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Page 208 Walker. Neuroimmunol Neuroinflammation 2020;7:194-214 I http://dx.doi.org/10.20517/2347-8659.2020.09

chemotaxis to sources that can include necrotic and apoptotic neurons. P2RY12 has also been identified
by most microglia gene profiling studies as being a specific marker for microglia. Its expression is highest
[30]
in homeostatic microglia with significant downregulation upon inflammatory activation . Recent studies
of P2RY12 expression by microglia in multiple sclerosis tissue sections showed downregulation in areas
of active white matter lesions, similar to TMEM119, but less in areas with gray matter lesions [28,115,116] .
In addition, it was observed that microglia around many Aβ plaques had reduced or no P2RY12
immunoreactivity [116] . However, our recent study further characterized P2RY12 expression by microglia in
AD brains, and observed that the pathological environment (diffuse or mature plaques) had an effect on
microglial P2RY12 expression [109] . Our results were noticeably different from others. We have concluded,
based on these observations of increased P2RY12 expressing microglia with activation morphologies,
particularly in AD brains, that this marker could be identifying other classes of microglia in addition to
homeostatic/resting microglia [109] . We observed that most P2RY12 positive microglia expressed CD68 and
progranulin. It was observed that highly activated appearing microglia, but also expressing P2RY12, were
frequently interacting with Aβ plaques. Although our results agreed that there was an overall decrease
in P2RY12 immunoreactivity, the positive cells suggest an additional function besides as a marker for
homeostatic microglia.

“Alternative activation” markers (CD200 receptor, CD206, CD163)
Additional markers that have been studied for defining microglial phenotypes are CD200 receptor
(CD200R), CD206 and CD163. These antigens have been defined as markers for alternative activation.
CD200R was shown to be induced in cultured microglia by IL-4 and IL-13, defining it as an alternative
activation marker [117,118] . CD200R has been the focus of a number of mechanistic and experimental
studies because activation of this microglia/monocyte by its ligand CD200 results in downregulation of
inflammatory activation signaling [119] . Activation of this receptor has significant neuroprotective effects.
We showed that there was significantly decreased expression of CD200R mRNA in AD brains [117] . Despite
having validated antibodies to CD200R, we could not detect CD200R protein in microglia in any brain
tissue sections examined [117] . A study employing tissue from multiple sclerosis cases also could not
demonstrate microglial staining of CD200R [120] . It appears then that freshly isolated microglia from human
brains have very low levels of CD200R expression [120,121] .

CD206 (macrophage mannose receptor C) is another widely used marker for alternative activation.
Increased levels of this receptor are associated with phagocytic activity. Preliminary studies of ND and AD
tissue sections with a validated antibody have failed to identify CD206-positive microglia. In our results,
we can detect strong staining of vascular macrophages and perivascular macrophages [Figure 5] but not in
microglia. These findings would suggest a deficit in levels of IL-4 in brain parenchyma.

However, the localization of another alternative activation marker, the phagocytic receptor CD163 has
been observed in microglia in AD and PD brains, and brains affected by human immunodeficiency virus
(HIV) [113,122,123] . CD163 is a high-affinity scavenger receptor for hemogloblin-haptoglobin and a low-affinity
receptor for hemoglobin alone. CD163 has been widely defined as a marker of macrophages rather than
microglia, particularly those that infiltrate brain tissue following stroke. It can be highly expressed by
macrophages but its role in neurodegeneration is unclear. Microglia strongly expressing CD163 were shown
to be plaque-associated, and CD163 immunoreactivity was present in CD68-positive microglia, suggesting
a phagocytic role, and also in TMEM119-positive microglia, a homeostatic role [122] . It should be mentioned
that the description of microglia based on morphology is unreliable and so, CD163 immunoreactive
microglia might be infiltrating macrophages.

SUMMARY
At present, it is unresolved from studies of human brain tissues whether “activated’ microglia, defined in