BackgroundThe cholinergic anti-inflammatory pathway (CAP) primarily functions through acetylcholine (ACh)-alpha7 nicotinic acetylcholine receptor (α7nAChR) interaction on macrophages to control peripheral inflammation. Interestingly, ACh can also bind α7nAChRs 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 1β (IL-1β) in vitro. In addition, the potential involvement of prostaglandins as mediators of nicotine was studied using cyclooxygenase 2 (COX-2) inhibition.MethodsCultured human fetal astrocytes were stimulated with human recombinant IL-1β and treated simultaneously with nicotine at different concentrations (1, 10, and 100 μM). Cell supernatants were collected for cytokine and cholinesterase profiling using ELISA and MesoScale multiplex assay. α7nAChR 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.ResultsNicotine treatment dose dependently limits the production of critical proinflammatory cytokines such as IL-6 (60.5 ± 3.3, %inhibition), IL-1β (42.4 ± 1.7, %inhibition), and TNF-α (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 μM. Expression of α7nAChR 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.ConclusionsActivation of the cholinergic system through α7nAChR 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.

The apolipoprotein E (APOE) gene remains the most strongly established risk factor for late onset Alzheimer's disease (LOAD). Recently the gene, TOMM40, which is in linkage disequilibrium with APOE, was identified to be associated with LOAD in genome-wide association studies. One of the identified polymorphisms in TOMM40 is rs10524523, which is located in intron 6 and composed of thymidine repeats varying between 14 to 36 base-pairs in length. Reported results are contradictory in regard to the very long poly-T variant that has been associated with both increased and decreased risk of LOAD. Our study aimed to elucidate the functional implication of rs10524523 in an in vitro model of human fibroblast cells obtained from cognitively healthy APOE ε3/ε4 carriers harboring very long or short poly-T variants coupled to their APOE ε3 allele. We have studied (i) expression levels of TOM40 protein and mRNA, (ii) TOM40 mRNA splicing, and (iii) mitochondrial function and morphology; and we have found no significant differences in regards to very long or short poly-T variant.

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