Capsaicin-sensitive vagal afferent neurons contribute to the detection of pathogenic bacterial colonization in the gut
Introduction
Gastrointestinal inflammation can arise from different factors including genetics, diet, and gastrointestinal infection, and is a major financial burden in industrialized countries (Danese et al., 2004, Hulisz, 2004). While the initiating causes may be diverse, they all eventually involve a dysregulation of the balance between pro- and anti-inflammatory cytokines that are essential for maintaining the immunological function of the gut (Maloy and Powrie, 2011). This dysregulation facilitates the initiation and propagation of pro-inflammatory pathophysiology that ultimately results in disease symptoms (Artis, 2008, Hooper and Macpherson, 2010).
The gastrointestinal tract and its mucosal lining provide an essential barrier from nearly continuous exposure to foreign pathogens. To maintain this protection, multiple systems have evolved to coordinate the release of pro- and anti-inflammatory cytokines which mediate the initiation and control of immune responses. Recently the significance of the parasympathetic nervous system, and specifically the vagus nerve, in regulating inflammatory responses in the intestinal tract has been demonstrated (Borovikova et al., 2000, Tracey, 2007, Van Der Zanden et al., 2009); leading to the concept of a vagally dependent cholinergic anti-inflammatory pathway (Tracey, 2002). This cholinergic anti-inflammatory pathway has been proposed to be part of a vago-vagal immune reflex in which signals arising from the gut activate vagal afferents, which in turn activate the descending cholinergic anti-inflammatory pathway (Tracey, 2009). The majority of work studying this reflex has concentrated on the cholinergic efferent branch. Much less is known regarding the afferent branch, and more specifically the mechanisms through which vagal afferents are activated in response to a localized immune challenge. The primary goal of the experiments presented in this communication is to delineate the role(s) of vagal afferent neurons in detecting gastrointestinal infection.
Numerous studies have used direct recordings from vagal afferent neurons or hindbrain c-Fos expression to demonstrate that vagal afferent neurons can be activated by direct peripheral injections of lipopolysaccharides (LPS) (Liu et al., 2007), the pro-inflammatory cytokines interleukin-1β (IL-1β) (Niijima, 1996, Ek et al., 1998) or tumor necrosis factor-α (TNFα) (Rogers et al., 2006). While the use of injections of purified pro-inflammatory agents may produce a reliable response, there are limits to this approach that may lead to effects not observed in the normal sequela of events that follow intestinal colonization by pathogenic bacteria. For example, the purified component may have access to sites other than those accessible in a normal host/pathogen response, the concentration of the exogenously applied component may exceed that in a normal host/pathogen response, and the normal host/pathogen response might include factors other than the purified components. Prior work by Goehler and colleagues employed inoculation with Campylobacter jejuni (Gaykema et al., 2004, Goehler et al., 2005) to initiate a gastrointestinal immune challenge. These studies demonstrated that oral inoculation with C. jejuni induced c-Fos in the nTS within hours of inoculation, consistent with a neural (vagally) mediated detection of pro-inflammatory agents. They further demonstrated neuronal activation in more rostral brain regions, such as the hypothalamic paraventricular nucleus and central amygdala, without any detectable increase in circulating cytokines (IL-1β, IL-6, TNFα). This suggests that vagal afferent detection is sufficient to activate higher-level brain structures that are involved in generating systemic immune responses (Konsman et al., 1999). However, in these studies the neuronal activation in the nTS is assumed to be due to direct activation of vagal afferents, but this was not definitively tested through lesions of the vagus nerve.
In a similar study Wang et al. (2002) inoculated rats with Salmonella typhimurium. Oral inoculation of rats with S. typhimurium resulted in increased hypothalamic c-Fos expression, which was attenuated by subdiaphragmatic vagotomy (Wang et al., 2002). This study indicates that immune information from the gut to the brain is partially transmitted by an intact vagus nerve. While this study strongly supports the role of vagal afferents in immune monitoring of the gut, key characterization of the neural circuitry was not described. Specifically, no results showing markers of neuronal activation in the hindbrain or in the nTS were presented. Thus, whether the attenuation of S. typhimurium-induced hypothalamic activation following sub-diaphragmatic vagotomy was a result of decreased vagal signaling to the nTS or from decreased circulating cytokine levels was not clear.
In this study we used systemic injections of capsaicin to destroy afferent neurons that express the transient receptor potential type V1 ion channel (TRPV1). Capsaicin is an agent that causes activation of TRPV1 (Caterina et al., 1997) and high concentrations of capsaicin can induce neuronal death due to excessive stimulation of ion influx. Expression of TRPV1 is commonly interpreted to be a marker for C-type sensory neurons that have unmyelinated axons (Holzer, 1991). Approximately 70% of the afferent vagal fibers express TRPV1 (Li and Schild, 2007). Capsaicin lesions have previously been shown to cause nearly complete elimination of intraganglionic laminar endings in the upper and lower small intestines, cecum, and colon, while leaving a significant proportion (60–90%) of the intraganglionic laminar endings as well as intramuscular arrays in the stomach and esophagus intact (Berthoud et al., 1997). Since the primary site for C. jejuni colonization is the cecum (Jesudason et al., 1989) and the primary site for S. typhimurium is the large intestine proximal to the cecum (Nevola et al., 1985), we hypothesized that neuronal activation in the nTS caused by intestinal colonization by these pathogenic bacteria would be blocked by capsaicin lesions of vagal afferent neurons. We found that capsaicin lesions did indeed prevent activation of neurons in the nTS following inoculations with C. jejuni and S. typhimurium, and further found that almost all vagal afferent neurons that are acutely responsive to TNFα or LPS are also capsaicin sensitive. These results demonstrate that capsaicin-sensitive vagal afferent neurons are a critical component in signaling the presence of pathogenic bacteria in the intestines to the brain.
Section snippets
Animals
Male BALB/c and CF-1 mice (4–8 weeks old) were purchased from Harlan Laboratories (Indianapolis, IN) and used as subjects for the bacterial inoculation experiments. All animals were housed individually in Association for Assessment and Accreditation of Laboratory Animal Care (AALAC)-accredited quarters under a 12:12 hour light:dark cycle with lights on at 7:00 am. Animals had ad libitum access to pelleted chow (Purina #5001) and water except when indicated.
Adult male Sprague-Dawley rats (200–240
Optimization of intestinal colonization by C. jejuni
We found in initial experiments that 14-week-old BALB/c mice treated with C. jejuni strain 11168 did not produce a reliable c-Fos signal in the nTS (data not shown). Thus we investigated various parameters to optimize the inoculations. We began to treat our mice with streptomycin in their drinking water prior to the inoculation to reduce competition from endogenous flora. Further, we also investigated the effectiveness of various strains of C. jejuni and different strains and ages of mice since
Discussion
In this study we found that oral inoculation of mice with either C. jejuni or S. typhimurium causes activation of neurons in the nTS via a capsaicin sensitive pathway. In comparison to a physical vagotomy, the use of capsaicin to induce a lesion not only confines the loss of afferent neurons to a specific subtype of afferent neuron, it also preserves vagal efferent neurons. This avoids complications in the interpretation of the effects of the lesion that might be caused by a loss of efferent
References (58)
- et al.
Capsaicin-resistant vagal afferent fibers in the rat gastrointestinal tract: anatomical identification and functional integrity
Brain Res.
(1997) - et al.
Lipopolysaccharide and interleukin-1 depress food-motivated behavior in mice by a vagal-mediated mechanism
Brain Behav. Immun.
(1995) - et al.
Inflammatory bowel disease: the role of environmental factors
Autoimmun. Rev.
(2004) - et al.
Tumor necrosis factor alpha increases cytosolic calcium responses to AMPA and KCl in primary cultures of rat hippocampal neurons
Brain Res.
(2003) - et al.
Interleukin-1 beta induced corticosterone elevation and hypothalamic NE depletion is vagally mediated
Brain Res. Bull.
(1995) - et al.
TNF-alpha-induced corticosterone elevation but not serum protein or corticosteroid binding globulin reduction is vagally mediated
Brain Res. Bull.
(1997) - et al.
Thermogenic and corticosterone responses to intravenous cytokines (IL-1beta and TNF-alpha) are attenuated by subdiaphragmatic vagotomy
J. Neuroimmunol.
(1998) - et al.
Brain response to cecal infection with Campylobacter jejuni: analysis with Fos immunohistochemistry
Brain Behav. Immun.
(2004) - et al.
Blockade of cytokine induced conditioned taste aversion by subdiaphragmatic vagotomy: further evidence for vagal mediation of immune-brain communication
Neurosci. Lett.
(1995) - et al.
Vagal paraganglia bind biotinylated interleukin-1 receptor antagonist: a possible mechanism for immune-to-brain communication
Brain Res. Bull.
(1997)