Guard of Delinquency? A Role of Microglia in Inflammatory Neurodegenerative Diseases of the CNS
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- Wu, W., Shao, J., Lu, H. et al. Cell Biochem Biophys (2014) 70: 1. doi:10.1007/s12013-014-9872-0
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Activation of microglia and inflammation-mediated neurotoxicity are believed to play an important role in the pathogenesis of several neurodegenerative disorders, including multiple sclerosis. Studies demonstrate complex functions of activated microglia that can lead to either beneficial or detrimental outcomes, depending on the form and the timing of activation. Combined with genetic and environmental factors, overactivation and dysregulation of microglia cause progressive neurotoxic consequences which involve a vicious cycle of neuron injury and unregulated neuroinflammation. Thus, modulation of microglial activation appears to be a promising new therapeutic target. While current therapies do attempt to block activation of microglia, they indiscriminately inhibit inflammation thus also curbing beneficial effects of inflammation and delaying recovery. Multiple signaling cascades, often cross-talking, are involved in every step of microglial activation. One of the key challenges is to understand the molecular mechanisms controlling cytokine expression and phagocytic activity, as well as cell-specific consequences of dysregulated cytokine expression. Further, a better understanding of how the integration of multiple cytokine signals influences the function or activity of individual microglia remains an important research objective to identify potential therapeutic targets for clinical intervention to promote repair.
Although the brain is considered immunologically privileged, it is now evident that innate immunity is present there and plays an important role in defending this organ against foreign antigens and clearing debris resulting from the normal age-related turnover. Microglial cells  can be activated to become phagocytic and represent the main cellular component of innate immunity in the brain.
The innate immune response in the central nervous system (CNS) is necessary to counter potential pathogenic insults. The response is usually brief and ensures that the insult is removed. Transient up-regulation of inflammatory events in the brain is natural and does not lead to neuronal cell death  due to endogenous factors, such as glucocorticoids, that limit this response by inhibiting pro-inflammatory gene expression . However, overactivation of innate immunity may lead to neurodegeneration.
An increasing evidence is available that microglia serve as resident immune cells and function as a bridge between the CNS and immune system and are prominently involved in the pathogenesis of multiple sclerosis (MS), as well as pathogenesis of Alzheimer’s and Parkinson’s diseases . Recent studies of newly formed MS lesions (i.e., when deaths occurred within 24 h of symptom onset) highlighted microglial reactivity at the earliest stage of disease prior to profuse T cell infiltration . In these early MS lesions, extensive oligodendrocyte apoptosis and microglial activation were found in the absence of proximal lymphocyte infiltration, suggesting that microglial activation can rapidly occur in response to the oligodendrocyte cell death at the onset of lesion formation. In extant acute and chronic active lesions, microglial cells are particularly abundant, on average out-numbering lymphocytes by 10–20 times. Microglia are also observed in demyelinated and remyelinated lesions, although their numbers are reduced compared with active lesions . The consistent observation of microglia throughout evolution of lesion highlights the importance of innate immune response and the spectrum of microglial activities at different stages of disease.
Relationship Between Inflammation, Neurodegeneration, and Environmental Exposures
It is thought that nutritional deficiencies, environmental toxins, heavy metals, chronic bacterial and viral infections, autoimmune immunological responses, vascular diseases, head trauma, and accumulation of fluid in the brain, changes in neurotransmitter concentrations are involved in the pathogenesis of various neurodegenerative diseases [7–12]. Epidemiological studies demonstrate an association of exogenous factors with the onset and progression of these diseases. While it is still uncertain, it is possible that bacterial or viral infections may be exogenous triggers of MS . Thus, it was demonstrated that lipopolysaccharide (LPS)-induced chronic inflammation in the rat brain causes microgliosis and up-regulation of interleukin (IL)-1β and tumor necrosis factor (TNF)-α mRNA expressions . This was followed by demyelinated foci.
Further, some studies provided an evidence for potential adverse effects of air pollution on the CNS. In one study, dogs that lived in a highly polluted region had an increase in brain inflammation compared with animals that lived in a less polluted area . Since inflammatory events have been associated with neurodegenerative processes, it is possible that extended exposure to the particulate matter present in the ambient air may aggravate the progression of these disorders. There is a correlation between MS relapse and the particulate matter content of ambient air. Since metals constitute a high proportion of particulate matter, it is possible that metals may account for some of the responses following exposure. Further, a direct causal link between exposure to metal and abnormal neurological symptoms was also observed [16, 17].
Under normal circumstances, microglia are activated only briefly followed by quick cessation of phagocytic activity. However, under hyperresponse, these cells can remain active for very long time creating considerable damage to neighboring tissue.
While environmental factors are capable of triggering inflammatory events in the CNS, individual genetic predisposition and the interaction of the body with environmental exposures may be more important in the pathogenesis of neurodegenerative diseases.
Individual Susceptibility to Diseases: Genetic Determinants of Microglial Activity
Not all microorganisms are recognized as dangerous entities by the host immune system. The homeostatic ecosystem of microorganisms in the body is named human microbiome and is thought to play an integral role in shaping our immune system and modulating health. Human survival depends on the ability of our immune system to co-exist with microbes in the environment. While genetic diversity of immune system is the basis for adequate adaptation to the environment, the reverse is also true. Our immune system requires input from the environment to ensure proper functioning . Industrialization and modernization of human lifestyle eliminated a great portion of microbial communities from our surroundings through childhood vaccination, frequent antibiotic use, and much improved hygiene standards. It is possible that this rapid change in the lifestyle inadvertently deprived us of important microbiological stimulation needed to prime the immune system tolerance. As a consequence, people became susceptible to a range of immune-related diseases that target innocuous antigens or self-antigens. The lack of early life infection may contribute to immune disorders that develop later in life .
There is growing evidence that immunologic dysfunction plays a major role in uncontrolled and excessive activation of microglia which leads to inflammation-mediated neurodegeneration.
Another important cause of impaired immunity may be associated with genetic polymorphism of the genes that cause the microglia-driven neurodegeneration .
It is further known that nutritional deficiencies can negatively affect immune system. Conversely, an excess of certain nutrients (e.g., omega-6 fats, monosodium l-glutamate, aspartame, sugar) can significantly interfere with immune function .
In addition, aging can slow the ability of immune system to generate antibodies. The efficiency of immune system is affected by aging eventually leading to the development of age-associated immune malfunctions . This set of immune deficiencies leads to direct cytokine activation of microglia through autoimmune-induced cytokine stimulation of microglial receptors. Another age-related immune deficiency is caused by low levels of C4B complement, which is important for eliminating viruses, mycoplasma, and fungi. This increases the likelihood of viral and mycoplasmal persistence in the brain. Both of these infections cause chronic activation of microglia .
As shown above, microglia are key participants in virtually all neuropathologies of the CNS. The critical role of microglia in regulating both pathogenic and repair processes is becoming increasingly evident.
Modest Activation of Microglia Leads to Reversible Pathological Processes
As outlined above, etiology of inflammation and neurodegenerative disorders is multifactorial and includes interactions between environmental exposures and genetic predisposition. When the resident innate immune cells of the CNS are confronted with stress factors, microglia become activated, and release cytokines and reactive oxygen species (ROS) that cause neurotoxicity. These mediators typically have a short half-life. However, if production of ROS is extended, endogenous stores of antioxidants are exhausted and cell damage can ensue . The moderately activated microglia function in a tightly regulated manner to eliminate the invader while minimizing the damage to surrounding normal tissue. When neuron degeneration or necrosis reaches a certain level, some endogenous ligands, such as high-mobility group box 1 (HMGB1), heat shock proteins, or extracellular matrix are released. These substances will bind to microglial Mac1 to activate either NF-κB or NADPH oxidase in microglia whose activation depends on the extent of structural damage and the amount of released endogenous ligands [23, 24].
MAC1 can also be considered as a phagocytosis receptor of microglia whose activation triggers cell phagocytosis to clear apoptotic T cells and oligodendrocytes and to remove myelin debris to facilitate resolution of the inflammatory response [26, 27]. Studies show that activation of phagocytic activity in microglia is accompanied by attenuated production of cytotoxic mediators and pro-inflammatory cytokines .
In general, MAC1 mediates the initial phase of microglial response. The MAC1-stimulated response alone cannot lead to a completely cytotoxic phenotype. Recent research indicates that PI3K inhibition in completely activated microglia does not prevent accumulation of nitrogen monoxide or cell death. However, this blocking effect is not as specific as blocking JAK-STAT pathway . This is in contrast to NF-κB inhibition that can reduce production of nitric oxide and facilitate cell survival. Therefore, a moderate stimulation through this pathway cannot lead to development of a full cytotoxic phenotype.
After phagocytic activity of microglia triggered by MAC1 receptor completes its mission, the apoptosis program is launched. Expression of pro-apoptotic proteins Fas (CD95), Fas ligand (CD95L), and Bax eventually leads to elimination of microglia and macrophages, representing a self-regulatory mechanism to limit release of cellular inflammatory mediators .
Fully Activated Microglia Contribute to Irreversible Pathological Phase
Microglia serve as key effectors of innate immunity in the CNS and act as the first line defense against pathogens. These cells also eliminate damaged, infected, or invading cells through the induction of inflammatory and phagocytic responses . The cell responses are determined by a respective pattern recognition receptor (e.g., toll-like receptor) that a given pathological stimulus activates in microglia. Yet, compared with the specificity of the adaptive immune response, these responses can be viewed as relatively non-specific [31, 32]. The balance between innate immunity-induced injury and protection may be defined by a net effect of extracellular signals acting on microglia. In neurodegenerative diseases, neurons experience a primary insult that makes them vulnerable, probably, through oxidative stress. Then, a secondary insult mediated by activated microglia may shift the balance toward irreversible injury .
Pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, that are secreted by microglia in response to STAT1 and NF-κB activation, play an important role in propagating cytotoxic pro-inflammatory response of microglia beyond the initial site of activation. The predominant signaling pathways implicated in microglial activation are the p38 and ERK members of the mitogen-activated protein kinase (MAPK) family, which control release of neurotoxic mediators and pro-inflammatory cytokines . The self-propagating cycle of inflammation is mediated to a large extent by the ability of these pro-inflammatory cytokines to potently activate microglia, which leads to amplification of the inflammatory response.
Microglia that Exhibit Immune Adjustment Effect Drive Pathological Process into Relapsing–Remitting Vicious Circle
Yet, microglia are not only involved in tissue destruction in inflammatory neurodegenerative diseases. Certain signaling mechanisms can change their phenotype from the pro-inflammatory to the immunomodulating and nerve repairing one.
Many researchers demonstrated that IL-10 induces SOCS-3 expression, and this may mediate anti-inflammatory effects of IL-10 [42–45]. IL-10 is also a potent activator of STAT-3 [25, 43, 46–48] whose activation is critical for SOCS-3 expression induced by IL-10 . IL-10 expression is necessary for optimal induction of SOCS-3 by LPS, especially at later time points of stimulation [44, 49]. Further, Staples et al.  recently demonstrated that IL-10 induces its own expression in monocyte-derived macrophages via activation of STAT-3. This positive autocrine feedback loop may allow IL-10 to enhance its effect on SOCS-3 gene expression.
There are also several negative feedback mechanisms that collaborate to limit pro-inflammatory response. These autoregulatory processes are critical for decreasing inflammation to minimize the risk of excessive tissue damage. The conversion of microglia from a pro-inflammatory to an immunomodulatory phenotype is a key component of the autoregulatory response; this transition initiates a switch from the production of pro-inflammatory to production of immunomodulatory and anti-inflammatory cytokines.
Some studies indicated that TLR-mediated effects involve immunoregulatory cytokines, such as IL-10 and transforming growth factor (TGF)-β, and different subsets of regulatory T cells, most notably CD4+CD25+FoxP3+ T cells for TLR4 agonists and NKT cells for TLR3 agonists. TLR2, TLR3, and TLR7 agonists preferentially stimulated IL-10 and TGF-β production which was not the case for the TLR4 agonist LPS .
TGF-β is a pleiotropic cytokine that plays an important role in regulating both innate and adaptive immune responses and is critical for the induction of immunological tolerance . An important mechanism by which TGF-β exerts anti-inflammatory effects is via inhibiting proliferation and effector function of Th1 cells . Treatment of antigen-specific T cells with TGF-β1 and TGF-β2 inhibits T cell activation, suppresses production of pro-inflammatory cytokines including Interferon (IFN)-γ, TNF-α, and LT .
The apparent dichotomous nature of microglial activity during inflammatory demyelination reflects the central role of this cell type in regulating the transition in the innate immune response from the disease-promoting phenotype to the repair-facilitating one. This postulate is consistent with histopathological data that demonstrate considerable heterogeneity in MS lesion pathology . In addition to degenerative processes, MS is also characterized by a spontaneous regenerative response that results in partial remyelination of denuded axons [55, 56]. In fact, the two processes are inseparably linked, with destruction and repair often occurring at the same time, depending on the type of infiltrating inflammatory cells and cytokine production in lesions. Since inflammation and repair process always alternate, this context-dependent activation of microglial cells and their secretion of pro-inflammatory cytokines transform pathological process into relapsing–remitting vicious circle.
There is growing evidence that the etiology of neurodegenerative disorders is multifactorial and consists of an interaction between aging, environmental factors, and genetic predisposition. Meanwhile immunologic dysfunction also plays a major role in uncontrolled and excessive activation of microglia , which release ROS that cause neurotoxicity. It is possible that the changes in our lifestyle limit beneficial early exposure of these innate immune cells to pathogens, and this results in occurrence of immunological disorders later in life .
Microglia-mediated neurotoxicity occurs through pattern recognition receptors when pathogen-associated molecular patterns trigger an excessive immune response, or when stimuli (environmental toxins, endogenous proteins, and neuron damage) are misinterpreted as pathogens, and multiple receptor-initiated signaling pathways are simultaneously activated to cause hyperactivation of transcription factors NF-κB and Ap-1 [37–39]. Given that a single ligand is often recognized by multiple pattern recognition receptors, the cumulative effect of various receptor combinations might define how microglia respond to neurotoxins and whether the activation is deleterious or beneficial.
The full cytotoxic phenotype of microglia becomes deleterious, fuelling further neuronal loss (reactive microgliosis) and resulting in a perpetuating cycle of neurotoxicity and a progressive neurodegenerative disease.
Fortunately, endogenous protective regulatory signals in the brain have been identified that inhibit microglial overactivation, such as anti-inflammatory cytokines (that is, IL-10 and TGF-β), glucocorticoids, and even microglial apoptosis. However, it has been proposed that when the ability to activate these protective mechanisms fails, or when they are overwhelmed by an excessive inflammatory response, microglia initiate neuronal death and drive the progressive nature of neurodegenerative disease. Moreover, this adjustment mechanism itself is flawed. For example, the Fas–Fasl signal pathway triggered by the excessive activation of the inflammatory response also leads to apoptosis of oligodendroglioma to further aggravate the condition after eradication of excessive activation of microglia/macrophages, and T lymphocytes [37, 58–60].
Untargeted suppression of microglia-mediated inflammation is unlikely to be an effective therapeutic strategy in MS and related diseases. Effective therapies will require a perfect combination of approaches to facilitate immunomodulation, neuroprotection, and remyelination [37, 61].
Future research will need to focus on delineating the mechanisms responsible for microglial overactivation in an effort to identify more specific markers and develop novel compounds or biologics with greater therapeutic efficacy.
This work was supported by the China National Natural Science Fund Project No. 812272791.