenotype with reduced homeostatic activities and increased neurotoxic potential (23
). During this phase, Treg engage activated microglia to induce apoptosis or affect a neurotrophic phenotype while showing a less robust affect on pro-inflammatory activities. Treg may also be, in part, reduced in numbers and/or function as a result of N-α-syn-mediated immunity. This results in more widespread nigrostriatal damage, recruitment of reactive immunocytes into brain, and pathogenic events that facilitate accelerated neuronal damage. In the aging rodent brain microglia show exaggerated neuroinflammatory responses that affect nitration of α-syn (43
). In addition, such aged microglia may be less susceptible to Treg regulation than similar cells from neonates, as was used in the current studies. However, data obtained using bone-marrow derived macrophages from adult mice suggest that Treg can modulate aged macrophage activation responses to inflammatory stimuli including N-α-syn or HIV-1 (29
) (J. Liu, N. Gong, A. D. Reynolds, X. Huang, R. L. Mosley, and H. E. Gendelman, manuscript submitted). Importantly, the effects of Treg on the macrophage activation and neurotoxicity can be recapitulated in human cells in response to inflammatory stimuli or viral human immunodeficiency virus infection (46
) (our unpublished observation). However, changes in percentages, phenotype, TCR repertoire, and function of Treg may preclude these effects in the aging populations (42
FIGURE 7 CD4+ T cells in the prevention and pathogenesis of PD prior to onset of symptoms and during overt disease. T cell-mediated immune surveillance has been proposed that may account for a neurotrophic phenotype of resident microglia. The neurotrophic capacity (more ...)
Previous works demonstrate that Treg and Teff modulate microglial activation and affect neuroprotection in laboratory and animal models of PD (29
), ALS (31
), and HIV-associated neurocognitive disorders (30
) (J. Liu, N. Gong, A. D. Reynolds, X. Huang, R. L. Mosley, and H. E. Gendelman, manuscript submitted). Previous studies revealed microglial activation within a few hours following MPTP-intoxication, prior to infiltration of CD4+ T cells (47
), which may be linked in significant manner to accumulation of nitrated and aggregated α-syn and DA neuronal death (21
). This inflammatory reaction then facilitates recruitment of adaptive immune responses to the brain that exacerbate microglial activation and DA neuron death (21
). Surprisingly, adoptive transfer of CD3-activated Treg significantly attenuated such neurodegenerative responses (29
). We therefore propose that Treg function to prevent robust microglial inflammation and promote DA survival through a concerted mechanism. First,
by suppressing primary microglia activation through induction of apoptosis. Second,
through modulation of adjacent microglia to suppress secondary activation in response to both innate and adaptive immunity. Third,
inducing a microglial phenotype to promote DA survival. This is supported in other model systems including a murine model of HAD (J. Liu, N. Gong, A. D. Reynolds, X. Huang, R. L. Mosley, and H. E. Gendelman, manuscript submitted). Moreover, in vivo
, Treg may modulate the adaptive immune responses within the nervous system.
Treg modulation of the N-α-syn-activated microglial proteome is linked to secretory, phagocytic, redox, and enzymatic cell functions. In a significant manner, Treg affected key microglial free radical clearance, glutamate metabolism, proteasome, and protease activities along with cell migration, vesicle transport, and bioenergetic function. These were substantive and paralleled diminished NF-κB activation and secretion of pro-inflammatory cytokines. As such, these results contrast what is well known concerning Treg effects on T cell proliferation in vitro
, which requires cell-cell contact (49
). Although the suggestion that a higher frequency of Treg could be linked to increased suppressive activity and accelerating neurodegeneration (41
) is not supported by the current data set, the precise mechanism for immune regulation of disease remains a new and actively investigated area of study.
Macrophage NF-κB blockade decreases pro-inflammatory responses without affecting phagocytic function (50
) and supports the current results. Treg diminished the pro-inflammatory response, and diminished ROS production was coincident with increased glutathione stores. These observations paralleled increased expression of redox-active proteins. The induction of antioxidants and their clearance of noxious oxygen free radicals provide new insights as to how Treg may control microglial neurotoxic responses in disease (51
). Indeed, redox effects on neuronal function are implicated in a broad range of neurodegenerative diseases regardless of etiology (54
). Thus, robust increases of 2-fold or greater in microglial biliverdin reductase B and glutaredoxin 1 amongst others suggest that Treg function to increase the cell's buffering capacity against oxidative stress.
Glutaredoxin 1 is important both for sustaining intracellular glutathione and for promoting mitochondrial respiration and oxidative phosphorylation (57
). Downregulation of glutaredoxin 1 results in loss of glutathione and mitochondrial complex I activity (58
). Moreover, environmental toxins that target mitochondria reproduce PD pathobiology such as DA neuronal loss and α-syn nitration and aggregation (21
). One example is the proneurotoxin MPTP, from which its metabolite, MPP+, engages complex I of the ETC and is linked to the formation of ROS (60
). Familial forms of PD are associated with mutations in the α-syn gene that influence cellular responses to mitochondrial stress (62
). Under oxidative and metabolic stress conditions α-syn translocates from the cytosol to the mitochondrial surface and may play a direct role in mitochondrial physiology (63
). Our results demonstrate that N-α-syn induced a functional decline of microglia mitochondrial function. Importantly, Treg promoted mitochondrial function and led to increases in proteins that are associated with the ETC, thus providing mechanisms for Treg-mediated neuroprotection for PD (29
). Such findings support the notion that Treg affect mitochondrial respiration leading to an enhanced energetic efficiency of N-α-syn microglial mitochondria. Changes in free radical clearance mechanisms, including increased expression of peroxiredoxins and catalase, further strengthen Treg-induced enhancement of redox processes in N-α-syn stimulated microglia. Treg also regulated expression of HSPs, which affect protein import to the mitochondrial matrix. Overall, these data suggest that Treg enhance microglial mitochondrial functional capacity, decreasing the amount of oxidative stress that results from mitochondrial dysfunction.
Our results indicate that inhibition of oxidative stress is but one Treg neuromodulatory effect. Another is linked to diminished cathepsin activity. Indeed, cathepsin activity is increased in activated microglia and contributes to the their neurotoxic potential (64
). In particular, CB is responsible, in part, for the terminal degradation of intracellular proteins (65
). It has been implicated in a variety of inflammatory diseases and secretion of both pro-CB and CB by activated microglia leading to the induction of neuronal death (64
). In support of this notion, CB inhibitors protect against microglial-mediated neurotoxicity (24
). We showed that Treg induced a greater than 2-fold decrease in CB in N-α-syn stimulated microglia along with decreased enzymatic activity, and may play an active role in attenuation of microglial neurotoxicity.
Treg pre-treatment also resulted in significant increases in proteins of the UPS in activated microglia. This is important to disease prevention as UPS functional inhibition of the UPS can affect the accumulation of ubiquinated proteins commonly seen in neurodegenerative diseases, and diminished UPS enzymatic activities are reported in PD (66
). Moreover, diminished function of the UPS is linked to α-syn nitration and aggregation (67
). Increased UPS function induced by Treg may be either reflective of enhanced clearance mechanisms of misfolded proteins, or protection from decreased proteasome function by aberrant α-syn.
Whereas pre-treatment with Treg resulted in significant diminution of cyto/chemokine secretion, post-treatment analysis was more variable in its effects. Similar to pre-treatment, Treg suppressed the production of inflammatory mediators TNF-α, MCP-1, and IL-12. In contrast, production of IFN-γ was enhanced. Interestingly, IFN-γ was shown to mediate neuroprotective T cell responses (69
). This result was coincident with previous reports suggesting that Treg act primarily on the afferent rather than the efferent end of the immune response.
Induction of apoptosis may be another mechanism by which Treg regulate microglial inflammation. Increased expression of the lysosomal proteases including cathepsins B and D may indicate that Treg induce autolysis (70
). Our observation that Treg-mediated apoptosis of activated microglia is acting, in part, by Fas-FasL interactions correlates with previous reported studies demonstrating that Treg upregulate FasL expression through which they induce target APC apoptosis (35
). Interestingly, N-α-syn stimulation induced transient resistant in microglia to Fas mediated apoptosis, that was reversed by Treg and not Teff, although both T cell subsets were shown in upregulate Fas expression on microglia. The effects of TNF-α were shown to increase cell resistance to Fas-mediated apoptosis (71
). Therefore, one possible explanation is that Treg, more so than Teff, decreased TNF-α production by stimulated microglia resulting in increased susceptibility to Fas-mediated apoptosis.
Overall, our data suggest that interactions between Treg and microglia affect microglial processes with conversion of microglia from a neurotoxic to neuroprotective phenotype or induction of apoptosis. This change is multifunctional as the microglial response to stimuli can induce reversion to its original function in maintenance of homeostasis and prevention of neuronal damage. The ability of Treg to regulate microglial inflammation, cell function, and specific enzymatic activities provide novel tools to manipulate ongoing microglial inflammatory responses. In light of these, and previously published findings regarding T cell populations in PD, we now propose a model for disease with regards to a role for Treg in both the prevention and pathogenesis of PD. As such, these data support the use of therapeutics that take advantage of Treg responses within the brain or that target specific protein changes linked to reversion of a neurotoxic microglial phenotype to neurotrophic.