This article is Part II of a 3-part series from the 2015 Keystone Symposium on “Neuroinflammation in Diseases of the Central Nervous System”. To read all coverage from the meeting, click here.
Microglia, the macrophages of the brain, took center stage at “Neuroinflammation in Diseases of the Central Nervous System,” a Keystone symposium held January 25-30 in Taos, New Mexico. Even though the spotlight has been shining on the shape-shifting glial cells for years, researchers are only now developing the tools to understand what they do in the normal brain, how they differ from macrophages that come from other parts of the body, and how they react to and influence the course of neurodegenerative disease. At the meeting, researchers presented a trove of findings that painted microglia as cells tailor-made for their brainy environment, yet quite capable of transformation—good and bad—when put under pressure. Part 2 of this Keystone series addresses microglial origins and how their local environment helps seal their fate. Part 3 will describe how the cells change in the diseased brain. Click here for Part 1.
Day in and day out, multitudes of macrophages live in drastically different environments throughout the body. Some, such as microglia in the brain or Kupffer cells in the liver, stay put in their home organs throughout life, whereas others, such as blood monocytes, are nomads, primed to invade any tissue on a whim. Early in development, progenitors of microglia and other tissue-resident macrophages migrate to their destination and continue to supply those organs with macrophages throughout life (see Gomez Perdiguero et al., 2013). This is in stark contrast to blood- borne monocytes, which are continuously being replaced by hematopoietic stem cells in the bone marrow. In recent years scientists discovered that brain-resident microglia derive from cells in the embryonic yolk sac, but at Keystone Frederic Geissmann of King’s College London claimed those progenitors make a pit stop on the way.
Using an arsenal of transgenic mice engineered to express fluorescent markers at pivotal times during development, Geissmann and colleagues found that erythro-myeloid progenitor cells arise in the yolk sac at embryonic day 8.5. Two days later, they colonize the fetal liver. There, they give rise to red blood cells and progenitors of tissue-resident macrophages, including microglia, which migrate to their respective organs, including the brain (see Gomez Perdiguero et al., 2014). Just after the blood-brain barrier walls off the brain from new immigrants, hematopoietic stem cells colonize the liver and take over the job of producing red blood cells and monocytes until the bone marrow matures.
Asked how the budding macrophages know which organs to home to, Geissmann said the cells could be pre-programmed in some way, or they may randomly end up in certain tissues and then adapt to their new environment. Richard Ransohoff of Biogen Idec in Cambridge, Massachusetts, expressed the concept metaphorically: “You may decide to move to Florida because you already have a bikini, or perhaps you randomly find yourself in Florida and then go buy one.”
Whichever is correct, tissue-resident macrophages must take on roles suited to their environment. Chris Glass of the University of California, San Diego, reported that tissue-specific signals shape gene-expression patterns in the incoming macrophages by triggering the activation of thousands of enhancer sequences (see Gosselin et al., 2014). Glass initially compared gene-expression patterns in microglial cells to two different types of macrophage that live in the abdomen—large and small peritoneal macrophages. Using RNA sequencing, he found that the two subsets of peritoneal macrophage expressed similar patterns of genes; however, the microglia expressed about 800 genes at levels more than 16-fold higher than both types of peritoneal macrophage, and vice versa. For this reason, he suggested that the tissue environment plays a strong role in dictating these gene-expression profiles. In support of this idea, the genes likeliest to be specifically expressed in particular macrophage types shut down when Glass transferred the cells into tissue culture dishes.
Glass next asked how the cellular environment dictates these patterns of gene expression, hypothesizing that the activation of enhancer regions likely plays a part. In a previous study, Glass and colleagues had reported that lineage-specific transcription factors that switch on early in development bind to enhancer regions and prime them for full activation. Once the cell arrives in its environment, other transcription factors, turned on by signals in the local milieu, bind adjacent to those poised enhancers, and turn on downstream genes. This two-hit scenario explains how gene activation can be specific to both a cell type and a unique environment (see image above and Heinz et al., 2013).
Glass hypothesized that the local signals in the peritoneum could be retinoic acid and in the brain, TGF-β, and that they might activate transcription factors in macrophages that would bind to PU.1-primed enhancer regions in the DNA. PU.1 is a macrophage-specific transcription factor. To test this idea, Glass first surveyed the landscape of enhancer and promoter activity throughout the genome by measuring histone methylation and acetylation, which usually coincide with areas where genes are active. Eight thousand promoters were marked in macrophages. The activity of about 300 of those was higher or lower in microglia compared to peritoneal macrophages. Starker differences emerged at enhancers. Glass found that about 50,000 were primed in macrophages and about a quarter of those were either more or less active in specific cell types. Glass also found evidence for around 500 “super-enhancers,” which, he said, are regions containing clusters of enhancers that work together to activate a single gene. Of those, around half were differentially activated between microglia and peritoneal macrophages.
Using a variety of biochemical and genetic techniques, Glass determined that signal-dependent transcription factors, such as SMAD (which is triggered by TGF-β in the brain), activated a subset of PU.1-poised enhancers and super-enhancers in microglia. For peritoneal macrophages, retinoic acid receptors elevated the activation of other PU.1-poised enhancers.
If the local environment plays such a major role, then could it effectively turn infiltrating monocyte-derived macrophages into microglia once they enter the brain? Researchers have been struggling to establish the provenance of macrophages and microglia in the brain. Glass speculated that because monocytes derive from hematopoietic stem cells in the bone marrow, rather than the yolk sac as microglia do, they would have some unique lineage-dependent transcription factors that would always make them a bit different than microglia. How different, and which factors stably indicate which lineage, is still up for debate.
Researchers at the meeting were impressed with Glass’s work, but also pointed out that the epigenetic findings need to translate into functional outcomes before it’s clear how important they really are. “These findings represent differences only in the ‘phenotypic potential,’ of macrophages,” commented Anne La Flamme from Victoria University of Wellington in New Zealand. Glass acknowledged that this is a problem that plagues all genetic and epigenetic studies. He sees epigenetics as a way to sift out what may be most important to the identity of each cell type.
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