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Page 308 Davis et al. Neuroimmunol Neuroinflammation 2020;7:300-10 I http://dx.doi.org/10.20517/2347-8659.2020.19

findings on the acute effects of β-FNA suggest that that the anti-inflammatory actions are likely mediated,
at least in part, by disrupting the NF-κB signaling pathway [26-28,37] . In this study, we found that NF-κB
signaling is also inhibited by chronic exposure to a lower concentration of β-FNA. Interestingly, the overall
level of NF-κB p65 in the nucleus was not impacted by β-FNA; rather, phosphorylation of NF-κB p65 was
reduced in the presence of β-FNA. Together, these findings suggest that β-FNA acts at a common factor in
the signaling pathways activated by these diverse stimuli, in turn implicating the NF-κB signaling pathway.
We hypothesize that β-FNA exerts these anti-inflammatory effects via alkylation of one or more lysines
of the signaling proteins in the NF-κB pathway, and we are currently testing this hypothesis using in vitro
approaches. Further studies are warranted to determine the mechanism by which β-FNA inhibits the
phosphorylation of NF-κB p65.

We have also determined that β-FNA exerts anti-inflammatory actions in vivo [29,37] . For instance, LPS-
induced neuroinflammation and sickness behavior in mice were attenuated by peripherally administered
[29]
β-FNA . More specifically, β-FNA inhibited LPS-induced expression of both CXCL10 and CCL2 in the
brain, but had no effect on IL-6 levels. Furthermore, β-FNA did not impact plasma levels of CXCL10,
CCL2, or IL-6. These in vivo findings are largely in line with our in vitro findings in human astrocytes,
except that CCL2 expression in NHA was inhibited by β-FNA. Certainly, the differential sensitivity of
CCL2 to β-FNA could be related to species differences or model systems (in vitro vs. in vivo). However, it
may also reflect cell type-specific differences in sensitivity to β-FNA. For example, microglial cells (at least
IL-1β-stimulated human microglial cells) are not sensitive to the anti-inflammatory actions of β-FNA.
Because relatively high concentrations of β-FNA had no effect on chemokine/cytokine expression in C20
microglial cells and because of the observed cytotoxicity, we did not pursue the chronic effects of β-FNA
on these cells at this point. However, in future experiments, we expect to assess chronic exposure to lower
concentrations of β-FNA. Together, it is conceivable that the protective effects of β-FNA in vivo are largely
due to modulatory effects on astrocytes; however, further investigation is needed to clearly establish the cell
types affected and mechanisms involved.

In summary, we advanced our understanding of the anti-inflammatory effects of β-FNA by demonstrating
that chronic exposure inhibits NF-κB p65 activation and CXCL10 expression in astrocytes more effectively
than does acute treatment. We also found that expression of neither CCL2 nor IL-6 in astrocytes is affected
by chronic β-FNA. Lastly, we provided evidence of cell type-specific effects of β-FNA, as indicated by the
relative resistance of C20 human microglial cells to the anti-inflammatory effects of β-FNA. Further study
is warranted and expected to advance the therapeutic potential of β-FNA, or related compounds, in the
treatment of brain disorders that involve neuroinflammation.

DECLARATIONS
Acknowledgments
We greatly appreciate the statistical expertise and insights provided by J. Thomas Curtis, PhD.

Authors’ contributions
Concept, experimental design, literature review, statistical analysis, manuscript preparation: Davis RL
Performed experiments, data acquisition and analysis, and manuscript editing: McCracken K, Buck DJ

Availability of data and materials
Data can be made available upon valid request.

Financial support and sponsorship
This work was supported in part by Oklahoma Health Research Program (HR 14-007, HR 18-033);
Oklahoma State University Center for Health Sciences, Intramural funds (RLD).