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Page 8 of 11 Davis et al. Neuroimmunol Neuroinflammation 2018;5:50 I http://dx.doi.org/10.20517/2347-8659.2018.60

results. Whereas, when 30 µg of protein from unstimulated C20 cells were electrophoresed, we did not detect
TMEM119. Further investigation is warranted to fully appreciate the expression profile of TMEM119 at the
mRNA and protein levels; and the modulatory influence of proinflammatory mediators. Additional insights
into the expression profile and functional role of TMEM119 in microglia are therefore needed and C20 cells
may prove to be a useful tool in this line of investigation.

Previously, during the initial characterization of this cell line, C20 cells were found to secrete proinflammatory
[37]
cytokines following stimulation with TNFα . We have added to these findings and advanced our
understanding of inflammatory signaling in C20 cells. More specifically, we provide the first evidence that
IL-1β potently induces expression of CXCL10, CCL2, and IL-6 in C20 cells. We have also demonstrated that
LPS differentially affects expression of these inflammatory mediators as evidenced by stimulation of CCL2
expression, but not CXCL10 or IL-6. Overall, it is clear that IL-1β is a much more effective inducer of cytokine/
chemokine expression in C20 cells compared to LPS. In contrast, mouse microglia (including the BV-2 cells)
[36]
are very responsive to LPS, but not to IL-1β due to the absence of IL-1R expression .
IL-1β levels are elevated in a range of CNS disorders and this proinflammatory cytokine has been implicated
[40]
as a key mediator of neuropathology . Thus, it is fundamentally important to fully understand IL-1β-
induced inflammatory signaling in human microglia. Furthermore, advancing our understanding of these
signaling events in C20 human microglia is critical for developing this research tool. Secretion of CXCL10,
CCL2 and IL-6 by C20 microglia in response to IL-1β exposure is a functionally relevant endpoint measure
given both the important neurophysiological and neuropathological roles of these cytokines/chemokines. For
instance, CXCL10 is initially neuroprotective in viral infections , but can also contribute to neuropathology
[58]
as evidenced in human immunodeficiency virus (HIV)-induced dementia [59,60] . CXCL10 also plays a role
[61]
in neuropathology associated with traumatic brain injury (TBI) , and emerging data suggest CXCL10
[62]
is involved in sickness behavior . CCL2 functions as a chemotactic cytokine, activating and directing
[61]
migration of numerous cell types ; and it is increasingly evident that increased levels of this chemokine in
[61]
the brain contribute to neuropathology of HIV-dementia, AD, ischemia, epilepsy, and TBI . The cytokine
[63]
IL-6 acts in the hypothalamus as a regulator of metabolism and has gained attention for its involvement in
[64]
autism , major depression [65,66] , and neurodegenerative diseases [67-69] . Therefore, C20 microglia are expected
to be a useful tool in discovery of pharmacologic agents that may modulate microglial activation and
subsequent release of proinflammatory factors.
We also provide the first evidence that IL-1β induces activation of key proinflammatory signaling molecules
in C20 cells, including IκBα, NF-κB p65, and p38 MAPK. The importance of these signaling molecules in
microglia activation is well established and these proteins are viable targets for pharmacologic modulation
of inflammation [29,34,70-73] . Therefore, by demonstrating that these signaling pathways are functional in C20
cells, it is expected that these cells will be instrumental in the identification and characterization of novel
pharmacologic agents intended to alter microglial function.

In conclusion, we have determined that IL-1β-activated C20 microglia express the microglia specific marker
TMEM119. Additionally, we have provided the first evidence that IL-1β induces activation of IκBα, NF-κB
p65, and p38 MAPK and subsequent secretion of CXCL10, CCL2 and IL-6 in C20 human microglia. These
findings support the use of this human microglial cell line as a research tool to advance our understanding
of microglia function and for the development of pharmacotherapies targeting a range of neuropathologies.

DECLARATIONS
Acknowledgements
We greatly appreciate the technical assistance of Robert W. Allen, PhD and Jun Fu, PhD at the Human
Identity Testing Laboratory in the School of Forensic Sciences at Oklahoma State University Center for
Health Sciences.