Revathikumar_2016_J.Neuroinflammation_13_256

BACKGROUND: The cholinergic anti-inflammatory pathway (CAP) primarily functions through acetylcholine (ACh)-alpha7 nicotinic acetylcholine receptor (alpha7nAChR) interaction on macrophages to control peripheral inflammation. Interestingly, ACh can also bind alpha7nAChRs on microglia resulting in neuroprotective effects. However, ACh effects on astrocytes remain elusive. Here, we investigated the effects of nicotine, an ACh receptor agonist, on the cytokine and cholinesterase production of immunocompetent human astrocytes stimulated with interleukin 1beta (IL-1beta) in vitro. In addition, the potential involvement of prostaglandins as mediators of nicotine was studied using cyclooxygenase 2 (COX-2) inhibition.
METHODS: Cultured human fetal astrocytes were stimulated with human recombinant IL-1beta and treated simultaneously with nicotine at different concentrations (1, 10, and 100 muM). Cell supernatants were collected for cytokine and cholinesterase profiling using ELISA and MesoScale multiplex assay. alpha7nAChR expression on activated human astrocytes was studied using immunofluorescence. For the COX-2 inhibition studies, enzyme activity was inhibited using NS-398. One-way ANOVA was used to perform statistical analyses.
RESULTS: Nicotine treatment dose dependently limits the production of critical proinflammatory cytokines such as IL-6 (60.5 +/- 3.3, %inhibition), IL-1beta (42.4 +/- 1.7, %inhibition), and TNF-alpha (68.9 +/- 7.7, %inhibition) by activated human astrocytes. Interestingly, it also inhibits IL-8 chemokine (31.4 +/- 8.5, %inhibition), IL-13 (34.243 +/- 4.9, %inhibition), and butyrylcholinesterase (20.8 +/- 2.8, %inhibition) production at 100 muM. Expression of alpha7nAChR was detected on the activated human astrocytes. Importantly, nicotine's inhibitory effect on IL-6 production was reversed with the specific COX-2 inhibitor NS-398.
CONCLUSIONS: Activation of the cholinergic system through alpha7nAChR agonists has been known to suppress inflammation both in the CNS and periphery. In the CNS, earlier experimental data shows that cholinergic activation through nicotine inhibits microglial activation and proinflammatory cytokine release. Here, we report similar anti-inflammatory effects of cholinergic activation on human astrocytes, at least partly mediated through the COX-2 pathway. These results confirm the potential for cholinergic neuroprotection, which is looked upon as a promising therapy for neuroinflammation as well as neurodegenerative diseases and stroke. Our data implicates an important role for the prostaglandin system in cholinergic regulatory effects.