The human immune system consists of various cells circulating in blood and lymph vessels that can localize to sites of damage, injury, or infection and help in repairing damaged cells and destroying foreign or unhealthy substances. The immune system is complex, involving innate mechanisms such as inflammation and fever as well as adaptive mechanisms like cells that recognize specific antigens and respond to them faster upon second exposure. In any case, the body’s immune system is largely immaterial when it comes to neurodegenerative diseases like Huntington’s Disease, as there is a blood-brain barrier that prevents immune cells from crossing from the body’s circulation into the brain’s bloodstream. There has been some research that suggests in HD, there is either some crossing over the blood-brain barrier or the brain and body’s immune systems are activated in synchronization (see Dr. Paul Muchowski’s research presentation at the UCSF research symposium here), when studying HD scientists have traditionally regarded the blood-brain barrier as intact, and focused on the components of the immune system specific to the brain and how they affect and are effected in HD. The potential of focusing therapies on immune mechanisms in the brain is an interesting new area of research in the search for HD treatments.
The Blood Brain Barrier^
While the blood-brain barrier seems an abstract concept, it has a tangible counterpart in reality: tight junctions in the capillaries separating the brain’s circulation from the rest of the body do not allow cells like immune system macrophages through but allowing for the passage of oxygen and other small molecules such as hormones. This makes it necessary for the brain to have its own immune system against foreign pathogens. It has been observed that in HD and other neurodegenerative diseases, cytokines, small signaling proteins involved in immune function, are found in increased levels in both the central nervous system of the body and in the brain. Although it is possible that the cytokines are flowing through the blood-brain barrier, scientists think it is more likely that the cytokines are being released from glial cells in the brain and immune cells in the body in concurrence, causing this correlation. The blood brain barrier’s seeming impermeability has implications for any potential drugs administered orally or intravenously for HD: these chemicals must be able to cross the blood-brain barrier if they are to be of any direct use in slowing disease progression.
Inflammation, a key immune response that can be harmful if prolonged chronically, plays a large role in the immune response to HD and recent research has shown that inflammation may lead to increased permeability of the blood-brain barrier, with potential harmful results for patients whose immune systems would be thus compromised. In inflammation, increased activity of microglia, the major immune cell of the brain, increases the permeability of the blood-brain barrier and allows in macrophages from the body’s immune system. While this may be helpful in combating the disease in the short-run, in the long run the loss of this protection of the brain from harmful foreign cells and substances in the body is threatening to the patient. The permeability of the blood-brain barrier also increases with age, furthering the risk of compromising the immune system of the brain. It has also been proposed that this increased permeability does not result from increased activity of the brain immune system but actually increased activity of the macrophages of the body’s immune system. As both macrophages and microglia promote inflammation when activated, either could be the potential cause of this increased permeability. Inflammation in the brain and heightened immune activity is more risky than in the body as the brain is more susceptible to disorganization and damage when inflamed.
Innate immune system and inflammation^
The innate immune system is the first line of defense against foreign pathogens or cancerous cells, and while rapid it is neither complete nor specific in the way the adaptive (also called acquired) immune system is. HD affects cells of the innate immune system; for instance it was seen that monocytes expressing mutant huntingin were hyperactive, which can cause an auto-immune response, where the immune system turns on and destroys healthy cells of the body, mistaking them as foreign or infected. The rise in cytokine levels in HD also suggested overactivation of the innate immune response, as the cytokines increased earliest are part of the innate response. This overactivation of the innate immune system in both the body and the brain (microglia are overactive in HD patients) corresponds to HD progression and so is a potential target for HD therapies.
One aspect of the innate immune system that plays a particularly prominent role in HD is inflammation, as mentioned above. In HD, molecules promoting inflammation are released in increased and sustained levels, causing irregularly high inflammation. As inflammation activates the adaptive immune system, including microglia, which release inflammatory factors, this cycle is only perpetuated as microglia activity is increased, in turn further increasing inflammation. Many neurodegenerative diseases are associated with chronic neuroinflammation, which contributes directly to neuronal death. For instance, normally neuroinflammation prevents the accumulation of amyloid plaques. However, in Alzheimer’s Disease, the accumulation of amyloid plaques may be a result of chronic increased neuroinflammation. In HD, inflammation is chronic in both the peripheral and central immune systems. Mutant huntingtin promotes inflammatory factors, such the cytokine IL-6, which promotes inflammation in glial cells.
While neuroinflammation in response to pathogens in usually beneficial, minimizing injury and expediating tissue repair, chronic neuroinflammation’s self-perpetuating nature causes it to persist long after the initial injury had rendered it beneficial. Whether neuroinflammation is beneficial or harmful in the brain clearly depends on its duration, with prolonged inflammation tipping the scale to harm. Inflammation also causes increased oxidative stress, another factor that leads to neurodegeneration. It is also a factor contributing to the compromise of the blood-brain barrier, which allows macrophages to infiltrate the brain, furthering inflammation even more. An important part of an inflammatory response is the activation of anti-inflammatory regulation that is not present in HD and other neurodegenerative diseases. The neuronal death in HD also further feeds inflammation, perpetuating the cycle.
One the major ways chronic inflammation leads to neurodegeneration is through oxidative stress and the damage this causes to neuronal cells. In diseased brains, peroxidases, enzymes that oxidize cells, are present in elevated level. Oxidizing agents such as hypochlorous acid (a precursor to hydrogen peroxide—a very harmful oxidizing agent) are produced by active immune cells, and in this way immune responses promotes oxidation that leads to neurodegeneration and cell death, to a harmful degree if the response is chronic. It is also important to remember that neuronal death leads to a less functional nervous system, less able to combat these oxidative stresses. While neuronal death is the direct cause of neurodegeneration, the pathways to neuronal death in HD and other diseases is still not completely clear. Oxidative stress is recognized as at least one of these pathways. Oxidative stress occurs when the cellular defenses against reactive oxygen species (oxidants such as hydrogen peroxide or peroxidases) are compromised. As the brain has the highest metabolic rate of any organ, it is the most sensitive to oxidative stress. Brain cells also have a high content of unsaturated fatty acids in their membranes as well as high levels of iron, both of which promote and are particularly susceptible to oxidative stress (for an article on omega-3 fatty acids and neuronal membranes, click here). The brain also has much fewer antioxidants than other organs.
In immune cells such as phagocytes, NADPH oxidase, an enzyme involved in electron transport chains in the membrane is activated in immune response, creating oxygen radicals that are precursors to hydrogen peroxide. Immune cells also produce nitric oxide, which activates hydrogen peroxide activity. In Alzheimer’s Disease, accumulation of amyloid plaques promotes NADPH oxidase in immune cells such as microglia and macrophages, stimulating hydrogen peroxide production. Again, the inflammatory response causes an increase in microglia and macrophage response in the brain, furthering oxidative stress. In Parkinson’s Disease, microglia also activate NADPH oxidation in dopaminergic neurons, the cells harmed and killed in PD. The extracellular matrix in brains suffering from neurodegenerative diseases is at risk from inflammation and oxidative stress, as are unsaturated lipids common to neuronal membranes (such as membranes containing omega-3 fatty acids).
The heat shock protein response to oxidative stress prevents misfolding and aggregation of damaged proteins. These proteins also reduce oxidative damage to lipids and DNA. In neurodegenerative diseases, this response is compromised and cannot keep up with increased oxidative stress. As oxidative stress is such an important cause of cellular death in neurodegenerative diseases, it and its causes, inflammation and chronic immune system activation, are promising targets for disease therapies.
Microglia have been mentioned above, and as the brain’s counterpart to macrophages, they are the primary cells of the central nervous system’s innate immune response. Unlike other cells in the brain, microglia are not derived from neuronal precursors but rather myeloid precursors, and have their own independent replication cycle. It has been shown that microglia are irregularly activated in HD, even before symptoms appear, and their activation is correlated with disease severity. Mutant huntingtin is expressed in microglia, which may be the reason for their increased activation in HD. The hyperactivation of microglia leads to the increased production of cytokines by the cells, which further increases immune activation. Microglia also produce oxidizing agents such as hydrogen peroxide.
In AD, it has been seen the microglia-mediated inflammation contributes to neurodegeneration, and anti-inflammatory drugs that decrease this inflammatory response has had positive results in Alzheimer patients. In Parkinson’s Disease as well it has been shown that microglial activation leads directly to the death of dopaminergic neurons. As mentioned above, the destruction of the blood-brain barrier means microglia overactivation is augmented by infiltrating macrophages from the body’s immune system.
As the principal cause of inflammation in diseased brains, microglia react to many external stimuli to cause neuronal dysfunction. In healthy brains, microglia have mechanisms that monitor their environment, and when activated, begin protection and repair of damaged tissue. In this controlled activation, microglia take up and destroy neurotoxins, remove dying neurons and debris, and secrete neurotrophic factors to promote neuronal survival. Pathogens and neurotoxins activate microglia either by damaging cells, which are recognized by the microglia or being recognized themselves as foreign agents. While not all microglia activation contributes to neurodegeneration, when overactivated, the microglia become neurotoxic and contribute to disease progression, accelerating and exacerbating disease mechanisms. Inflammation shifts microglia activity from plaque removal to plaque deposition in AD, and a similar switch occurs in other neurodegenerative diseases, showing that the brain’s environment and circumstances influence whether microglia are helpful or harmful.
Interestingly, patients with PD have elevated microglia activity in many brain regions regardless of how long the disease has progressed. Similarly, in HD, microglia are activated before symptoms are manifest. Once symptoms are observed, microglial activation corresponds to disease severity. Microglia regulate both inflammation levels and adaptive immune response, and are responsible for scavenging cellular debris. Microglia cells are also the reason for graft rejection in the central nervous system, and this immune rejection would have to be taken into account with any potentials for tissue regeneration treatments for HD. The importance of microglia in the brain’s immune system cannot be overstated, and their chronic activation in HD contributes directly to disease progression.
The microglia’s counterpart in the body’s immune system are phagocytic cells, specifically monocytes. Though HD pathology mainly affects the brain and its functions, it has been shown that monocytes in HD patients also express the mutant huntingtin allele. As mentioned above, even before HD symptoms show up monocytes in HD positive patients can behave abnormally, increasing production of the cytokine IL-6. This overactivity of monocytes is harmful to the body’s immune system and makes the cells more able to cross the blood brain barrier and turn into microglia, further overactivating the brain’s immune system.
The first indicators that immune activity was upregulated in HD patients came from the observation of cytokine levels. Cytokines, as protein signalers of the immune system, play many roles in activating various components of the immune response. It was seen that in HD patients the cytokines IL-6 and IL-8 increased enormously, and IL-4 and IL-10 increased substantially as well. The increase in IL-6 was shown to happen approximately sixteen years before motor symptoms of HD appeared, which indicates IL-6 may be a potential early indicator of disease progression. IL-8 levels corresponded directly with disease progression, and all the cytokines were upregulated not only in the brain, but all around the body. While in healthy subjects there is low levels of cytokine production that activate the immune system when necessary, the neuroinflammation in HD causes the release of cytokines to spiral out of control, worsening the disease. Activated microglia secrete cytokines that perpetuate inflammation and activation of microglia. HD is not the only neurodegenerative disease connected to cytokine activity; in Alzheimer’s Disease plaque aggregation increases the secretion of IL-6 and IL-8 as well, which in turn lead to increased aggregation, and in Parkinson’s patients the cerebrospinal fluid has been seen to have high levels of IL-6. In HD patients, the increases of cytokines were most marked in the striatum, where the disease pathology is the worst. Cytokines are an indispensible part of the chronic inflammation and immune activation indicative of neurodegenerative diseases, and their heightened levels before symptoms occur in HD have potential to be used in HD therapies.
Many of the immune system changes in HD occur many years before motor symptoms, such as the increase in cytokine levels and inflammation. Because of this occurrence, the immune activation in HD must be better characterized so there are markers of disease progression (state biomarkers) that connect immune activation to their stage in HD. The changes in the body’s immune system are easy to monitor and most likely mirror the neurodegeneration in the brain that is much less easy to track. Biomarkers in AD in the peripheral immune system have already been characterized, with the goal of finding therapies that can treat the disease in its earlier stages, before symptoms even occur. Immune activation in neurodegenerative diseases occurs before significant neuronal death, and the prospect of arresting disease progression in these early stages holds much promise for these diseases. Anti-inflammatory treatments given early on to HD patients may alter the disease pathology and slow its progression. In this way, the immune system and inflammatory agents are both useful in the detection of HD and as potential targets for its preemptive treatment.
The complement system^
The complement system is yet another component of the innate immune system that overactivates in neurodegenerative disease. The complement system recognizes and kills foreign pathogens or infected cells, as part of the immune system’s distinction between self and non-self. If it is activated in inappropriate regions or overactivated, it can attack host tissue and become harmful. The tissues of the brain create a complete complement system in parallel to the body’s system, and also produce inhibitors to the complement system to prevent overactivation and lysis by the complement system. Complement activation induces a cell to lyse, especially neurons, and also influences inflammation, alternatively in pro and anti-inflammatory activities. Research has shown that complement activation may also allow tissue remodeling to repair cells after injury, especially in the brain and the central nervous system, clearing toxic and damaging deposits. A major role of the complement system is recognizing and clearing away cell debris and lysed cells by microglia that have receptors recognizing components of the complement system. This prevents further inflammation that is caused by an excess of toxic debris. It can additionally form a membrane attack complex (MAC) on a target cell, that destroys the cell membrane by creating pores into the cell and lysing the cell. Inflammation by the complement system causes neuronal apoptotic activity as well.
Because the complement system initiates inflammation and lysis, if unregulated, it can lead to severe tissue damage. While complement activity is helpful in repairing tissue after injury, its roles are potentially threatening, especially if upregulated. For this reason, the system is normally tightly regulated by soluble and membrane proteins produced by the liver and immune and endothelial cells. In AD, amyloid plaques further activate the complement system and in HD microglia increase production of complement proteins and receptors. In healthy brains complement activation is minimal, as it is inhibited by the specific inhibitory proteins produced by the brain, to avoid the self-destructive activity of an overactivated system. If a healthy brain becomes infected, its cells can synthesize complement proteins to kill the pathogen while preserving healthy, uninfected cells. Glial cells and neurons in are capable of producing a full complement system, and increase complement expression after brain infection. Neurons are extremely susceptible to attack by the complement system, especially by membrane attack complexes, as neurons, unlike all other brain cells, do not express high levels of complement inhibitor proteins in their membranes. Increased complement synthesis and activation can in this way lead to neuronal loss in HD and other neurodegenerative disease, though it is under debate to what extent complement activation is a cause of neurodegeneration or an effect of it.
In neurodegenerative diseases, the complement system is particularly increased in the areas of the brain most affected by the disease. Complement inhibitors do not increase their levels in these diseases, leaving the brain vulnerable to the increased complement activity. Some research has suggested that the complement system plays a role in the apoptosis of neurons caused by mutant huntingtin and also causes damage to surrounding cells by lysing them as well. The complement system is further activated by the intracellular components that are released in lysed cells, such as nucleic acids and mitochondrial membranes. While normally microglia with complement cells aid in the removal of pathogens and toxic debris, overactive complement system can be signaled by inflammatory activity. Activation of the membrane attack complex has been shown to increase complement regulators and inhibitors and protect against overactivation of the complement system. Once this mechanism is further elucidated, it may hold potential towards treating the overactivation of the complement system in neurodegenerative diseases. Inhibitors of the complement system would have to be specifically delivered or expressed in the brain, as the complement system continues to perform a beneficial role in the body during neurodegenerative diseases.
A different immune system overactivation in neurodegenerative diseases is overactivation of endocannabinoids. Normally, endocannabinoids act protectively in the brain, reducing neurodegenerative and inflammatory damage. In HD, the peripheral endocannabinoid system (ECS) mirrors the problem occurring in the ECS of the central nervous system. Because of these parallels, blood ECS levels may serve as an early, non-invasive diagnosis for HD and other neurological diseases, like cytokines with which the ECS is intimately linked. Also like cytokines, the ECS may be a potentially promising target for HD therapies. Endocannabinoid receptors, which come in two primary types, CB1R and CB2R, are activated by the neurotransmitter endocannabinoid anandamide (AEA). In neurodegenerative diseases, AEA production is increased and flows through the blood brain barrier into the brain. AEA activity depends greatly on its life span which begins when it is taken up by a cell and ends when it is degraded in the cell. It is not known whether there is a transport protein that ferries AEA across the blood-brain barrier. CB1R is the cannabinoid receptor primarily found in the central nervous system, and CB2R is predominantly expressed by immune cells in the body. When the ECS is overactivated, it increases neuroinflammation and displays autoimmune behavior, harming healthy neurons. ECS activation has been studied in Parkinson’s Disease, where it becomes overactive in dopaminergic neurons, elevating AEA levels, which is also true in multiple sclerosis (MS). ECS activation is connected to the neuroinflammation that precedes neuronal loss and onset of symtoms of many neurological diseases. CB1 receptors are densely expressed in the striatum, the main region of the brain affected by HD. A severe loss of CB1 receptors has been seen in HD patients. This loss correlates directly to the length of the CAG repeats, which determines the onset and severity of disease pathology. In mouse models, preventing this loss of CB1 receptors slowed progression of HD, which may point to potential therapies for HD involving the CB receptors.
Although it has been seen that the blood-brain barrier is not immutable, the brain’s immune system still poses a formidable barrier to HD therapies, as even a compromised brain can recognize drugs as foreign and may destroy cell and tissue therapies that are intended to treat the disease. It was formerly thought that the brain was an “immunologically privileged site”, that does not reject implants of foreign cells and tissue as it does not recognize them as foreign. This has been shown to be false, for although the blood-brain barrier does allow a degree of “ignorance” (unawareness that foreign material is present), and “tolerance” (incomplete ability of the immune system to reject tissue), it does not display absolute immunological privilege as activated lymphocytes can cross the blood brain barrier. There are multiple factors that contribute to the brain’s ability to reject grafts of tissue, such as the type of tissue transplanted, the degree of immunological difference between the host and introduced tissue, and the way in which the tissue is transplanted. Interestingly, transplantation to a site where microglia are activated to produce inflammation results in more successful grafts, most likely due to the microglia’s production of growth factors. As this characterizes HD brains, tissue transplantation therapies may not face as many obstacles in HD and other neurodegenerative diseases. But there have been no successful neural transplantations for HD yet; twelve patients have been grafted with embryonic neural pig tissue, with no benefit and in fact harm done to the patients’ brains.
Cell therapies hold promise to combat the mass injury done in neurodegenerative brains, and the need for successful transplantation is vital. Using multiple donors has shown to be beneficial to transplantations. Transplantation surgery will inevitably damage the blood-brain barrier. T cells are the main cellular causes of immune rejection of transplants, as are microglial cells. Microglia in a healthy brain are typically immature and ineffective, but when activated they mature into active macrophages. Healthy brains also have a much lower level of antibodies, complement proteins, and other immune proteins than is found in the body’s bloodstream, but in response to a transplant these levels increase dramatically. For drugs taken orally or through the bloodstream, the blood-brain barrier poses a obstacle towards these therapies reaching their targets in the brain. The blood-brain barrier is a capillary barrier of tight junctions restricting cell passage, as well as enzymes that degrade substances before they can pass through. Active transport proteins across the blood-brain barrier are highly specific and do not easily carry drugs.
Peripheral immune activation^
HD symptoms do not occur exclusively in the brain, and the body’s innate immune system is abnormally activated in HD. The earliest blood abnormality associated with HD is an increase in IL-6 levels sixteen years before symptoms occur. The parallel central nervous system and peripheral pathways of immune activation in HD occurs because the mutant protein is expressed all over the body. It is unknown what immune effects (such as cytokine levels rising) is due directly to the mutant protein and what is due to inflammation that is itself caused by the mutant protein. Complement proteins are also up-regulated peripherally by mutant huntingtin or inflammation. Because monocytes in the body display immune dysfunction in premanifest HD patients, they offer a non-invasive way to track disease progression.
Anti-inflammatory drugs have reduced onset of Alzheimer’s Disease, with a five-year use of ibuprofen protecting against disease development. These drugs inhibit excessive microglia activation and the inflammation this causes. Unfortunately, clinical trials of anti-inflammatory drugs have yielded inconclusive results, as the key inflammatory proteins of AD have yet to be indentified and as such cannot be directly targeted. Drugs must also be delivered more effectively to the brain across the blood-brain barrier, and the beneficial effects of neuroinflammation cannot be shut down with the administration of anti-inflammatory drugs. Therapies must cross the blood-brain barrier and target destructive inflammatory mediators without compromising beneficial survival-promoting effects and overall immune function.
While this is a tall order for any drug, the importance of the immune system’s activation in HD points to its promise as a target for potential treatments. As the role of the immune system in HD, constantly fluctuating between beneficial and harmful, is better understood, its manipulation to track disease progression and slow the disease by arresting harmful immune responses may become a subject of HD research that leads to the next stage of knowledge about HD and how it affects the brain and body’s function.