Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and a7nAChR-/- Peritoneal macrophage Transcriptomes with Nicotine and/or LPS treatment

Purpose: The cholinergic anti-inflammatory pathway (CAP) links the nervous and immune systems and modulates innate and adaptive immunity. The goals of this study are to identify the new downstream signaling of a7nAChR in macrophages. Methods: Peritoneal macrophages isolated from a7nAChR+/+ and a7nAChR-/- mice were treated with nicotine (10 µM) and/or LPS (100 ng/ml), then RNA-seq was performed. Results: Genes were selected that had more than 4-fold relative gene expression in nicotine-treated cells compared to the control group (vehicle-treated). The same calculation was applied to nicotine+LPS-treated cells and LPS-treated cells and 264 genes were identified as genes commonly induced by nicotine based on these two comparisons. Then relative gene expression was compared between a7nAChR+/+- and a7nAChR-/- -derived cells. 18 genes were finally selected whose expressions are suppressed (<1/2) in a7nAChR-/- -derived peritoneal macrophages. Conclusions: Our study represents the first detailed analysis focused on the new downstream signaling of a7nAChR in macrophages, generated by RNA-seq technology. We newly revealed the important anti-inflammatory role of Hes1 in the CAP using some functional experiments. Overall design: Peritoneal macrophage's mRNA profiles of wild type (WT) and a7nAChR-/- mice treated with Nicotine and/or LPS were generated by deep sequencing.

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Inoue T, Abe C, Kohro T, Tanaka S, Huang L, Yao J, Zheng S, Ye H, Inagi R, Stornetta RL, Rosin DL, Nangaku M, Wada Y, Okusa MD