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Page 6 of 11 Davis et al. Neuroimmunol Neuroinflammation 2018;5:50 I http://dx.doi.org/10.20517/2347-8659.2018.60

8,000 600,000 400,000 b
7,000 b c 350,000
500,000 300,000
(pg/mg protein) 6,000 400,000 IL-6 (pg/mg protein) 250,000
5,000
CXCL10 3,000 CCL2 (pg/mg protein) 200,000 150,000
4,000
200,000
300,000
2,000
50,000
1,000 100,000 100,000
300 a a 20,000 a b 1,000 a a
0 0 0
Unstim. LPS IL-1β Unstim. LPS IL-1β Unstim. LPS IL-1β

Figure 2. Cytokine/chemokine expression in C20 human microglial cells. C20 cells were exposed to media alone (unstimulated; Unstim.)
or media containing either lipopolysaccharide (LPS) (1 µg/mL) or interleukin-1β (IL-1β) (20 ng/mL) for 24 h. CXCL10, CCL2, and IL-6
levels in the culture medium were determined by ELISA and normalized to total cellular protein (as determined by the bicinchoninic acid
method). Data represent mean ± SEM (n = 4-7). Bars with different letters are significantly different (P < 0.01) as determined by one-way
ANOVA and Tukey’s pairwise comparisons

Unstim. IL-1β Unstim. IL-1β
5 1.0
IL-1β b
US a
p-IκBα 40 kDa 0.6 a
3
IκBα 39 kDa p-IκBα/IκBα (relative density) 4 p-IκBα/β-tubulin (relative density) 0.8
β-tubulin 55 kDa 2 b 0.4
10 30 60 b 0.2
Minutes 1 b
b
a 0.0
0 10 30 60
10 30 60
Minutes Minutes
Figure 3. Interleukin-1β (IL-1β)-induced inhibitory kappa B alpha (IκBα) activation in C20 human microglial cells. C20 cells were exposed
to media alone (unstimulated; US) or media containing IL-1β (20 ng/mL) for 10-60 min. Western blot analysis was used to measure
levels of p-IκBα, IκBα, and β-tubulin in cytoplasmic protein extracts. The blots presented are representative of independent experiments
(n = 4-5) and the data represent mean ± SEM. Bars with different letters are significantly different (P < 0.05) as determined by one-way
ANOVA and Tukey’s pairwise comparisons

The expression of phospho-p65 in the nucleus rapidly increased within 10 min in response to IL-1β treatment
(P < 0.0001), before declining to baseline levels by 30 min [Figure 4]. Constitutive expression of p65 in the
nucleus of C20 cells was evident and levels remained unchanged (P = 0.37) throughout the 60 min exposure
to IL-1β.

Phosphorylation of p38 in the cytoplasm occurred rapidly following treatment with IL-1β as indicated by a
significant (P < 0.0001) increase in phospho-p38/p38 after 10 min [Figure 5]. The levels of phospho-p38 then
rapidly declined by 30 min (P < 0.05), and returned to baseline by 60 min (P = 0.19). Constitutive expression
of p38 in the cytoplasm of C20 cells was robust and the expression levels remained constant (P = 0.58)
throughout the 60 min exposure to IL-1β.

DISCUSSION
Microglia are a key component of the innate immune system with critical roles in response to injury and
[1]
infection . Furthermore, microglia are instrumental in neurodevelopment and physiological functions
[52]
necessary for maintaining CNS homeostasis . Microglia have been implicated in a multitude of CNS
[8]
disorders, and modulation of microglia function has emerged as a potential therapeutic strategy .