- Theoretical Neurosciences Research, LLC, Neurosurgeon (Ret), Ridgeland, MS
Russell L. Blaylock
Theoretical Neurosciences Research, LLC, Neurosurgeon (Ret), Ridgeland, MS
DOI:10.4103/2152-7806.118349Copyright: © 2013 Blaylock RL This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
How to cite this article: Blaylock RL. Immunology primer for neurosurgeons and neurologists part 2: Innate brain immunity. Surg Neurol Int 18-Sep-2013;4:118
How to cite this URL: Blaylock RL. Immunology primer for neurosurgeons and neurologists part 2: Innate brain immunity. Surg Neurol Int 18-Sep-2013;4:118. Available from: http://sni.wpengine.com/surgicalint_articles/immunology-primer-for-neurosurgeons-and-neurologists-part-2-innate-brain-immunity/
Over the past several decades we have learned a great deal about microglia and innate brain immunity. While microglia are the principle innate immune cells, other cell types also play a role, including invading macrophages, astrocytes, neurons, and endothelial cells. The fastest reacting cell is the microglia and despite its name, resting microglia (also called ramified microglia) are in fact quite active. Motion photomicrographs demonstrate a constant movement of ramified microglial foot processes, which appear to be testing the microenvironment for dangerous alteration in extracellular fluid content. These foot processes, in particular, interact with synapses and play a role in synaptic function. In event of excitatory overactivity, these foot processes can strip selected synapses, thus reducing activation states as a neuroprotective mechanism. They can also clear extracellular glutamate so as to reduce the risk of excitotoxicity. Microglia also appear to have a number of activation phenotypes, such as: (1) phagocytic, (2) neuroprotective and growth promoting, or (3) primarily neurodestructive. These innate immune cells can migrate a great distance under pathological conditions and appear to have anatomic specificity, meaning they can accumulate in specifically selected areas of the brain. There is some evidence that there are several types of microglia. Macrophage infiltration into the embryonic brain is the source of resident microglia and in adulthood macrophages can infiltrate the brain and are for the most part pathologically indistinguishable from resident microglia, but may react differently. Activation itself does not imply a destructive phenotype and can be mostly neuroprotective via phagocytosis of debris, neuron parts and dying cells and by the release of neurotrophins such as nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF). Evidence is accumulating that microglia undergo dynamic fluctuations in phenotype as the neuropathology evolves. For example, in the early stages of neurotrauma and stroke, microglia play a mostly neuroprotective role and only later switch to a neurodestructive mode. A great number of biological systems alter microglia function, including neurohormones, cannabinoids, other neurotransmitters, adenosine triphosphate (ATP), adenosine, and corticosteroids. One can appreciate that with aging many of these systems are altered by the aging process itself or by disease thus changing the sensitivity of the innate immune system.
Keywords: Immune surface receptors, immunoexcitotoxicity, innate immunity, microglia, microglial priming
SUMMARY OF PART I: IMMUNITY PRIMER
In part one, we explored the anatomy and functional activation of the various components of the systemic immune system. Basically, systemic immunity is divided into an innate system and an adaptive system. These two systems do not act independently, but rather exhibit intimate interactions so as to initiate a coordinated attack. The innate system reacts rapidly, acting as the first line of defense when the interior of the body is invaded, whereas the adaptive system reacts slower, but with far greater specificity – that is, it can identify individual molecular differences in invading organisms and other antigens.
Next to the central nervous system (CNS), the immune system is one of the most complex systems in the body. This complexity extends to its individual components, especially the various cells involved in immunity. The innate immune system contains a number of specialized immune cells located in tissues lining the respiratory air passages, the gastrointestinal (GI) tract's mucosa and the mucosal lining of the urogenital tract. It is here that most invasions take place. Combined with the physical barriers of the skin and mucosal cell layers, these innate immune cells provide a formable protective defense.
The adaptive immune system utilizes a system of presentation of invading antigens that involves specialized molecules such as complement, to aid in molecular identification for future reference. Macrophages within tissues play a major role in transporting the antigen to the regional lymph nodes so that they can be presented to lymphocytes where tagging for future reference occurs. Certain lymphocytes store memories of the antigen for later identification, should the same antigen reinvade the body. This then allows for very rapid responses, since the complex identification and recognition process can be avoided.
Of special importance to this discussion is the communication between the systemic immune system and the brain's own special innate immune system. Previous beliefs about the brain being an immunologically privileged site have now been modified once it was realized that this immune barrier was incomplete.
The brain does not contain its own adaptive immune system, but rather can transport antigens to regional cervical lymph nodes that can process the antigen and then direct activated lymphocytes and other immune cells back to the brain substance. Immune chemokines from microglia and astrocytes can direct systemic immune cells to the brain as well. In addition, the vagus nerve acts as a rapid response system to turn on brain innate immune cells when inflammation occurs within the GI tract or abdominal cavity. This two-way communication between the brain and the systemic immune system is critical. One can easily see that when the body is invaded, the brain must be rapidly informed of the invasion so that it can gear up its innate immune system in preparation of a possible invasion.
This gut – brain and systemic immune axis each play a major role in a number of neurological disorders, especially in the case of neurodegenerative diseases. We now know that when an infection or inflammatory condition occurs systemically, microglia are activated within specific brain areas within minutes. In fact, even minor surgical procedures have been shown to trigger microglial activation within the brain. This is especially important in treating patients with multiple injuries, since microglial activation worsens the neurological picture if prolonged or too intense.
Along this line, it should be appreciated that the immune system, both systemic and within the CNS, contains a great number of fail-safe systems and feedback controls to prevent over-activation of the immune process. In addition, complex cell signaling mechanisms are utilized to switch the brain's microglia so as to tailor the immune cell to the particular function that is needed. For example, with brain trauma microglia migrate to the site of the injury and are switched to a phagocytic phenotype so as to remove cellular debris. They can then be switched to a reparative phenotype to aid in repairing the damage. It is now apparent that dysfunction of this switching mechanism can play a major role in a number of neurological disorders should the microglia become stuck in a neurodestructive mode.
It is also important to appreciate that systemic immune cells, particularly macrophages, frequently enter the brain from the blood stream and once inside the brain take on the appearance and function of resident microglia. Under certain conditions, such as with brain trauma and CNS infections, large numbers of neutrophils, lymphocytes, mast cells, and macrophages enter the CNS. The functional state of these immune cells can make the difference between progressive brain destruction or a reversal of immunoexcitotoxicity. For example, macrophages can assume a primarily cytotoxic phenotype called M1 or an antiinflammatory phenotype called M2. Likewise, lymphocytes can be either cytotoxic or suppress brain cell inflammation and injury. The antiinflammatory lymphocytes, called regulatory T-cells or Tregs, are now recognized to play a critical role in protecting the brain. In fact, we now know that early assumptions concerning the pathological role of invading lymphocytes in experimental allergic encephalomyelitis (EAE), for example, were mistaken and that these were mostly Tregs attempting to combat the out of control inflammatory response.
Finally, several hormones and neurotransmitters also play a critical role in controlling immune reactions. One of the critical control systems is the immune–hypothalamo–pituitary axis, with corticosteroids playing a major role. Links between systemic immune activation and microglia within the hypothalamus trigger a release of corticosteroids, which under most conditions, dampens the immune reaction, both systemically and within the brain – but not always. Under certain conditions, these hormones can worsen inflammation.
Frequently forgotten is the fact that norepinephrine, epinephrine, and acetylcholine play a major role in dampening inflammation, especially within the CNS. A loss of acetylcholine levels in the Alzheimer's brain, for example, would significantly enhance any inflammatory reaction that is ongoing and may explain in part why acetylcholine agonists improve symptoms. With this brief review, now let us examine more closely the innate immune reactions within the CNS.
PART II: MICROGLIA
In 1932 Pio del Rio-Hortega introduced the concept of the microglia with incredible accuracy in the publication Cytology and Cellular Pathology of the Nervous system, citing many characteristics that would not be agreed upon until over half a century later. For instance, he accurately postulated that microglia entered the brain during early neurodevelopment and that these cells were of mesodermal origin. Further, that the guiding structures for the embryonic precursors included blood vessels and white matter tracts, which allowed these cells to migrate to all areas of the brain.
He was also the first to suggest that in the undisturbed brain microglia assumed a ramified (resting) morphology and upon disturbance or encountering specific brain pathology, they were morphed into an amoeboid phenotype. Del Rio-Hortega suggested that these cells could not only migrate within the brain toward areas of injury, but also could proliferate and carry out phagocytosis. The only error he made was to assume that microglia were evenly distributed throughout the brain. We now know that there is a heterogeneous distribution, with the highest concentrations in the gray matter and particularly within the limbic areas (hippocampus, amygdala, and entorhinal cortex) and substantia nigra, which has the highest microglial density of any area of the brain.[
Largely ignored until quite recently, microglia have now become the focus of a great deal of research. Newer studies suggest that microglia form a part of the tripartite synapse along with astrocyte foot processes and the synapse itself. Time lapse two-cell microphotography, extending up to 10 hours of observation, demonstrate that ramified microglia, the so-called resting phenotype, actually are quite active, with continuous extension and retraction of foot processes around the synaptic neuropile.[
These newer studies also demonstrate that microglia are not uniform in their activity but exist as a number of subtypes, which offers an extensive program of activation states in response to a wide number of dynamic pathological conditions. In addition, microglia, as mentioned, are heterogenous in their distribution. Microglia found along brain–blood vessels are often found in an activated state and form a particular immunological barrier for the brain in conjunction with the blood–brain barrier (BBB).[
Under pathological conditions, resident microglia may not only proliferate, but also can migrate great distances and accumulate near the sites of pathologic damage. Activation stages of microglia are not all or none, rather they are capable of assuming gradations of activation, which also includes priming phenotypes [
We are learning that much like the peripheral immune system, the brain's innate immune system is enormously complex. Further complexity is added when we consider the interaction between the innate immune system and the adaptive immune system composed of invading immune cells and immune factors from the periphery.
Developmental anatomy of microglia and of the innate CNS immune system
It is now accepted that microglia are derived from mesodermal/mesenchymal tissues, primarily myeloid cells from the bone marrow. These cells migrate to the CNS during the first trimester and throughout the early part of the second trimester in the human. There is evidence that during these stages microglia consist of two populations, which include (a) myeloid/mesenchymal cells and (b) a developmental and transitory forms of fetal macrophages.[
Clinical Correlation: Fractalkine, a cytokine modulator of microglial proinflammatory activation, and its receptor, located on microglial cell membranes, in general suppress inflammatory signaling. In the Paolicelli et al. study they found that microglia via stimulation by fractalkines, modulated synaptic pruning during neural development and that deficiencies in fractalkine or its receptor led to excessive synaptic connectivity and abnormal brain development. This condition also produced excessive sensitivity to excitatory neurotransmission and excitotoxicity-related disorders, such as seizures. While other factors are playing a role in embryonic brain pruning, such as the compliment cascade components C1q and C3, during the early stages of neurodevelopment, fractalkine is the primary factor.
While macrophages enter the developing brain in significant numbers; in the adult brain such an invasion is quite rare in the undisturbed brain. Under pathological conditions invasion of leukocytes is dramatically increased.
Molecular pathophysiology of microglia
Microglia membrane receptors, second messengers, and cell signaling
There many cell signaling pathways from receptors on the cell surface connecting to the genes in the nucleus. These signaling pathways are called second messengers, a collective term referring to a number of molecules in the signaling pathway. When the cell surface receptor is engaged by its specific stimulating extracellular activator, the intracellular signaling pathway is activated leading directly to the nuclear genes. These genes or gene, when activated, lead to the production of proteins that are made in the cytoplasm directed by mRNA, which copies the sequence of the gene in the nucleus and then travels to the cytoplasm to participate in the manufacture of the gene-specific protein on the ribosomes. These proteins then direct certain activities of the cell. These signaling pathways exist in all cells but there are some that are very important in microglia, which are discussed as follows.
Signaling Pathways and MyD88, a major coordinating signaling protein: Immune cell receptors, like most cell membrane receptors, are linked to intracellular signaling molecules that carry the messages to effector systems and nuclear gene activators. This process is called cell signaling and is central to the activation and suppression of molecular pathways in all cells. Cells contain thousands of cell signaling molecules, which are linked into functional pathways and when stimulated cause the cell to perform specific physiologic roles. Central to immune activation responses is the cell signaling adaptor molecule, myeloid differentiation primary-response protein 88 (MyD88), which acts like a coordinating molecule (called an adaptor molecule) linking surface receptors to immune cell signaling pathways within the cytoplasm. Mice lacking MyD88 cannot produce tumor necrosis factor-alpha (TNF-α) or Interleukin 12 (IL-12) when exposed to bacterial antigens.[ Signaling Pathways containing Janus kinases: Another cell signaling pathway in immune cells and microglia involve the Janus kinases. Critical to immune function are the Janus family of kinases, Jak1, Jak 2, Jak3, and Tyk2, which are involved in cell growth, survival, development, and differentiation of immune cells.[ Signaling Pathway From Cell Membranes to a gene through NFkB: One essential cell signaling link from the cell membrane is directed to Nuclear Factor kappa B (NFkB), which activates genes controlling the production and secretion of proinflammatory cytokines such as TNF-α. Activation of toll-like receptors (TLRs) and other immune receptors on the surface of microglia, macrophages, and other immune cells are linked to NFkB activation. This cell signaling molecule represents a major controlling mechanism of inflammation. Signaling Pathways that reduce the inflammatory response of microglia and other immune cells: Other cell signaling mechanisms play an important role in reducing inflammation and potentially dangerous states of immune activation. For instance, MAPK phosphatase 1 (MKP1), is a major negative modulator of TLR-induced inflammation. You will recall the TLRs are a family of receptors on the cell membrane of immune cells that interact with antigens and other immune chemicals to initiate immune activation. By modulating these receptors, MAPK phosphatase 1 can fine-tune the immune reaction. Another immune response regulator is called a suppressor of cytokine signaling 1 (SOCS1), which is an encoded gene. When the SOCS1 gene is activated by extracellular produced cytokines, such as IL-2, IL-3, interferon-gamma (INF-γ), or GM-CSF, the microglial SOCS1 gene reduces the activation of proinflammatory cytokine cell signaling. That is, it also dampens the immune response. Another such immune inhibitory molecule TREM2, is found on the surface of myeloid cells (primarily monocytes and macrophages) from the bone marrow. This protein interacts with another protein produced by the TYROBP gene, which in turn reduces the proinflammatory signaling of microglia, macrophages and dendritic cells. Mutations of this gene, which have been found to be common in Alzheimer's cases, impairs this immune dampening effect, thus leading to chronic brain inflammation, as seen in many neurodegenerative diseases.[
Signaling Pathways and MyD88, a major coordinating signaling protein: Immune cell receptors, like most cell membrane receptors, are linked to intracellular signaling molecules that carry the messages to effector systems and nuclear gene activators. This process is called cell signaling and is central to the activation and suppression of molecular pathways in all cells. Cells contain thousands of cell signaling molecules, which are linked into functional pathways and when stimulated cause the cell to perform specific physiologic roles. Central to immune activation responses is the cell signaling adaptor molecule, myeloid differentiation primary-response protein 88 (MyD88), which acts like a coordinating molecule (called an adaptor molecule) linking surface receptors to immune cell signaling pathways within the cytoplasm. Mice lacking MyD88 cannot produce tumor necrosis factor-alpha (TNF-α) or Interleukin 12 (IL-12) when exposed to bacterial antigens.[
Signaling Pathways containing Janus kinases: Another cell signaling pathway in immune cells and microglia involve the Janus kinases. Critical to immune function are the Janus family of kinases, Jak1, Jak 2, Jak3, and Tyk2, which are involved in cell growth, survival, development, and differentiation of immune cells.[
Signaling Pathway From Cell Membranes to a gene through NFkB: One essential cell signaling link from the cell membrane is directed to Nuclear Factor kappa B (NFkB), which activates genes controlling the production and secretion of proinflammatory cytokines such as TNF-α. Activation of toll-like receptors (TLRs) and other immune receptors on the surface of microglia, macrophages, and other immune cells are linked to NFkB activation. This cell signaling molecule represents a major controlling mechanism of inflammation.
Signaling Pathways that reduce the inflammatory response of microglia and other immune cells:
Other cell signaling mechanisms play an important role in reducing inflammation and potentially dangerous states of immune activation. For instance, MAPK phosphatase 1 (MKP1), is a major negative modulator of TLR-induced inflammation. You will recall the TLRs are a family of receptors on the cell membrane of immune cells that interact with antigens and other immune chemicals to initiate immune activation. By modulating these receptors, MAPK phosphatase 1 can fine-tune the immune reaction.
Another immune response regulator is called a suppressor of cytokine signaling 1 (SOCS1), which is an encoded gene. When the SOCS1 gene is activated by extracellular produced cytokines, such as IL-2, IL-3, interferon-gamma (INF-γ), or GM-CSF, the microglial SOCS1 gene reduces the activation of proinflammatory cytokine cell signaling. That is, it also dampens the immune response. Another such immune inhibitory molecule TREM2, is found on the surface of myeloid cells (primarily monocytes and macrophages) from the bone marrow. This protein interacts with another protein produced by the TYROBP gene, which in turn reduces the proinflammatory signaling of microglia, macrophages and dendritic cells. Mutations of this gene, which have been found to be common in Alzheimer's cases, impairs this immune dampening effect, thus leading to chronic brain inflammation, as seen in many neurodegenerative diseases.[
There are a growing number of second messenger cell signaling molecules essential for modulating immune reactions. For a detailed discussion of immune cells signaling, see reference.[
Molecular pathophysiology of microglia
Immune receptors and microglia reactions
Immune Receptors leading to molecular activation: A great number of surface membrane receptors are seen on microglia, which not only act as “on/off” signals, but also modulate their responses to the microenvironment. For example, proinflammatory cytokines and glutamate can activate microglia toward a cytotoxic role and lead to destruction of synapses, dendrites, and whole neurons if not interrupted [ One can easily see that either chronic activation of the brain's innate immune cells or even a condition where the cells are unable to switch off their immune activation, can lead to progressive destruction of synapses, dendrites or even the entire neurons or complex networks of neurons, as we see in neurodegenerative diseases. Examples of chronic microglial activation would include long-term systemic immune activation, as with autoimmune diseases and chronic infections (periodontal infections, chronic viral infections, Lyme disease, etc.). Neurotoxic metal accumulation within microglia or astrocytes, as seen with aluminum, lead, triethyl tin, and mercury exposure, also leads to continuous activation of microglia/astrocytes and macrophages. Immune receptors leading to microglia phagocytosis: Stimulation of PRRs by PAMPs and DAMPs can also activate microglia phagocytosis.[ Upregulation of Fc receptors by pathogens: Exposure to pathogenic organisms also upregulates Fc receptors on microglia and macrophages, which aid phagocytosis by opsonizing the antigen. Opsionization is a process whereby antigens are attached to larger molecules (opsonins) that make phagocytosis easier by giving the phagocytic microglia a larger target to grasp. Microglial responses to cellular debris from dying cells either mounts a phagocytic response without a release of proinflammatory cytokines or combines phagocytosis with proinflammatory cytokine release.[ Toll-Like Receptors as Antigen-Specific Receptors: Human microglia recognize bacteria, fungi, viruses, parasites, and the host itself, by utilizing specific toll-like receptors 1-9 (TLRs 1-9) located on the microglia cell membrane. Like peripheral macrophages, brain microglia demonstrate specific ligand reactions to TLR subtypes. For example, lipopolysaccharides (LPS), which make up the outer wall of Gram-negative bacteria, react specifically with TLR4, petidoglycan (PGN) (from cell walls of Gram-positive bacteria) reacts with TLR2, unmethylated CpG-DNA (viruses) reacts with TLR9, polyI: C (synthetic double-strand RNA) responds with TLR3 and West Nile virus RNA signals via TLR3[ In experimental studies, vaccinations and in human infections, TLR4 is of vital importance. In conjunction with CD14, TLR4 makes up the primary receptor for LPS the cell wall component of Gram-negative organisms [ Cellular debris has a different molecular pattern and operates through Damage Associated Recognition Receptors (DAMPs). What activates the DAMPs is that the debris contains either internal cellular components that normally would not be in contact with the immune cell surface receptor or a mutated molecule is formed from the damaging process that is then recognized as a foreign molecule. Again, enzymes within the proteasome and lysomes digest the foreign protein and debris and either remove it from the body or utilize the proteins for cell repair. In some instances, as with persistent viruses and neurotoxic metal accumulation, the offending antigen cannot be removed and thus acts as a constant source of immune stimulation. We see this with aluminum accumulation, where the highest concentration is within microglia. This would keep the microglia in an activated proinflammatory state, which could lead to a chronic state of immunoexcitotoxicity and subsequent neurodegeneration. Clinical Correlation: There is growing evidence that excessive or overreactive responses to certain vaccines or groups of vaccines are responsible for many of the complications associated with vaccinations, including encephalomyelitis, seizures, autism spectrum disorders, and the recently described macrophagic myofascitiis.[ Both metals can cause prolonged microglial activation and mercury has been shown to kill astrocytes, its primary site of accumulation.[ Neuropathic Pain: Clinical Correlation: TLR4 has also been implicated with neuropathic pain, for example, as that associated with transection of the L5 nerve root, thus suggesting microglial reactions to DAMPs. A number of studies have linked the production of chronic pain to a triggering of immune-excitotoxic pathways, which are initiated by activated microglia.[ Microglia and TLR Molecular specificity: Free gangliosides and sialic acid-containing glycosphingolipids found in neural membranes, are known to activate microglia via TLR4, offering another way cellular debris can activate microglia.[ TLR7 is vital for microglial reactions to viruses such as the herpes simplex virus[ Other Microglial actions: Microglia also contain a number of scavenger receptors, which can identify modified lipoproteins and various polyanionic ligands. These include scavenger receptors of class A1 (SR-A1), SR-B1, and CD36 that are differentially regulated by microglia in response to various pathologies.[ · Scavenger receptors. Scavenger receptors are upregulated in Alzheimer's disease (AD) in reaction to amyloid beta (Aß).[ Another important scavenger receptor in neurodegenerative diseases, receptor for advanced glycation end-products (RAGE), recognizes advanced glycation end products produced as a reaction to elevated glucose levels in the brain. The RAGE receptors on microglia are upregulated in the presence of Aß, which enhances plaque phagocytosis.[ MAC1 Receptor (The Integrin or Compliment Receptor) The MAC1 receptor (macrophage antigen complex 1), also called integrin CD11b/CD18 or complement receptor 3 (CR3), functions both as an adhesion molecule and a PRR capable of recognizing diverse ligands.[
Immune Receptors leading to molecular activation: A great number of surface membrane receptors are seen on microglia, which not only act as “on/off” signals, but also modulate their responses to the microenvironment. For example, proinflammatory cytokines and glutamate can activate microglia toward a cytotoxic role and lead to destruction of synapses, dendrites, and whole neurons if not interrupted [
One can easily see that either chronic activation of the brain's innate immune cells or even a condition where the cells are unable to switch off their immune activation, can lead to progressive destruction of synapses, dendrites or even the entire neurons or complex networks of neurons, as we see in neurodegenerative diseases. Examples of chronic microglial activation would include long-term systemic immune activation, as with autoimmune diseases and chronic infections (periodontal infections, chronic viral infections, Lyme disease, etc.). Neurotoxic metal accumulation within microglia or astrocytes, as seen with aluminum, lead, triethyl tin, and mercury exposure, also leads to continuous activation of microglia/astrocytes and macrophages.
Immune receptors leading to microglia phagocytosis: Stimulation of PRRs by PAMPs and DAMPs can also activate microglia phagocytosis.[
Upregulation of Fc receptors by pathogens: Exposure to pathogenic organisms also upregulates Fc receptors on microglia and macrophages, which aid phagocytosis by opsonizing the antigen. Opsionization is a process whereby antigens are attached to larger molecules (opsonins) that make phagocytosis easier by giving the phagocytic microglia a larger target to grasp. Microglial responses to cellular debris from dying cells either mounts a phagocytic response without a release of proinflammatory cytokines or combines phagocytosis with proinflammatory cytokine release.[
Toll-Like Receptors as Antigen-Specific Receptors: Human microglia recognize bacteria, fungi, viruses, parasites, and the host itself, by utilizing specific toll-like receptors 1-9 (TLRs 1-9) located on the microglia cell membrane. Like peripheral macrophages, brain microglia demonstrate specific ligand reactions to TLR subtypes. For example, lipopolysaccharides (LPS), which make up the outer wall of Gram-negative bacteria, react specifically with TLR4, petidoglycan (PGN) (from cell walls of Gram-positive bacteria) reacts with TLR2, unmethylated CpG-DNA (viruses) reacts with TLR9, polyI: C (synthetic double-strand RNA) responds with TLR3 and West Nile virus RNA signals via TLR3[
In experimental studies, vaccinations and in human infections, TLR4 is of vital importance. In conjunction with CD14, TLR4 makes up the primary receptor for LPS the cell wall component of Gram-negative organisms [
Cellular debris has a different molecular pattern and operates through Damage Associated Recognition Receptors (DAMPs). What activates the DAMPs is that the debris contains either internal cellular components that normally would not be in contact with the immune cell surface receptor or a mutated molecule is formed from the damaging process that is then recognized as a foreign molecule. Again, enzymes within the proteasome and lysomes digest the foreign protein and debris and either remove it from the body or utilize the proteins for cell repair.
In some instances, as with persistent viruses and neurotoxic metal accumulation, the offending antigen cannot be removed and thus acts as a constant source of immune stimulation. We see this with aluminum accumulation, where the highest concentration is within microglia. This would keep the microglia in an activated proinflammatory state, which could lead to a chronic state of immunoexcitotoxicity and subsequent neurodegeneration.
Clinical Correlation: There is growing evidence that excessive or overreactive responses to certain vaccines or groups of vaccines are responsible for many of the complications associated with vaccinations, including encephalomyelitis, seizures, autism spectrum disorders, and the recently described macrophagic myofascitiis.[
Both metals can cause prolonged microglial activation and mercury has been shown to kill astrocytes, its primary site of accumulation.[
Neuropathic Pain: Clinical Correlation: TLR4 has also been implicated with neuropathic pain, for example, as that associated with transection of the L5 nerve root, thus suggesting microglial reactions to DAMPs. A number of studies have linked the production of chronic pain to a triggering of immune-excitotoxic pathways, which are initiated by activated microglia.[
Microglia and TLR Molecular specificity: Free gangliosides and sialic acid-containing glycosphingolipids found in neural membranes, are known to activate microglia via TLR4, offering another way cellular debris can activate microglia.[
TLR7 is vital for microglial reactions to viruses such as the herpes simplex virus[
Other Microglial actions: Microglia also contain a number of scavenger receptors, which can identify modified lipoproteins and various polyanionic ligands. These include scavenger receptors of class A1 (SR-A1), SR-B1, and CD36 that are differentially regulated by microglia in response to various pathologies.[
· Scavenger receptors.
Scavenger receptors are upregulated in Alzheimer's disease (AD) in reaction to amyloid beta (Aß).[
Another important scavenger receptor in neurodegenerative diseases, receptor for advanced glycation end-products (RAGE), recognizes advanced glycation end products produced as a reaction to elevated glucose levels in the brain. The RAGE receptors on microglia are upregulated in the presence of Aß, which enhances plaque phagocytosis.[
MAC1 Receptor (The Integrin or Compliment Receptor)
The MAC1 receptor (macrophage antigen complex 1), also called integrin CD11b/CD18 or complement receptor 3 (CR3), functions both as an adhesion molecule and a PRR capable of recognizing diverse ligands.[
Illustration of lipopolysaccharide (LPS) Gram-negative cell wall molecular component interacting with TLR on microglial membrane surface, which along with co-stimulatory molecule CD14, activates defensive cell signaling. The IL-1 type pro-inflammatory cytokine receptor TIR, plays a major role in microglial activation
How microglia are activated: Molecular mechanisms behind “microglial priming”
Cell signaling pathways can set in motion a series of molecular reactions that activate microglia. When an antigen is recognized by a receptor on the surface of the microglia cell, the receptor, through a series of conformational changes which requires energy, sets off a chain of molecular reactions that signal the genes in the microglia to produce proteins that activate the microglial cells to respond by producing proteins that react with the antigen and or initiate phagocytosis. For example, when PRRs in the microglial membrane recognize an antigen, NADPH oxidase is activated in response to antigen stimulation of the immune receptor. NADPH oxidase generates high levels of superoxide, which as discussed previously, combines with free NO to produce the powerful radical peroxynitrite. Superoxide can also break down into other powerful radicals, such as the hydroxyl radical.
Several PRRs can activate NADPH oxidase, the major source of microglial reactive oxygen species (ROS), especially the superoxide radical. NADPH oxidase is composed of several subunits that must be assembled for activity. These subunits are distributed between the cytosol and the membrane of intracellular vesicles. Once activated by complexing, they are translocated to the cell membrane. MAC1 appears to be crucial for assembly and activation of NADPH oxidase.[
Through elevated levels of ROS
The morphology and proliferation of microglia are to a large degree regulated by H202 generated by NADPH oxidase and, as a result, higher levels of ROS, in most instances, amplifies inflammatory responses.[
Through chemokines and cytokines
Chemokines are a family of molecules released from microglia that attract other microglia or immune cells, even at great distances. Microglia contain a variety of receptors for chemokines and cytokines.[
Clinical Correlation: Elevated levels of chemokines within the CNS are seen with a number of neurological conditions, including AD, amyotrophic lateral sclerosis (ALS), human immunodeficiency virus (HIV) dementia, ischemia, viral encephalitis, multiple sclerosis, and Parkinson's disease (PD).[
Microglia contain receptors for a number of cytokines, both proinflammatory and antiinflammatory. One of the critical types of cytokine receptors for microglia activation is the IL-1ß receptors, which includes the subtypes IL1RI, IL-1RII, and IL-RIII.[
How glutamate is produced by activated microglia
Another proinflammatory cytokine, TNF-α not only plays a critical role in inflammatory brain pathology, but is integral to immunoexcitotoxicity itself as it links the immune response to glutamate excitotoxicity[
Schematic demonstrating the link between TNF -α and an enhancement of excitotoxicity via its interaction with glutamate transport proteins, upregulation of glutaminase, suppression of glutamine synthetase, increased trafficking of AMPA calcium-permeable receptors to the synaptic membrane and endocytosis of GABA receptors
Clinical Correlation: The concept of immunoexcitotoxicity is based on the interaction between inflammatory cytokines and glutamate excitotoxicity. In a previous paper, it was demonstrated that TNF-α had a number of effects on glutamate neurotransmission and excitotoxicity, including upregulation of astrocyte glutaminase (which converts glutamine into glutamate), increased trafficking of calcium-sensitive AMPA receptors to the synaptic membrane, inhibition of glutamate transport proteins (excitatory amino acid transporters [EAATs]), suppression of glutamate removal by Kreb's cycle enzymes and conversion of glutamate to glutamine (glutamate dehydrogenase and glutamine synthase) and increased trafficking of NMDA receptors.[
Based on this central effect of TNF-α, Tobinick et al. demonstrated dramatic, rapid improvement among 629 consecutive stroke patients following injection of the TNF-α-blocking drug etenercept given perispinally (Batson's plexus).[
In a second group of patients, Tobinick and Gross, utilizing the same technique found that blocking of CNS TNF-α in a single case of severe AD produced rapid and sustained improvements in speech, agitation, and tests of spatial function.[
The final effect of TNF-α depends on which TNF receptor is activated. Microglia contain predominantly TNFR2 type receptors that when stimulated by high levels of TNF-α, are protective, thus protecting the microglial cell from its own released cytokine. Neurons, in contrast, contain mostly TNFR1 subtype, which when stimulated by high levels of TNF-α triggers a series of cell signaling pathways that are neurodestructive. Very low, constitutive concentrations of TNF-α are considered neurotrophic.
Neurotransmitter and neuroregulatory receptors on microglia
Microglia contain a number of receptors for neurotransmitters and other neuroregulatory molecules, including those for glutamate, acetylcholine, dopamine, gamma-aminobutyric acid (GABA), adrenergic compounds, cannabinoids, opioids, substance P, vasoactive intestinal peptide, histamine, glucorticoid, somatostatin, angiotensin II, platelet activating factor (PAF), and neurotrophin.[
Ionotropic glutamate receptors
Glutamate is the most abundant neurotransmitter in the brain, contributing 90% of cortical neurotransmission and 50% of all brain neurotransmission. These receptors include three subtypes named NMDA receptors, AMPA receptors, and kainite receptors [
Illustration demonstrating the trafficking of AMPA calcium-permeable receptors from the endoplasmic reticulum to the synaptic lipid raft, thus increasing glutamate sensitivity and increasing the internal flow of calcium. We see a condition of crosstalk between TNFR1 receptors and AMPA receptor trafficking mechanisms
Glutamate receptors on neuronal dendrites and synapses play a major role controlling the strength of the synaptic impulse and does so by a variety of mechanisms. Of special importance is the role glutamate neurotransmission plays in learning and memory and behavioral control.
Interestingly, glutamate receptors are not limited to neuronal dendrites and synapses. Over the past decade, researchers have demonstrated a significant role for glutamate receptors in regulating microglial behavior. One role for microglia as a member of the tripartite synapse is to modulate extraneuronal concentrations of glutamate, especially in the vicinity of the synapse. This helps to sharpen the signal and prevent interfering “noise” caused by excess diffused glutamate from surrounding synapses. Cell surface receptors for glutamate are divided into inotropic receptors and metabotropic receptors. The ionotropic receptors regulate opening of various ionic channels, principally those for sodium and calcium and the metabotropic receptors act through G-protein-coupled receptors to modulate the inotropic receptors. Microglial inotropic glutamate receptors play a role in microglial migration as well as microglial activation. When sites of injury or pathology result in the release of high levels of glutamate, the diffused extracellular gradient attracts surrounding microglia to the site of injury.
Of the ionotropic glutamate receptors, AMPA receptors appear to be the most abundant and critically important for microglial reactivity. There is some evidence that NMDA receptors exist on microglia, most of which is indirect evidence. For example, blocking NMDA receptors in experimental models has been found to reduce microglial activation associated with ischemic insult or exposure to HIV protein.[
Stimulation of microglial AMPA receptors induces the release of TNF-α, which in a paracrine manner stimulates further microglial activation and migration. Glutamate receptors, in particular AMPA receptors, play a major role in microglial chemotaxis. By following gradients of extraneuronal glutamate, microglial cells migrate toward sites of pathological injury or damage.[
Also of interest is the finding that under certain conditions, AMPA receptor stimulation can be neuroprotective and promote brain repair after a stroke.[
Metabotropic glutamate receptors
Metabotropic glutamate receptors (mGLuRs) are classed as noniontropic receptors and rather than utilizing ionic pores, such as the calcium pore seen with ionotropic glutamate receptors, this group of receptors is coupled to membrane G-proteins that act through cytoplasmic second messengers. Cloning studies have grouped them into three basic types. Group I mGLuRs, composed of metabotropic glutamate receptor subtypes 1 and 5 (mGluR1 and mGluR5), operate through activation of phospholipase C. In general, these enhance the activity of the iontropic glutamate receptors (NMDAR, AMPAR, and kainite receptors).
Group II metabotropic receptors include mGluR2 and mGluR3 and through their G-protein receptors inhibit adenylate cyclase. Most often they are inhibitory of the ionotropic receptors, but not always. Group III contains mGluR4, mGluR6, mGluR7, and mGluR8. They also inhibit adenyl cyclase and are considered to be inhibitory of ionotropic glutamate receptors. They are linked to the ionotropic receptors though cell-signaling molecules.
From a potential therapeutic viewpoint, mGLuRs exhibit a number of interesting properties. Group I mGluRs regulate LPS-induced microglial activation in primary cultures.[
In essence, the mGLuRs are acting as modulators of inotropic receptors either enhancing or dampening their response. This adds an important layer in controlling the excitatory response to glutamate, so as to prevent excessive brain excitation, which can lead to neurodegeneration or seizures. In addition, they also aid in sharpening the signals during glutamate neurotransmission. As with the ionotropic glutamate receptors, the metabotropic receptors play an important role in controlling other neurotransmitter function and levels of activity.
Frequently overlooked in discussion on neurodegeneration is the critical role played by cholinergic systems in controlling inflammation, especially the α7-nicotinic receptors (α7nAchRs). Cholinergic pathways are impaired in both AD and PD, most likely as a result of the immunoexcitotoxic reactions in Ach-controlling areas of the brain, such as the nucleus basalis of Meynart.[
Clinical Correlation: Any condition that reduces brain acetylcholine, such as brain trauma, dietary restriction of choline, advanced aging or chronic neurodegenerative diseases that lowers brain acetylcholine, could worsen brain inflammation. One can see that a combination of poor diet and advanced aging in particular can worsen Alzheimer's dementia by removing or suppressing this antiinflammatory system. Likewise, improvements in some AD patients taking acetylcholine-enhancing medications, such as donepezil (Aricept) may be as a result of Ach's antiinflammatory effect. Chronic activation of these receptors, as occurs with smoking or nicotine patches, could impair microglial function in cases of cerebral infections, strokes, and other inflammatory pathologies. It would also explain how smoking reduces one's risk of PD and why nicotine patches may reduce risk.[
Norepinephrine is known to suppress the release of NO, TNF-α, and IL-6 following immune stimulation with LPS and plays a critical role in protecting the brain from neuroinflammation.[
Adrenergic receptors and the locus ceruleus
This small collection of neurons, located in the posterior area of the rostral pons along the floor of the fourth ventricle, sends adrenergic (norepinephrine and epinephrine) projections widely throughout the CNS, thus making it the central controlling adrenergic nucleus (referred to as the locus ceruleus-noradrenergic [LC-NA] system). Stimulation of these neurons is generally excitatory for the brain, producing an arousal effect. Degeneration of the LC is an early event with AD and PD.[
Functional dopamine receptors on microglial membranes have been identified in both human brain tissue and mouse brain. Chronic stimulation of microglial dopamine receptors enhanced migratory activity, and like ARs, attenuated LPS-induced NO release.[
Clinical Correlation: ALS: A number of new studies considerably strengthen the case for an interaction between inflammation and excitotoxicity as the central mechanism in ALS. It has been determined that in experimental models of the disease microglial-triggered inflammation occurs in the presymptomatic stage of the disease, and microglial number increase as the disease progresses.[
Scanning studies of ALS patients demonstrate widespread microglial activation in the motor cortex, pons, dorsolateral prefrontal cortex, and thalamus.[
The reason for the prolonged microglial activation in the affected areas of the brain and spinal cord in ALS is not specifically known, but we know that the most commonly linked initiating factors, such as pesticide/herbicide exposure, aluminum and/or mercury accumulation, repeated minor injuries and certain persistent viruses all are known to cause prolonged microglial activation in a neurodestructive mode.
The relationship of superoxide dismutase-1 to ALS
In the past, it was thought that there were two distinct ALS disease forms, one sporadic with normal levels of the antioxidant enzyme SOD1 and a familial form with mutated SOD1. Newer studies have offered some important surprises. First, the loss of motor neurons was not related to a loss of functional SOD1, an antioxidant molecule that neutralizes superoxide.[
One might conclude incorrectly that motor neuronal death in ALS was caused by the immune activation alone, yet mouse models of ALS in which IL-1ß and TNF-α have been deleted still demonstrated continued neurodegeneration, indicating that another pathological process was in operation and linked to the immune effect.[
Excitotoxicity and its relation to neurological diseases and ALS
The high level of ROS/RNS and lipid peroxidation resulting from the widespread inflammation impairs the glutamate transporters, in particular GLT-1 (EAAT2), and impairs other essential metabolic enzymes and enzymes used to clear excess glutamate, such as glutamine synthetase. A number of studies have demonstrated elevated CSF glutamate levels in ALS patients as compared with controls.[
Impairment of EAAT2 expression in the ventral horn of the spinal cord of experimental models occurs in the presymptomatic period. Interestingly, EAAT2 levels were almost completely abolished at the end-stage of the disease.[
Overexpression of EAATs, thus increasing the efficiency of glutamate removal, in experimental animal models of ALS delays the onset of motor loss and prolongs survival. An analysis of motor cortex and spinal cord extracts from ALS patients demonstrated nearly complete loss of EAAT2 in 25% of patients and some impairment in up to 80%.[
The cannabinoid system plays a critical role in modulating excitatory neurotransmission. Rodent microglial cells contain CB1 and CB2 receptors on activated microglia but are very low in ramified (resting) microglia.[
Experimental allergic encephalomyelitis
EAE is an animal model of multiple sclerosis and related inflammatory demyelinating disorders. A number of antigens have been used to produce the immune demyelination, including spinal cord homogenates, various myelin proteins (MBP, PLP, and MOG) or peptides of these proteins. The pathology produced varies to some degree based on the antigen used.
In the case of EAE, a model for human multiple sclerosis in animals, one sees upregulation of CB2 receptors induced by the synergistic action of IFN-γ and GM-CSF.[
From this abbreviated review of microglial receptors, it becomes obvious that brain immune reactions are under a great number of regulatory controls and it is the interaction of these various receptors that determines the dynamic changes in microglial phenotypes and thus their function.
Microglial migration and motility
Microglia are not fixed immune cells, but are characterized by a high degree of motility in the ramified (resting) state and are capable of extensive migration upon activation. As demonstrated previously, motility of resting microglial foot processes allows the microglia to conduct ongoing surveillance of the synaptic microenvironment and make adjustments of the concentrations of excitatory neurotransmitters such as glutamate and aspartate. This surveillance is critical not only for protection against excitotoxicity, but also for signal sharpness, as previously mentioned.
At the interface of cerebral blood vessels, microglial motility allows rapid identification of invasion by microorganisms. This rapid identification process is especially important near zones where the BBB is deficient, as with the CVOs, which includes the area postrema, subfornical organ, organum vasculorum of the laminia terminalis, pineal region, subcommissural organ, median eminence, and neurohypophysis. These areas contain abundant microglia, which when exposed to invading organisms or systemic proinflammatory cytokines, are rapidly activated and migrate over great distances in an effort to protect the rest of the brain.[
Motility appears to be mostly controlled through purinergic receptors on the microglial surface membrane. Purinergic receptors respond to free ATP and adenosine and on neurons can initiate an excitatory response. A number of subtypes of purinergic receptors have been identified. ATP and adenosine, both stimulate motility in ramified (resting microglia) microglia and act through P2Y12 subtype purinergic receptors.[
In the face of pathological injury, activated microglia migrate to the site of damage mostly by following a chemical gradient. Like motility, microglial migration is controlled by a set of receptors. Studies suggest that a combination of P2X4 and P2Y12 receptors, subtypes of purinergic receptors, are involved in microglial chemotaxis.[
Other neurotransmitters are also involved in microglial migration, such as glutamate, dopamine and epinephrine via AMPA receptors, mGLuRs, dopamine receptors, and ARs, respectively.[
Chemokines are also potent chemoattractants and are released from neurons and microglia. Damaged neurons express the chemokine CCL21, which attracts microglia. Monocyte chemoattractant protein-1 (MCP-1) regulates migration of microglia, monocytes, and lymphocytes to the sites of inflammation in the CNS.[
A number of other factors also regulate microglial migration, but of special interest is nerve growth factor (NGF) and transforming growth factor-ß (TGF-ß) as both are involved in brain repair and reduction of neuroinflammation as well.[
Microglial cells are the main phagocytic cell type of the brain and can not only phagocytize dead cells, but also parts of neurons, for example, dendrites, and debris such as myelin and amyloid deposits. Microbes are recognized by TLRs and other PRRs, whereas apoptotic neurons utilize other receptors such as vitronectin and phosphotidylserine-mediated receptors.[
A major control mechanism for microglial phagocytosis is by way of the P2Y6 receptor, which is upregulated with neuronal damage.[
Synaptic stripping and synaptic building
States of inflammation are often associated with removal of synapses and the process is driven by activated microglia. Neuronal electrical activity normally suppresses MHC class II activation on both astrocytes and microglia.[
When in a reparative phenotype, microglia can also promote synaptogenesis by secreting thrombospondins (TSPs), an extracellular matrix protein critical for synaptic formation.[
Systemic immunity interactions with innate CNS immunity (Sickness behavior)
In the past it was assumed that the neurological and behavioral effects of viremia and sepsis were secondary to the infectious agent acting within the brain itself. It is now accepted that peripheral inflammation and immune activation secondarily effect brain function during the infectious process.[
There are four major links between peripheral immunity and CNS immunity, one a rapid system and the other three of slower onset. The slower-onset systems involve a passive diffusion of proinflammatory cytokines into the brain via the CVOs – organum vasculosum of the lamina terminalis, subfornical organ, neurohypophysis, pineal gland, median eminence, and dorsal vagal complex.[
The BBB has an energy-dependent, saturable, carrier-mediated transport system for cytokines, primarily IL-1, IL-6, and TNF-α.[
The rapid system operates through the vagus. Several lines of evidence implicate the vagus nerve in this fast process. For example, subdiaphragmatic sectioning of the vagus mitigates sickness behavior induced by peripheral immune activation.[
Activation of brain microglia following peripheral immune stimulation is most intense in the face of preexisting brain pathology and some feel that without this preexisting pathology peripheral immune stimulation will not lead to neurodegeneration. For example, using a ME7 model of prion disease, researchers found that injecting this protein into the brain prior to peripheral immune activation, produced an exaggerated proinflammatory response with intense microglial activation, extreme sickness behavior, and acceleration of neurodegeneration.[
Clinical Correlation: This may be the case in such conditions a chronic traumatic encephalopathy (CTE) and would explain why the phenomenon does not occur in every person suffering repeated minor concussions.[
As we see in peripheral immune cells, in particular macrophages, microglia can switch from a resting phenotype to a primed state by an initial immune stimulus that is not excessively intense. For example, a mild head injury or episode of hypoxia can switch microglia from its resting state to a functional condition in which the enzymes and genetic activation is upregulated, but the active immune molecules, primarily proinflammatory cytokines and chemokines, are not released. We see priming of microglia also with aging of the brain.
With a second immune stimulus, these primed microglia began to release proinflammatory cytokines and chemokines in concentrations much higher than would microglia that have not been primed. Systemic immune stimulation can prime brain microglia, which means that either subsequent brain disturbances or systemic immune activation would trigger a magnified immune response within the brain. Is essence, it strongly indicates that systemic immune activation can worsen and speed up CNS degenerative disorders, such as ALS, AD, and PD.
We see similar enhancement of neurodegeneration by peripheral infections in rodent models. The destructive process develops secondary to local microglial priming by the preexisting pathological process, such as a stroke, closed head injury, multiple sclerosis, AD, PD, prior brain surgery, or penetrating injury. The peripheral immune stimulation by way of the interacting process discussed earlier, fully activate the primed microglia in an exaggerated manner.[
Because humans are exposed to a number of immune events throughout life, such as multiple infections, persistent viruses, exposure to neurotoxic metals, exposure to pesticides/herbicides and fungicides, head injury, microinfarctions, chronic stress and aging, one can see that each of these episodes is associated with microglia priming and activation, leading to a progressive loss of neurons in the most vulnerable parts of the CNS, such as the hypothalamus, temporal lobes (hippocampus, striatal area, amygdala, and entorhinal cortex) and prefrontal cortex.
Those with the least efficient protective systems, such as glutamate transport systems, antioxidant network, cellular glutathione, low CD200 and fractakline and tissue iron binding would be at the greatest risk for one of the neurodegenerative diseases. Under such conditions, the microglia would remain activated in the affected zones and immunoexcitotoxicity would be triggered over a long period leading to neurodegeneration.
Aging and immunoexcitotoxicity
Since aging itself is associated with priming of microglia and elevations in inflammatory cytokines, one would expect peripheral immune stimulation under such conditions to worsen any coexisting clinical picture significantly.[
Immunoexcitotoxicity and behavioral disorders
Immunoexcitotoxicity is thought to play a significant, if not central role in many behavioral disorders in humans as well[
Illustration of the combined release of pro-inflammatory cytokines and chemokines along with excitatory amino acids during neurodestructive microglial activation. This demonstrates the interaction between immune factors, reactive oxygen and nitrogen species, lipid peroxidation species and excitotoxicity
Immunoexcitotoxicity and other diseases
A growing number of neurodegenerative disorders, including strokes, schizophrenia, autism, CTE, and neuropsychiatric disorders are associated with primed or activated microglia.[
Immunoexcitotoxicity is enhanced by vaccines
Also of concern is the present vaccine schedule for children, which entails a repetitive injection of concentrated powerful adjuvants during a period of intense brain pathway formation. The present US vaccine schedule recommended by the Center for Disease Control and Prevention (CDC), now mandated for most states, consist of giving small children up to age 6 years as many as 5-8 vaccines per office visit every 2 months for as total of 28 vaccines during the first year of life.[
It is not the antigens that are of most concern, but rather the adjuvants, which initiate the most intense and prolonged immune stimulation. These adjuvants are designed to magnify the immune response, as the antigens (viruses and bacteria) themselves are too weak to provide protection. In addition, adjuvant additives, such as aluminum and mercury, are known powerful microglial activators and are neurotoxic, even in very small concentrations.[
Priming of the microglia
In the infant or small child, the priming event may come from a number of sources, such as vaccination of the mother during pregnancy or with intrauterine or early postbirth infections.[
While natural infections can also produce this neurodestructive response, vaccinations produce higher levels of immune activation and the immune response can persist longer than natural infections – sometimes lasting years. In the case of the elderly, already having primed microglia, repeated, closely spaced, sequential vaccination can also present a potential hazard, since an exaggerated microglial response could worsen any preexisting neurological disorder (even silent ones) or initiate such a disorder.[
Control of microglia activation and deactivation
A great number of factors can activate microglia, which makes sense when you consider that it is the main defensive arm of the innate immune system and must react rapidly to a number of stimuli. Microglial activation occurs rapidly upon disturbance of the brain's homeostasis, usually within minutes. The major activator of microglia is IL-1ß, which can be released locally with pathological damage within the CNS or can be transported from the systemic circulation.[
Recent studies have shown that microglial activation can be anatomically selective in terms of both the brain anatomy affected and the microglial phenotype being expressed.[
Newer studies have shown that many of the traditionally used antipsychotic and antidepressive medications can suppress microglial activation and reduce inflammation and excitotoxicity.[
Of equal importance in brain inflammation are mechanisms utilized to downregulate or terminate microglial activation. Of principle importance are the cytokines, IL-10, IL-4, fractalkine (CX3CL1), and CD200 (cluster of differentiation 200) and its receptor CD200R. Suppression of the last two modulators is necessary for microglial activation. The immune suppressant cytokine IL-4 modulates CD200 so as to reduce microglial activation.[
IL-10 reduces microglial activation and lowers secreted levels of TNF-α, NO, ROS, and superoxide following LPS exposure (immune activation).[
Recently, TREM2, a DAP12-associated receptor and modulator of TLRs, has attracted a lot of attention as a regulator of microglial activation.[
Should we be taking microglial inhibitors in our diets?
Streit et al. put forth the hypothesis that with aging, microglia become dystrophic and therefore lose much of their ability to protect the brain utilizing both their immune and neurotrophic functions.[
With the finding of abnormal functioning of CD200-CD200R regulatory control in neurodegenerative diseases, one can appreciate the effect of age-related priming on accelerating neurodegenerative pathology since the normal switching off of the microglia would be impaired [
While microglial activation would appear to be a useful target in treating a number of neurological diseases, recent studies indicate that general suppression of microglia may not be a good idea, as certain phenotypes of microglia are neuroprotective. The timing is important when considering suppressing microglial activation. For example, it is thought that in the early course of ALS, microglia and macrophages are in the M2 protective mode of activation and as the disease progresses, they switch to an M1 cytotoxic mode.[
Safer targets would be downstream mechanisms, such as inhibiting glutaminase, enhancing neuronal glutathione, antioxidant protection of critical enzymes, such as glutamine synthase and glutamate dehydrogenase; suppression of NADPH oxidase or iNOS, stimulation of group III mGLuRs (inhibitory), enhancing EAATs and suppression of prostaglandin E2 production. A number of studies have been done using these various approaches with evidence of considerable success in animal models and some limited human studies. A growing number of natural products, most extracted from plants, have shown an ability to alter immunoexcitotoxicity.
Immune cell adaptive responses in the CNS
Over 90 years ago it was shown that an implanted tumor in the brain of a rat was resistant to systemic immunity, thus establishing the idea that the brain was an “immunologically privileged site” and therefore isolated from systemic immunity. Over the past several decades growing evidence has supported the idea that this immune isolation is not absolute. It is true that among lymphocytes only activated lymphocytes can enter the CNS, but do so with reduced efficiency.[
Under normal conditions, once within the CNS parenchyma, the activated CD8+ cytotoxic T-cells are treated as being hostile and most undergo apoptosis or conversion to regulatory T-cells (Tregs). Neurons protected themselves from activated lymphocytes by upregulating and secreting TGF-ß, an inflammation-suppressing cytokine that converts CD8+ cytotoxic T-cells to immune suppressant regulatory T-cells (Tregs). Newer studies have shown a preponderance of Tregs in the face of CNS inflammation.[
Lymphocyte migration into the CNS is regulated by chemokines and their receptors. In the normal brain, few lymphocytes are found in the brain parenchyma. Dramatic increased infiltration of T-cells occurs with strokes, brain trauma, and CNS infections. Lymphocytic infiltration into the CNS is increased in neurodegenerative disorders but not to the scale of brain trauma or infection. Gemechu and Bentivoglio found increased CNS lymphocyte infiltration with brain aging.[
Aging is associated with structural and functional alteration in the BBB, including alteration in the carrier-mediated mechanisms.[
The fate of these entering T-cells depends on the state of brain inflammation and particular responses of lymphocyte interaction with glia cells. It appears that in most cases of prolonged brain inflammation, even with aging, T-cells are in a neuroprotective phenotype (Tregs).[
Over the past several decades we have learned a great deal about microglia and innate brain immunity. While microglia are the principle innate immune cells, other cell types also play a role, including invading macrophages, astrocytes, neurons, and endothelial cells. The fastest reacting cell is the microglia and despite its name, resting microglia are in fact quite active. Motion photomicrographs demonstrate a constant movement of ramified (resting) microglial foot processes, which appear to be testing the microenvironment for dangerous alteration in extracellular fluid content. These foot processes, in particular, interact with synapses and play a role in synaptic function. In event of excitatory overactivity, these foot processes can strip selected synapses, thus reducing activation states as a neuroprotective mechanism. They can also clear extracellular glutamate so as to reduce the risk of excitotoxicity.
Microglia also appear to have number of activation phenotypes, such as: principally phagocytic, principally neuroprotective and growth promoting or primarily neurodestructive. These innate immune cells can migrate a great distance under pathological conditions and appear to have anatomic specificity. There is some evidence that there are several types of microglia. Macrophage infiltration into the embryonic brain is the source of resident microglia and in adulthood macrophages can infiltrate the brain and are for the most part indistinguishable from resident microglia, but may react differently pathologically.
Activation itself does not imply a destructive phenotype and can be mostly neuroprotective via phagocytosis of debris, neuron parts and dying cells and by the release of neurotrophins such as NGF and brain BDNF. Evidence is accumulating that microglia undergo dynamic fluctuations in phenotype as the neuropathology evolves. For example, in the early stages of neurotrauma and stroke, microglia play a mostly neuroprotective role and only later switches to a neurodestructive mode.
A great number of biological systems alter microglia function, including neurohormones, cannabinoids, other neurotransmitters, ATP, adenosine, and corticosteroids. One can appreciate that with aging, many of these systems are altered by the aging process itself or diseases, thus changing the sensitivity of the innate immune system.
The realization that CNS microglia can be activated by stimulation of the systemic immune system is of great concern both in terms of natural disease and attempts to protect against disease by vaccination. It appears that not only the intensity of the immune stimulation is important, but also how closely spaced apart it occurs. Closely spaced, repetitive immune stimulation maximizes CNS innate immune activation and immunoexcitotoxicity.
The author would like to thank Dr. James Ausman for his invaluable suggestions concerning readability and continuity of the discussion. The author also thanks Dr. Dennis Malkasian for four of the illustrations, which have added significantly to the paper.
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