Similar to in vitro data in

Similar to in vitro data, in vivo studies have also revealed modulation of TLR4-induced inflammatory responses by AEA. The proposed AEA reuptake inhibitor AM404 has been shown to attenuate TLR4-induced increases in plasma levels of IL-6 and IL-1β, the latter effect mediated by CB1 receptor activation (Roche et al., 2008). Furthermore, AM404 or enhancing AEA tone via pharmacological inhibition of FAAH, augmented TLR4-induced increases in plasma TNFα levels, an effect at least partially mediated via activation of PPARγ (Roche et al., 2008). In the brain, AEA activation of hypothalamic CB1 receptors has been shown to facilitate (De Laurentiis et al., 2010), while antagonism of the central CB1 receptors attenuates (Steiner et al., 2011), TLR4-induced increases in plasma TNFα levels. In addition, work from our laboratory has demonstrated that enhancing AEA levels following FAAH inhibition was associated with attenuation of TLR4-induced increases in IL-1β, and increases in expression of suppressor of cytokine signalling (SOCS3), in the hypothalamus (Kerr et al., 2012). It should be noted that in addition to AEA, related fatty APO866 amides, N-oleoylethanolamide (OEA) and N-palmitoylethanolamide (PEA), are also metabolised by (FAAH) and shown to be increased following FAAH inhibition. These N-acylethanolamines have been shown to exert potent biological effects on satiety, pain and inflammation (Esposito and Cuzzocrea, 2013, Mattace Raso et al., 2014, Sayd et al., 2015, Skaper et al., 2015, Suardiaz et al., 2007, Thabuis et al., 2008) and so it cannot be ruled out that some of the effects of FAAH inhibition may be due in part to activity of OEA or PEA, alone or in combination, with AEA. In addition to AEA, OEA has activity at the TRPV1 (Ahern, 2003, Almasi et al., 2008, Gonzalez-Aparicio and Moratalla, 2014, Movahed et al., 2005, Starowicz et al., 2013, Wang et al., 2005) and increasing evidence supports an important physiological role for TRPV1 in the brain (Edwards, 2014, Madasu et al., 2015, Martins et al., 2014). Furthermore, FAAH inhibition can lead to indirect activation/desensitization of TRPV1 and subsequent analgesic effects, anti-inflammatory effects and central effects on mood (Maione et al., 2007, Rubino et al., 2008, Starowicz et al., 2013). Taken together, data indicate that enhancing AEA (and related fatty acid amides) in vivo can modulate neuroinflammatory and behavioural responses. However, it remains to be determined if immunomodulatory effects occur due to indirect modulation of peripheral TLR4-induced immune responses, or directly at the level of the brain. Recent work from our group has demonstrated an important role for central FAAH substrates in attenuating the neuroinflammatory response to TLR3 activation (Henry et al., 2014). However, it is unknown if a similar effect is observed following activation of TLR4, or the receptor and molecular mechanisms involved. Furthermore, it remains to be determined if modulation of TLR4-induced neuroinflammatory responses results in concomitant alterations in behaviour. As such the aim of the current study was to investigate if enhancing FAAH substrate, tone in the brain directly modulates TLR4-induced neuroinflammatory responses, sickness behaviour and anhedonia, and examine the potential receptor and molecular mechanism(s) mediating these effects.
Materials and methods
Results
Discussion The data presented herein have demonstrated that the inhibition of FAAH and subsequent increases in levels of AEA and related N-acylethanolamines in the brain, is associated with modulation of neuroinflammatory responses following TLR4 activation. Furthermore, the data demonstrate that the effect of FAAH inhibition on neuroinflammation is mediated directly within the brain and does not involve CB1 or CB2 receptor activation. Rather, a role for central TRPV1 receptors in mediating, at least partially (IL-6), the effects of increased FAAH substrate levels on TLR4-induced neuroinflammation was observed. Furthermore, despite the pronounced effects of increasing FAAH substrate levels on TLR4-induced neuroinflammation, this was not associated with a change in sickness behaviour but rather tended to attenuate anhedonic-like behaviour. Overall these findings demonstrate an important role for FAAH substrates within the brain in the modulation of TLR4-induced inflammatory responses which may have implications in the treatment of neuroinflammatory disorders.