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. 2014 Jan 29;9(1):e87785.
doi: 10.1371/journal.pone.0087785. eCollection 2014.

Neuro-anatomical evidence indicating indirect modulation of macrophages by vagal efferents in the intestine but not in the spleen

Cathy Cailotto  1 , Pedro J Gomez-Pinilla  2 , Léa M Costes  1 , Jan van der Vliet  1 , Martina Di Giovangiulio  2 , Andrea Némethova  2 , Gianluca Matteoli  2 , Guy E Boeckxstaens  2
Affiliations

Neuro-anatomical evidence indicating indirect modulation of macrophages by vagal efferents in the intestine but not in the spleen

Cathy Cailotto et al. PLoS One. .

Abstract

Background: Electrical stimulation of the vagus nerve suppresses intestinal inflammation and normalizes gut motility in a mouse model of postoperative ileus. The exact anatomical interaction between the vagus nerve and the intestinal immune system remains however a matter of debate. In the present study, we provide additional evidence on the direct and indirect vagal innervation of the spleen and analyzed the anatomical evidence for neuroimmune modulation of macrophages by vagal preganglionic and enteric postganglionic nerve fibers within the intestine.

Methods: Dextran conjugates were used to label vagal preganglionic (motor) fibers projecting to the small intestine and spleen. Moreover, identification of the neurochemical phenotype of the vagal efferent fibers and enteric neurons was performed by immunofluorescent labeling. F4/80 antibody was used to label resident macrophages.

Results: Our anterograde tracing experiments did not reveal dextran-labeled vagal fibers or terminals in the mesenteric ganglion or spleen. Vagal efferent fibers were confined within the myenteric plexus region of the small intestine and mainly endings around nNOS, VIP and ChAT positive enteric neurons. nNOS, VIP and ChAT positive fibers were found in close proximity of intestinal resident macrophages carrying α7 nicotinic receptors. Of note, VIP receptors were found on resident macrophages located in close proximity of VIP positive nerve fibers.

Conclusion: In the present study, we show that the vagus nerve does not directly interact with resident macrophages in the gut or spleen. Instead, the vagus nerve preferentially interacts with nNOS, VIP and ChAT enteric neurons located within the gut muscularis with nerve endings in close proximity of the resident macrophages.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Injection sites of the neuronal tracer and deep cervical lymph nodes.
Panel A shows the site of dextran amine injection at the level of the DMV, 19 days after injection. The tracer was revealed by DAB staining. B. Epifluorescent picture shows the distribution of Texas-red dextran amines after lateral ventricle injection. In the panels C & D, arrow heads show the presence of dextran amines (revealed by phosphatase alkaline staining to amplify the Texas red signal of the tracer) in the deep cervical lymph node. Of note, the presence of the tracer was found in all LV injected mice (D) and occasionally in DMV-injected mice (C). 3 V: third ventricle. LV: lateral ventricle. DMV: dorsal motor nucleus of the vagus. Scale bar represents 0.1 mm.
Figure 2
Figure 2. Distribution of dextran-labeled vagal fibers in the mesenteric ganglion and spleen coronal section.
No biotin dextran-labeled fibers or terminals were found on coronal section of mesenteric ganglion (A) or spleen (B). Of note, similar observations were obtained with Texas red dextran amine tracer. The brown spots on the spleen section were found in injected and non-injected mice, indicating of a strong endogenous biotin expression. WP: white pulp, RP: red pulp, T: trabeculae.
Figure 3
Figure 3. Sympathetic fibers and no vagal innervation of the spleen.
A. Tyrosine hydroxylase (TH) staining was used to reveal the sympathetic innervation of the spleen. The central arteriole showed high TH positive fibers (blue) that are in close proximity of the T cells (green). B. Texas red amine signal (red) was found occasionally in the B cells area (green). This dextran amine signal was found in mice that exhibit also dextran amine in deep cervical lymph node, indicating that the spleen signal is the result of tracer leakage into the cerebrospinal fluid. The scale bar represents 20 µm.
Figure 4
Figure 4. Vagus nerve efferent fibers and terminals reach myenteric plexus region in the intestine.
A.1 Vagal motor efferent fibers (red; arrow head) were labeled using fluorescence conjugated streptavidin on 5 µm thin coronal section of small bowel. Labeled vagus fibers were found between circular and longitudinal muscle layers at the level of the myenteric plexus. A.2. High power magnification field showing localization of vagal motor efferent fibers (red, arrow head) between nuclei (blue) of circular (CM) and longitudinal (LM) smooth muscle. A.3. The presence of intrinsic biotin was found in cells of the submucosal crypts. B. Labeled-fibers of the vagal nerve were revealed with ABC/DAB staining protocol while cuprolinic blue was used as pan-neuronal marker to visualize enteric neurons (blue). Labeled vagal efferent fibers and terminals with a basket-like shape terminals were found within myenteric ganglia (arrow head). C. Confocal image showing the presence of the dextran amine in the inter-ganglionic fibers (arrow). D. Epifluorescence image corresponding to vagal efferent fibers (green) densely found at the level of myenteric ganglion (PGP 9.5, red). The scale bar represents 50 µm.
Figure 5
Figure 5. Vagus nerve efferent is fully cholinergic in nature.
Epifluorescence images collected for the identification of the neurotransmitters (red) and dextran-labeled vagus efferent fibers and terminals (Green). Choline acetylfransferase (ChAT, A), neuronal nitric oxide synthase (nNOS, B), Tyrosine hydroxylase (TH, C), substance P (SP, D), vasoactive intestinal peptide (VIP, E) and Calcitonin gene related peptide (CGRP, F). Arrow heads point discrete co-localization between Vagus nerve and VIP or CGRP positive structures. Vagus nerve efferent fibers and terminals are only positive for ChAT and located in close proximity to ChAT and nNOS enteric neuronal bodies. Scale bars represent 20 µm.
Figure 6
Figure 6. Intestinal resident macrophages are located in proximity to enteric neurons.
A. Staining of F4/80 positive intestinal resident macrophages (brown) surrounding a myenteric ganglion (blue). Efferent vagus nerve fibers are shown in black. B.1 Regular distribution of resident macrophages (F4/80) in the muscularis of the murine small bowel. B.2 & C. Confocal image showing the distribution of the resident macrophage (F4/80, blue) close to enteric neurons (PGP9.5, red) in the muscle layers of the small intestine. D. Epifluorescence image showing the presence of resident macrophages (F4/80, blue) in close proximity to ChAT positive enteric ganglion (red). Scale bar represents 25 µm, except for B.1 it represents 0.1 mm.
Figure 7
Figure 7. Vagus nerve efferent fibers and terminals are close to cholinergic and nitrergic enteric neurons.
A. Epifluorescence image shows dextran-labeled vagal efferents (green) that co-localize with ChaT (yellow), and are in close contact with ChaT positive enteric neurons (red). B. Epifluorescence image shows labeled vagal efferent fibers (green) making contact with nNOS positive neurons (red). Of note, cholinergic neurons, and to lesser extent nitrergic neurons, are the main population targeted by the vagal efferent fibers. C. Confocal image of VIP (red) and nNOS (green) myenteric neurons. Most of the cells bodies exhibit co-localization of these two neurotransmitters (arrow head). D. Confocal image of VIP (red) and ChaT (green) myenteric neurons. Occasionally myenteric neurons showed immunoreactivity for both neurotransmitters (arrow head). C1 and C2 show the distribution for the nNOS and VIP positive cells bodies, respectively. D1 and D2 show the distribution for ChaT and VIP positive cells bodies. Scale bar represents 20 µm.
Figure 8
Figure 8. Neuronal fibers and intestinal resident macrophages: neurotransmitter and receptor expression.
Epifluorescence images show F4/80 positive intestinal resident macrophages (blue) located in close proximity to inter-ganglionic enteric fibers positive for ChAT (A), nNOS (B) and VIP (C). D. Intestinal resident macrophages (F4/80, red, D.1) expressing α7 nicotinic receptor (green, arrow heads, D.2). E. Resident macrophages of the gut muscularis (F4/80, red) express VPAC1 receptors (green). Asterisk corresponds to intestinal resident macrophages located at the level of the myenteric plexus region. The other macrophage is located at the level of the submucosal plexus. Scale bar represents 10 µm from A–C and 20 µm from D–E.
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Grants and funding

This work was supported by grants from the Research Foundation - Flanders (FWO) (Odysseus program to G.E.B.), by a FWO postdoctoral research fellowship (to G.M. and P.J.G.), and by FWO Ph.D. fellowship (to M.D.G.). This work was partly supported by grant from the European Union 7th Framework Program (to A.N.), by the Netherlands Organization for Scientific Research (NWO Vici grant 918-76 623), and by the Maag Lever Darm Stichting (W09-30). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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