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Davis et al. Neuroimmunol Neuroinflammation 2020;7:300-10 I http://dx.doi.org/10.20517/2347-8659.2020.19 Page 301

Conclusion: β-FNA differentially affects inflammatory cytokine/chemokine expression in human astrocytes and
microglia. These findings warrant further investigation into the novel anti-inflammatory actions of β-FNA, with
a particular focus on astrocytes. These insights should contribute to the development of strategies to treat brain
disorders that involve neuroinflammation.

Keywords: Neuroinflammation, brain, opioid, nuclear factor-kappaB, pro-inflammatory

INTRODUCTION
Neuroinflammation is present in brain infection, trauma, neurodegenerative diseases, and psychiatric
[1-5]
disorders as well . Glial cells, including astrocytes and microglia are instrumental in neuroinflammation.
The therapeutic effectiveness of certain antidepressant and antipsychotic drugs reportedly results, in part,
[6-9]
from anti-inflammatory actions . Astrocytes and microglia have integral roles in metabolic/neurotrophic
support, ion and neurotransmitter homeostasis, synaptic plasticity, and modulation of neuronal excitability;
and they respond to infection and cellular insults associated with neuropathology [10-13] . Astrocytes have
a fundamental role in neuropathogenesis, in part, through the release of neurotoxic/neuroinflammatory
factors including cytokines [i.e., interleukin (IL)-1β and IL-6] and chemokines [interferon-γ inducible
protein-10 (CXCL10) and monocyte chemoattractant protein-1 (CCL2)]. These secreted factors can be
directly neurotoxic or can induce further glial activation/dysregulation [14-17] . Microglia are resident CNS
macrophages and thus play an important role in neuroinflammation, in part, through phagocytosis of
cellular debris and release of inflammatory mediators including cytokines/chemokines. Importantly,
emerging evidence highlights the importance of astrocyte/microglia dysregulation in a wide range of CNS
disorders, thus implicating glial cells as potential therapeutic targets [18-21] .

We, and others, are therefore interested in identifying novel, anti-inflammatory agents that are
therapeutically effective in the treatment of neurological disorders. We are interested in the previously
identified, novel anti-inflammatory actions of beta-funaltrexamine (β-FNA). As a fumaramate methyl
ester derivative of naltrexone, β-FNA is most notably recognized as a selective, irreversible antagonist
at the mu-opioid receptor (MOR) [22,23] . In both behavioral and in vitro assays, β-FNA acts initially as a
reversible kappa-opioid receptor agonist, and then later results in MOR antagonism [24,25] . As an alkylating
[23]
agent, β-FNA irreversibly antagonizes MOR by covalently binding at Lys233 on the receptor . However,
we discovered that β-FNA also has novel anti-inflammatory actions which seem to be mediated through
MOR-independent actions [26-28] . For instance, neither naltrexone (a nonselective opioid receptor antagonist)
nor D-Phe-Cys-Tyr-D-Trp-D-Arg-Pen-Thr-NH2 (CTAP) inhibits pro-inflammatory-induced CXCL10
expression in human astroglial cells [27,28] . Additionally, we predicted that if the anti-inflammatory actions of
β-FNA were due to alkylation, this covalent modification should then remain after washout. Indeed, pre-
treatment of astroglial cells with β-FNA for 60 min, followed by drug washout prior to stimulating with
IL-1β (or tumor necrosis factor α), resulted in inhibition similar to 24 h co-exposure (cytokine stimulus
+ β-FNA). These findings suggested that β-FNA-induced modifications (i.e., alkylation) are persistent
and lead to the disruption of signal transduction. Notably, our in vitro findings also showed that β-FNA
reduces inflammatory signaling in astroglia, regardless of whether the stimulus is tumor necrosis factor α,
IL-1β or bacterial lipopolysaccharide (LPS). We also determined that β-FNA inhibits LPS-induced pro-
inflammatory cytokine expression in mouse brain (but not in plasma) . Furthermore, treatment with
[29]
[29]
β-FNA reduced LPS-induced sickness behavior in mice suggesting important translational implications .
The primary goal of the present study was to determine the effect of chronic β-FNA treatment on
inflammatory signaling in NHA. Additionally, we assessed for the first time the effects of β-FNA on
inflammatory signaling in human microglial cells.