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Page 12 of 15 Slattery et al. Neuroimmunol Neuroinflammation 2018;5:11 I http://dx.doi.org/10.20517/2347-8659.2018.05

DISCUSSION
Our data indicate that the inhibitory effect of p-aminophenol and AM404 on NO secretion by stimulated
BV-2 microglia is not caused by COX inhibition, or interaction with CB2 or TRPV1 receptors.
Furthermore, enzymatic conversion of p-aminophenol to AM404 by FAAH is not required for its
pharmacological activity in BV-2 cell cultures. Stimulation of microglia with LPS has been shown to induce
the expression of several genes that are regulated by mitogen-activated protein kinases (MAPKs) and NF-
κB, including such cytokines as TNF-α, interleukin (IL)-1b, interferon (IFN)-γ, and their receptors, as
well as the stress proteins superoxide dismutase (SOD) 2, COX-2, and thioredoxin interacting protein [62,63] .
Moreover, it has already been documented that stimulation of BV-2 murine microglia with LPS induces the
expression of iNOS in a MAPK- and NF-κB-dependent manner [64-66] . Previous studies have also determined
that AM404 prevents the activation of transcription factors NFAT and NF-κB but preserves extracellular
signal-regulated kinases (ERK)1/2 MAPK signaling in vivo [30,67] . Moreover, as NFAT is not directly activated
by LPS, the only signaling pathway in this study that is both inhibited by AM404 and activated in microglia
following stimulation with LPS is NF-κB [30,68] . AM404 has been shown to attenuate NF-κB activation by
inhibiting the phosphorylation, and subsequent degradation, of nuclear factor of kappa light polypeptide gene
[30]
enhancer in B-cells inhibitor, alpha (IκBα) . Intact IκBα sequesters NF-κB in the cytoplasm by interfering
[69]
with the function of the nuclear localization signal, thereby blocking NF-κB gene activation . Considering
that NF-κB activity is directly linked to the LPS-induced expression of iNOS and secretion of NO, it can be
concluded that AM404 most likely modulates the secretion of NO from LPS-stimulated microglia through
its inhibitory action on the NF-κB pathway [70,71] . This conclusion is consistent with previous studies where
AM404 was demonstrated to block the overexpression of iNOS in models of neuropathic pain, supporting
a mechanism of AM404 action where it inhibits NO secretion at the transcription level [23,72] .

DECLARATIONS
Authors’ contributions
Conceived the study and wrote the manuscript: Slattery WT, Klegeris A
Conducted experiments and analyzed the data: Slattery WT

Data source and availability
Data in this study were obtained by experimentation and are original. All primary data used to construct
the summary figures are available by contacting the authors of this study.

Financial support and sponsorship
This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada
(NSERC) and the Jack Brown and Family Alzheimer’s Disease Research Foundation.

Conflicts of interest
The authors declare that they have no conflicts of interest.

Patient consent
Not applicable.

Ethics approval
Not applicable.

Copyright
© The Author(s) 2018.