Figure 8.

ACh reduces cytokine release from cultured resident macrophages. (A) GFP-expressing macrophages were isolated by flow cytometry from the colonic muscularis propria of Cx3cr1^GFP;CCR2^RFP mice. (B) Cx3cr1^GFP macrophages were cultured in vitro and formed colonies in the presence of CSF-1 (dashed box enlarged in inset). (C) Transcript levels of Tnf in the presence of LPS (100 ng/mL) were reduced significantly by the addition of ACh in a dose-dependent manner (n = 3 per group). Channelrhodopsin-expressing cholinergic neurons (ChAT^ChR2-tdT) and muscularis macrophages (Cx3cr1^tdT) were co-cultured in vitro. (D) After addition of LPS, IL1β can be seen expressed by tdT+/CD11b+ macrophages (white arrows), and not by tdT+/CD11b-negative cholinergic neurons (yellow arrows). (E) Quantification of IL1β immunofluorescence (MFI, mean fluorescence intensity) is shown in control conditions, in the presence of LPS, and with LPS after BLS to activate ChAT-expressing neurons (n = 8 per group). Scale bars: 200 μm (B), 50 μm (D). Data are shown as means ± SEM. ∗∗P < .01, ∗P < .05.