The blood-brain barrier is a layer of cells that covers the blood vessels in the brain and regulates the entry of molecules from the blood into the brain.
Increases in blood-brain barrier “permeability,” or the extent to which molecules leak through, are observed in several neurological and psychiatric disorders; therefore, understanding the regulation of blood-brain barrier permeability is crucial for developing better therapies for such disorders.
In a study recently published in Nature Communications, a research team led by Prof. Hiroaki Wake of Nagoya University Graduate School of Medicine shows that microglia – the resident immune cells of the brain – initially protect the blood-brain barrier from damage due to “systemic inflammation,” a condition of chronic inflammation associated with factors like smoking, ageing, and diabetes, and leading to an increased risk of neurodegenerative disorders. However, these same microglia can change their behavior and increase the blood-brain barrier permeability, thereby damaging it.
“It has long been known that microglia can become activated due to systemic inflammation,” remarks Prof. Wake, “so we became interested in the question of whether microglia can regulate blood-brain barrier permeability.”
To explore this, Prof. Wake’s team worked with mice that were genetically engineered to produce fluorescent proteins in the microglia.
This “fluorescent labeling” allowed the investigators to use a technique called “two-photon imaging” to study the interactions of microglia and the blood-brain barrier in living mice.
The investigators also injected the mice with fluorescent molecules that can pass through the blood-brain barrier only if the barrier is damaged enough to be sufficiently permeable.
By observing the locations of these fluorescent molecules and the interactions of microglia, the research team could study microglial interactions with the blood-brain barrier and the permeability of the blood-brain barrier under various conditions.
Microglia express tight junction molecule (CLDN5) to maintain BBB integrity in early phase and molecule for phagocytosis (CD68) to impair the BBB function in late phase of systemic inflammation. Microglia phagocytose astrocyte end feet which is one of the components of BBB. The image is credited to Hiroaki Wake.
A key point of interest was the systemic inflammation induced by injecting the mice with an inflammation-inducing substance.
Such injections resulted in the movement of microglia to the blood vessels and increased the permeability of the blood-brain barrier within a few days.
Then, the microglia initially acted to protect the blood-brain barrier and limit increases in permeability, but as inflammation progressed, the microglia reversed their behavior by attacking the components of the blood-brain barrier, thus increasing the barrier’s permeability.
The subsequent leakage of molecules into the brain had the potential to cause widespread inflammation in the brain and consequent damage to neurons (cells of the nerves).
These results clearly show that microglia play a dual role in regulating the permeability of the blood-brain barrier. In describing his team’s future research objectives, Prof. Wake comments, “We aim to identify therapeutic targets on the microglia for regulating blood-brain barrier permeability, because drugs designed for such targets can be used to treat neurological and psychiatric diseases by curbing inflammatory responses in the brain.”
As the scientists note in their study, uncontrolled inflammatory responses in the brain can cause a range of cognitive disorders and adverse neurological effects, and drugs that target microglia may help patients avoid such problems by preserving the integrity of the blood-brain barrier.
More studies are required to understand more about the processes underlying the microglial behaviors observed in this study. Nevertheless, the study’s results offer hope for the development of therapies that could “force” microglia to promote blood-brain barrier integrity and prevent microglia from transitioning to behaviors that damage the barrier.
This can range from acute severe illness and fever causing malaise and poor cognition, from sepsis causing subsequent neurological disease and cognitive failure3, through to chronic systemic inflammation associated with smoking, diabetes, chronic periodontitis and even aging, leading to an increased risk of dementia or neurodegenerative disorders4,5.
Characterizing the physiological and molecular mechanisms by which systemic inflammation impacts on brain function may improve understanding of brain pathophysiology and open up new therapeutic targets.
One important pathway by which systemic inflammation or infection may initiate neuronal consequences is via communication across the blood–brain barrier (BBB) between systemic inflammatory or immune molecules and the neuronal elements involved in maintenance of neural circuits.
The BBB is a tightly regulated syncytium of endothelial cells with low transcellular and paracellular transport properties that surround cerebral vessels and protect the delicate neuronal microenvironment from neurotoxic substances.
Endothelial transport is strictly regulated through interactions with astrocytes, pericytes, microglia, and the basement membrane—together forming a neurovascular unit that constitutes the BBB6.
Astrocytes and pericytes directly encircle endothelial cells and not only help link blood supply to metabolic demand but also secrete a number of molecules that enhance and maintain BBB integrity7–10.
Microglia are part of the neurovascular unit although their ablation in the mature mouse does not increase BBB permeability11. Nevertheless, their ablation can adversely influence BBB integrity via release of cytokines or reactive oxygen species12,13.
Increases in BBB permeability are seen in many neurological and psychiatric disorders, including stroke, epilepsy, amyotrophic lateral sclerosis, Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and schizophrenia, suggesting BBB compromise is involved in the pathogenesis and/or severity of brain disease7,14.
Reactive microglia (and astrocytes) are likely to contribute to the leaky BBB observed in these diseases through downregulation of paracellular tight-junction proteins such as Claudin-5 (CLDN5), occludin, and zonula occludens-1 (refs. 7,9).
Conversely, microglia can be driven towards reactive phenotypes in these diseases by direct neuronal damage, via systemic factors, or through neutrophils that invade the damaged BBB15 making it difficult to correlate specific microglia phenotypes with changes in BBB.
Systemic inflammation and infection can also result in microglial phenotype changes and disruption of BBB integrity in the absence of any precipitating neuronal damage or neuronal infection14.
Given the importance of physiological microglia in monitoring and sculpting neural circuits in development and adult learning, the activation of microglia into different phenotypes may potentially disrupt their homeostatic function and contribute to the development of adverse cognitive effects due to systemic inflammation11,18.
Indeed, we have recently reported that microglia activated by experimentally induced systemic inflammation (using lipopolysaccharide [LPS] injections) lose their capacity to synchronize local neural circuits19. Furthermore, in a recent study using a mouse model that replicates the pathogenic inflammation in systemic lupus erythematosus (SLE), reactive microglia phagocytosed cortical synapses which correlated to observed cognitive deficits20.
An important initial step in identifying how systemic infection or inflammation signals convert resident physiological microglia, and how changes in BBB permeability impact this process, is to correlate the temporal changes in BBB leakage with resulting microglial responses.
In this study, we attempted to achieve this goal by simultaneously measuring BBB permeability and microglia dynamics during both acute and chronic systemic infection using in vivo two-photon imaging. We demonstrated that microglia respond to inflammation by migrating towards and accumulating around cerebral vessels, and that this begins before any detectable change in BBB permeability. Surprisingly, our data suggest that the initial microglial contact with cerebral blood vessels actually protect BBB integrity.
Further prolonged inflammation results in a dominance of a more activated microglial phenotype, resulting in phagocytosis of astrocytic end-feet and a loss of BBB permeability. Our results implicate microglia as playing a dual role in BBB permeability during systemic infection and inflammation, with important implications for understanding how systemic diseases may adversely impact on neural circuits and brain functions.
Microglia are active surveyors of brain parenchyma with important roles in sculpting and coordinating neural circuits in healthy brains that respond rapidly to form a range of reactive phenotypes in brain infection and damage19,25,26,41.
Activated microglia play roles in a range of acute and neurodegenerative diseases, where they can help clear neuronal damage by phagocytosis, but can also contribute to disease progression by releasing molecules that can initiate a neuroinflammatory states12,42,43. Microglia can also respond to peripheral inflammatory diseases.
A key question is how microglia change phenotypes when the primary pathological insult resides in the peripheral organs and systemic circulation. Determining how these systemic and neuronal inflammatory responses are linked may help reduce the deleterious impact of systemic immune activation and inflammation on cognitive function and susceptibility to brain disease. The BBB represents a major pathway by which systemic inflammation and immune responses potentially interact with the brain microenvironment.
The goal of this study was to examine the role of microglia in responding to systemic infection and inflammation, and its contribution to BBB integrity. Using two different models of peripheral inflammation, MRL/lpr mice and mice treated for 7 days with LPS, we demonstrated that resident brain microglia migrate to cerebral vessels during systemic inflammation in response to the release of the chemokine CCL5 from endothelial cells.
This triggers microglial cells to express CLDN5 and to infiltrate through the neurovascular unit, thus contacting endothelial cells and forming tight junctions to maintain BBB integrity.
Consistently, partial microglial ablation or blocking CCL5 signaling, actually increased BBB permeability during the early stages of inflammation. Additional factors may also trigger microglial migration to blood vessels and phenotype changes under different conditions. Fibrinogen, for example, can attract microglia to vessels in murine models of experimental autoimmune encephalomyelitis44.
Sustained inflammation causes microglia to further transform into a phagocytic phenotype associated with morphological changes, engulfment of astrocytic fragments, and leakage across the BBB. Partial ablation of these vessel-associated microglia reduced the BBB leakage, as did inhibition of reactive microglia with minocycline.
Minocycline reduces BBB leak associated with rodent models of brain diseases, such as hypoxia, ischemia, and Alzheimer’s disease45–47 and our results extend this to BBB leak resulting from systemic inflammation.
Minocycline can also improve cognitive function in both mouse models of ischemia and human stroke48. Protection from microglia-induced initiation or exacerbation of BBB leak may contribute to such therapeutic effects38,49.
Indeed, limiting BBB leakage can reduce access to the brain microenvironment for a range of toxic circulating molecules, such as inflammatory cytokines, ions, and immune cells. These mediators will further (directly and indirectly via neuronal damage) activate microglia and astrocytes and exacerbate neuroinflammatory damage and BBB integrity50.
Our data indicate opposing actions of microglia in regulating BBB integrity with distinct time courses and underlying signaling pathways. The CCL5-CCR5-mediated increase in microglial CLDN5 expression, and the evidence of microglia infiltrating through the neurovascular unit, is consistent with a novel tight-junction connection between endothelial cells and microglia to mediate protective sealing of the BBB. CLDN5 forms tight junctions between adjacent endothelial cells in a complex of other claudin proteins and occludins32.
Upregulation of members of the CLDN family (Cldn4, Cldn1) in reactive astrocytes has also been shown to contribute to new tight junctions in inflammatory diseases, and furthermore to reduce the extent of neuropathology51.
Indeed, our microarray data also suggest that astrocytes adopt a more reactive phenotype, which may also contribute to regulating the permeability of the BBB, either independently or in concert with microglia. Astrocytes, BBB integrity, and blood flow regulation are all closely linked, and astrocyte reactivity may be a key component in both acute and chronic brain diseases associated with altered BBB permeability52.
Altered CLDN5 expression also regulates BBB integrity, where a decrease in CLDN5 increases BBB leakage. For example, microglia activated in response to epileptic seizures release interleukin-1β (IL-1β) which downregulates the tight-junction protein CLDN5 in endothelial cells that then disrupts BBB function53.
Our results suggest novel CLDN5 upregulation in microglia maintains BBB integrity via proposed microglial tight junctions with endothelial cells. Our results contradict the notion that microglia do not express this tight-junction protein.
For example, whole-brain RNA seq data suggest microglial expression is almost negligible54 (see http://www.brainrnaseq.org/). However, CCL5-CCR5-dependent Cldn5 expression is likely to be restricted to a small subset of vessel-associated microglia in response to specific and possibly transient inflammatory signaling pathways, and hence challenging to detect in whole brain samples. Indeed, more recent single-cell RNA sequencing clearly shows Cldn5 expression in some genotypically characterized microglial subsets, even in healthy brain55 (see also https://myeloidsc.appspot.com/).
The second aspect of the microglia–BBB interaction we observed was transformation of vessel-associated microglia to a CD68-expressing phagocytic phenotype with loss of BBB integrity. Activated microglia are known to contribute to BBB leakage in neuroinflammatory diseases, via cytokine release or by upregulating adhesion molecules to facilitate invasion of circulating immune cells15,56,57.
For example, systemic inflammation induced by bacterial infection and stress activate microglia which then release cytokines tumor necrosis factor-α and IL-1β15,58. These trigger endothelial cells into expressing adhesion molecules that facilitate invasion of systemic immune cells and subsequently enable more pronounced neuroinflammation.
Our results add an additional facet in explaining how reactive microglia can cause neuroinflammation. By phagocytizing the astrocytic end-feet during more sustained systemic inflammation, the integrity of the BBB is compromised. Disruption of astrocytes in Alzheimer’s disease and Parkinson’s disease also results in a leaky BBB59.
However, the increased permeability of the BBB in our LPS and MRL/lpr models is more subtle than seen in some Alzheimer’s and hypoxia or ischemia models, where much larger molecules like albumin-conjugated Evan’s Blue dye (~66 kDa) and IgGs (~150 kDa) can permeate the BBB, and the coverage of vessels by pericytes can also be substantially decreased in these diseases45–47. Type I interferon (IFN) has been suggested to mediate the link between systemic inflammation in SLE mice, and the conversion of resident parenchymal microglia to a phagocytic phenotype20, and IFN may have also contributed to the activation of microglia we observed in our study.
A small cytokine (such as IFNα) is consistent with the modest leakiness of the BBB we observed. We did not detect peripheral macrophage accumulation around blood vessels. Indeed, our EM analysis showed that microglial processes protruded through the basement membrane and even occasionally appeared to obtrude through to the vessel wall where IFNα or another systemic cytokine may directly initiate microglia transformation into a reactive phenotype that then amplifies the inflammatory response. A cellular communication pathway involving endothelial cells may also be involved.
In conclusion, our study sheds light on how brain microglia respond to systemic inflammation and interact with the BBB. They initially migrate to the BBB and protect its integrity, before then transforming into a reactive phenotype that phagocytose BBB components to initiate leakage of systemic substances into the parenchyma and cause widespread neuroinflammation. We also identified some key molecules and signaling pathways involved in instigating the opposing microglial responses.
This may lead to strategies that will maintain the BBB integrity during systemic disease, and thereby reducing susceptibility to cognitive disorders and/or the adverse neural effects associated with peripheral infection, and systemic stress and inflammation.