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Benusa et al. Neuroimmunol Neuroinflammation 2020;7:248-63 I http://dx.doi.org/10.20517/2347-8659.2020.03 Page 249

INTRODUCTION
Microglia, the resident immune cells of the central nervous system (CNS), were first characterized
[1]
100 years ago by Pio del Rio Hortega (reviewed in ) but our understanding of their function remains
incomplete. The best-known function of microglia is CNS surveillance whereby cell debris are scavenged
during periods of pathology to maintain and re-establish a healthy homeostatic environment. However,
this limited view of microglia function has evolved to include a list of other potential functions designed
to establish, maintain, and when necessary, re-establish CNS homeostasis following both pathologic events
and in the developing and mature healthy brain. This new appreciation for the plethora of microglial
functions in both health and disease has resulted in a renewed interest in these enigmatic and mercurial
cells.

At present, it is unclear how microglia are capable of mediating a wide range of activities that, in some
cases, are seemingly in contrast to each other. For example, during development, microglia regulate
neuronal numbers by both driving cell death [2-7] and promoting proliferation and survival [8-11] . This
[12]
dichotomy of neurogenesis regulation is not limited to the developing brain since microglia both enhance
[13]
and deplete the number of neural progenitor cells in the adult brain. Similarly, microglia regulate synapse
numbers by both stripping/pruning [14-18] and stabilizing [19,20] dendritic spines and inhibitory synapses
both in development and adulthood by potentially distinct mechanisms [21-24] . Furthermore, microglial
regulation of cell populations is not limited to neurons as similar observations have also been reported for
oligodendrocytes and astrocytes. Additionally, under pathologic conditions in the adult brain, microglia
influence astrocytic phenotypes by ranging from neuroprotective to neurotoxic [25,26] and have been
implicated in angiogenesis including regulation of the structure and function of the neurovasculature [27,28] .
Taken together, it is becoming apparent that microglia oversee a vast array of events in the developing,
healthy and diseased CNS although how such a single cell type can manage such a multitude of functions
remains to be determined. Strong evidence is now emerging that microglia present as distinct subclasses
but it remains to be determined if these subclasses represent intrinsically distinct cell populations, or if
intrinsically similar cells are driven into functional heterogeneity dictated by changes in environmental
[29]
cues provided by a highly dynamic CNS .
In addition to providing a brief review of several parameters and subclasses that define microglial
heterogeneity, we also present novel RNA expression profile data that are consistent with the development
of distinct microglial phenotypes as a consequence of distinct inflammatory environments. As presented
in more detail below, we isolated cortical microglia from mice in three commonly used models to study
various aspects of multiple sclerosis - cuprizone, lipopolysaccharide (LPS) and experimental autoimmune
encephalomyelitis (EAE). Orally administered cuprizone results in CNS demyelination secondary to
oligodendrocyte death. Intraperitoneal injection of the endotoxin LPS mediates a peripheral immune
response that results in widespread CNS neuroinflammation. Similarly, EAE is induced by a peripheral
injection of a bacterial exotoxin that is accompanied by Complete Freund’s Adjuvant and a myelin antigen
resulting in breakdown of the blood brain barrier. Although microglia from all three models presented pro-
inflammatory profiles, the microglia from each expressed a unique set of factors suggesting environmental-
specific responses. Although these observations are consistent with environmental cues driving
heterogeneity, it remains possible, and perhaps likely, that microglia also represent intrinsically distinct
populations.

Microglial heterogeneity
Currently, a prevailing thought is that microglia, which derive from the embryonic yolk sac, develop
initially as a single-cell type lineage and subsequently, into a heterogeneous population in the adult brain
[30]
as a result of local environmental cues that define their differentiation and functional specificity [31-35] . For
example, in the injured adult brain, neurons can express or secrete “find me” signals such as fractalkine/