Nicotine Attenuates Activation of Tissue Resident Macrophages in the Mouse Stomach through the β2 Nicotinic Acetylcholine Receptor

<div><p>Background</p><p>The cholinergic anti-inflammatory pathway is an endogenous mechanism by which the autonomic nervous system attenuates macrophage activation via nicotinic acetylcholine receptors (nAChR). This concept has however not been demonstrated at a cellular level in intact tissue. To this end, we have studied the effect of nicotine on the activation of resident macrophages in a mouse stomach preparation by means of calcium imaging.</p><p>Methods</p><p>Calcium transients ([Ca²⁺]_i) in resident macrophages were recorded in a mouse stomach preparation containing myenteric plexus and muscle layers by Fluo-4. Activation of macrophages was achieved by focal puff administration of ATP. The effects of nicotine on activation of macrophages were evaluated and the nAChR involved was pharmacologically characterized. The proximity of cholinergic nerves to macrophages was quantified by confocal microscopy. Expression of β2 and α7 nAChR was evaluated by β2 immunohistochemistry and fluorophore-tagged α-bungarotoxin.</p><p>Results</p><p>In 83% of macrophages cholinergic varicose nerve fibers were detected at distances <900nm. The ATP induced [Ca²⁺]_i increase was significantly inhibited in 65% or 55% of macrophages by 100µM or 10µM nicotine, respectively. This inhibitory effect was reversed by the β2 nAChR preferring antagonist dihydro-β-eryhtroidine but not by hexamethonium (non-selective nAChR-antagonist), mecamylamine (α3β4 nAChR-preferring antagonist), α-bungarotoxin or methyllycaconitine (both α7 nAChR-preferring antagonist). Macrophages in the stomach express β2 but not α7 nAChR at protein level, while those in the intestine express both receptor subunits.</p><p>Conclusion</p><p>This study is the first <i>in</i> <i>situ</i> demonstration of an inhibition of macrophage activation by nicotine suggesting functional signaling between cholinergic neurons and macrophages in the stomach. The data suggest that the β2 subunit of the nAChR is critically involved in the nicotine-induced inhibition of these resident macrophages.</p></div

Confocal analysis of the proximity of macrophages to cholinergic varicosities in mouse stomach myenteric plexus-circular muscle layer region.

<p>F4/80 positive macrophages are labeled in red, VAChT positive acetylcholine-releasing sites are labeled in green. <i>A</i>, Two-dimensional maximal intensity projection of 6 confocal images (1.1 μm each, interval of 0.5 μm) showing close apposition of macrophages and cholinergic varicosities. <i>B</i> and <i>C</i>, Sections #-2 (B) and #-6 (C) show representative images of a confocal stack taken to evaluate proximity of macrophages to VAChT-positive nerve fibers. The two sections display two macrophages, each at less than 0.9 μm from VAChT-positive nerve fibers (marked by arrows). </p

Labeling of β2 and α7 nAChR subunits in tissue resident macrophages in the stomach and ileum.

<p>Tissue resident macrophages were immunohistochemically labeled for F4/80 (red). A; Resident macrophages in stomach muscularis of a wild-type mouse were β2 nAChR-immunoreactive (green). B; Lack of β2 nAChR-immunoreactivity (green) in resident macrophages of β2 nAChR knock-out mouse confirmed the specificity of the anti-mouse β2 nAChR antibody. C; Vital labeling with Cy5-conjugated α-bungarotoxin (ABGT; false color coded in green) revealed no α7 nAChR expression in resident macrophages in mouse stomach muscularis. D; In the mouse ileum ABGT labeled (false color coded in green) muscularis macrophages. This staining was used as a positive control for the α-bungarotoxin vital labeling protocol.</p

Pharmacology of the inhibitory effect of nicotine on ATP-evoked macrophage activation.

<p><i>A</i>, In presence of hexamethonium (Hexa), mecamylamine (Meca), different concentrations of α-bungarotoxin (ABGT) or different concentrations of methyllycaconitine (MLA), 100 µM nicotine still had a significant inhibitory effect on ATP-evoked [Ca²⁺]_i signals in macrophages (Wilcoxon Signed Rank test, * <i>P</i> < 0.001). <i>B</i>, In contrast, DHBE reversed the inhibitory effect of nicotine (Wilcoxon Signed Rank test, <i>P</i> = 0.156). <i>C</i>, ABGT did not reverse the inhibitory effect of 10 µM nicotine on ATP-evoked macrophage activation (Wilcoxon Signed Rank test, * <i>P</i> < 0.001). Please note that the scatter plots illustrating the inhibitory effect of 10 µM nicotine on ATP responses are identical to those in Figure 2 D. We show them here again as we used the same preparations, but different regions, to test the effect of 100 nM ABGT.</p> <p>For all panels: ATP_ctr labels the maximum [Ca²⁺]_i amplitude to a control ATP administration. ATP_nic labels the maximum [Ca²⁺]_i amplitude to ATP application immediately after a 10 sec nicotine administration 10 minutes after the control ATP application. Numbers in parenthesis indicate number of macrophages / number of preparations (equal to number of animals).</p

Vagal nerve stimulation reduces colonic and systemic inflammation.

<p>(A-B) Bar graphs represent Th1 (A) and Th2 (B) gene expression in colonic tissue isolated from sham- and VNS-treated mice challenged with oxazolone 6 hours after colitis induction. (C) Bar graphs represent serum levels of IL6, KC and TNFα in sham- and VNS-treated mice challenged with oxazolone 6 hours after colitis induction as determined by the Mann-Whitney test. Data are expressed as mean ± SEM; *p < 0.05; ns, not significant; (n = 21–27 mice per group).</p

Oxazolone exposure leads to early induction of systemic cytokines.

<p>(A-C) Bar graphs represent cytokines in serum and isolated splenocytes of oxazolone-treated mice in both early (6h) and late (24h) phases of the disease. Data are expressed as mean ± SEM as determined by the repeated-measures two-way ANOVA test. ns, not significant; *p < 0.05; (n = 4 mice per group). (D) TNFα and IL6 immunofluorescence on spleen and colon section of naïve and oxazolone-treated mice 6 hours post colitis induction. Scale bars are 40 μm.</p

Characterization of oxazolone-induced colitis model.

<p>(A) Wild type BALB/c mice were sensitized by epicutaneous application of 3% oxazolone at a dilution of 4:1 in a mixture of acetone and olive oil (100<b>μ</b>l) on day 0, followed by intracolonic administration of 1% oxazolone dissolved in 50% ethanol (100μl) (or 50% ethanol for control group EtOH-treated mice) on day 7 using an intravenous catheter inserted 3 cm in the colon. Mice were sacrificed 6, 24 hours and 5 days post oxazolone administration and tissue samples were collected for different analysis. (B) Survival proportion was assessed daily. Mortality is expressed as survival rate and shown by Kaplan-Meier survival curves; statistical significance of Kaplan-Meier survival curves was determined with Gehan-Breslow-Wilcoxon test. *p < 0.05; (n = 12–18 mice per group). (C) Repeated measurements of body temperature of EtOH- and oxazolone-treated mice were taken every 30 minutes and followed until 6 hours after treatment with oxazolone. Body temperature is shown as mean ± SEM, as determined by the repeated-measures two-way ANOVA test. ****p < 0.001; (n = 8 mice per group). (D) Bar graphs represent serum levels of HMGB1 in naïve, EtOH- and oxazolone-treated mice 6h post colitis induction as determined by the Mann-Whitney test. Ns, not significant; (n = 3–4 mice per group). (E) Paraffin-embedded colon sections were stained with hematoxylin and eosin (H&E) assessment of tissue alteration. Representative images of colonic sections stained with H&E from control naïve, EtOH- and oxazolone-treated mice at 6 and 24 hours after colitis induction. Scale bars are 40 μm in the upper panel and 100 μm in the lower panel. (F) Th1, chemokines and Th2 (G) gene expression was quantified in colonic tissue isolated from EtOH- and oxazolone-treated mice. Data are expressed as mean ± SEM of fold increase versus naïve mice as determined by the repeated-measures two-way ANOVA test. Ns, not significant; *p < 0.05; (n = 3–6 mice per group).</p

Histological assessment of colitis parameters (HAC).

<p>Histological assessment of colitis parameters (HAC).</p

Immune profile of oxazolone-induced colitis.

<p>(A) Representative expression of DAPI and CD11b (left column), Ly6G and CD11b (mid-left column), SiglecF and CD11b (mid-right column) and Ly6C and MHCII (right column) from the colon of naïve, EtOH- and oxazolone-treated mice 6 hours post colitis induction. Frequencies (B) and absolute numbers (C) of Ly6G⁺CD11b⁺, Ly6C⁺MHCII^- and Ly6C^-MHCII⁺ and SiglecF⁺CD11b⁺ cells among CD11b⁺, SiglecF^-CD11b⁺, Ly6G^-CD11b⁺ cells from colon of naïve, EtOH- and oxazolone-treated mice 6 hours post colitis induction. Data are expressed as mean ± SEM as determined by the Mann-Whitney test. ns, not significant; *p < 0.05; (n = 3–6 mice per group).</p

Induction of Th2 and NK T cells is specific to oxazolone-induced colitis treatment.

<p>(A) Representative expression of DAPI^-CD45⁺ cells (left column), CD49b⁺ and FceRIa⁺ cells (mid-left column), CD3⁺CD45⁺ cells (mid-right column) and Ly49c⁺CD3⁺ cells (right column), and DAPI^-CD45⁺ cells (left column) and CD4⁺IL5⁺ cells (right column) from the colon of naïve, EtOH- and oxazolone-treated mice 6 hours post colitis induction. Frequencies (B) and absolute numbers (C) of CD49b⁺ FceRIa⁺, CD3⁺CD49c⁺, CD4⁺IL5⁺ cells from colon of naïve, EtOH- and oxazolone-treated mice 6 hours post colitis induction. Data are expressed as mean ± SEM as determined by the Mann-Whitney test. Ns, not significant; *p < 0.05; **p < 0.005; (n = 3–6 mice per group).</p