Lactobacillus rhamnosus strain JB-1 reverses restraint stress-induced gut dysmotility.

BACKGROUND: Environmental stress affects the gut with dysmotility being a common consequence. Although a variety of microbes or molecules may prevent the dysmotility, none reverse the dysmotility. METHODS: We have used a 1 hour restraint stress mouse model to test for treatment effects of the neuroactive microbe, L. rhamnosus JB-1™ . Motility of fluid-filled ex vivo gut segments in a perfusion organ bath was recorded by video and migrating motor complexes measured using spatiotemporal maps of diameter changes. KEY RESULTS: Stress reduced jejunal and increased colonic propagating contractile cluster velocities and frequencies, while increasing contraction amplitudes for both. Luminal application of 10E8 cfu/mL JB-1 restored motor complex variables to unstressed levels within minutes of application. L. salivarius or Na.acetate had no treatment effects, while Na.butyrate partially reversed stress effects on colonic frequency and amplitude. Na.propionate reversed the stress effects for jejunum and colon except on jejunal amplitude. CONCLUSIONS & INFERENCES: Our findings demonstrate, for the first time, a potential for certain beneficial microbes as treatment of stress-induced intestinal dysmotility and that the mechanism for restoration of function occurs within the intestine via a rapid drug-like action on the enteric nervous system.

The gut microbiome restores intrinsic and extrinsic nerve function in germ-free mice accompanied by changes in calbindin.

BACKGROUND: The microbiome is essential for normal myenteric intrinsic primary afferent neuron (IPAN) excitability. These neurons control gut motility and modulate gut-brain signaling by exciting extrinsic afferent fibers innervating the enteric nervous system via an IPAN to extrinsic fiber sensory synapse. We investigated effects of germ-free (GF) status and conventionalization on extrinsic sensory fiber discharge in the mesenteric nerve bundle and IPAN electrophysiology, and compared these findings with those from specific pathogen-free (SPF) mice. As we have previously shown that the IPAN calcium-dependent slow afterhyperpolarization (sAHP) is enhanced in GF mice, we also examined the expression of the calcium-binding protein calbindin in these neurons in these different animal groups. METHODS: IPAN sAHP and mesenteric nerve multiunit discharge were recorded using ex vivo jejunal gut segments from SPF, GF, or conventionalized (CONV) mice. IPANs were excited by adding 5 µM TRAM-34 to the serosal superfusate. We probed for calbindin expression using immunohistochemical techniques. KEY RESULTS: SPF mice had a 21% increase in mesenteric nerve multiunit firing rate and CONV mice a 41% increase when IPANs were excited by TRAM-34. For GF mice, this increase was barely detectable (2%). TRAM-34 changed sAHP area under the curve by -77 for SPF, +3 for GF, or -54% for CONV animals. Calbindin-immunopositive neurons per myenteric ganglion were 36% in SPF, 24% in GF, and 52% in CONV animals. CONCLUSIONS & INFERENCES: The intact microbiome is essential for normal intrinsic and extrinsic nerve function and gut-brain signaling.

Spatiotemporal maps reveal regional differences in the effects on gut motility for Lactobacillus reuteri and rhamnosus strains.

BACKGROUND: Commensal bacteria such as probiotics that are neuroactive acutely affect the amplitudes of intestinal migrating motor complexes (MMCs). What is lacking for an improved understanding of these motility effects are region specific measurements of velocity and frequency. We have combined intraluminal pressure recordings with spatiotemporal diameter maps to analyze more completely effects of different strains of beneficial bacteria on motility. METHODS: Intraluminal peak pressure (PPr) was measured and video recordings made of mouse ex vivo jejunum and colon segments before and after intraluminal applications of Lactobacillus rhamnosus (JB-1) or Lactobacillus reuteri (DSM 17938). Migrating motor complex frequency and velocity were calculated. KEY RESULTS: JB-1 decreased jejunal frequencies by 56% and 34% in colon. Jejunal velocities increased 171%, but decreased 31% in colon. Jejunal PPr decreased by 55% and in colon by 21%. DSM 17938 increased jejunal frequencies 63% and in colon 75%; jejunal velocity decreased 57%, but increased in colon 146%; jejunal PPr was reduced 26% and 12% in colon. TRAM-34 decreased frequency by 71% and increased velocity 200% for jejunum, but increased frequency 46% and velocity 50% for colon; PPr was decreased 59% for jejunum and 39% for colon. CONCLUSIONS & INFERENCES: The results show that probiotics and other beneficial bacteria have strain and region-specific actions on gut motility that can be successfully discriminated using spatiotemporal mapping of diameter changes. Effects are not necessarily the same in colon and jejunum. Further research is needed on the detailed effects of the strains on enteric neuron currents for each gut region.

The microbiome is essential for normal gut intrinsic primary afferent neuron excitability in the mouse.

BACKGROUND: The role of intestinal microbiota in the development and function of host physiology is of high interest, especially with respect to the nervous system. While strong evidence has accrued that intestinal bacteria alter host nervous system function, mechanisms by which this occurs have remained elusive. For this reason, we have carried out experiments examining the electrophysiological properties of neurons in the myenteric plexus of the enteric nervous system (ENS) in germ-free (GF) mice compared with specific pathogen-free (SPF) control mice and adult germ-free mice that have been conventionalized (CONV-GF) with intestinal bacteria. METHODS: Segments of jejunum from 8 to 12 week old GF, SPF, and CONV-GF mice were dissected to expose the myenteric plexus. Intracellular recordings in current-clamp mode were made by impaling cells with sharp microelectrodes. Action potential (AP) shapes, firing thresholds, the number of APs fired at 2× threshold, and passive membrane characteristics were measured. KEY RESULTS: In GF mice, excitability was decreased in myenteric afterhyperpolarization (AH) neurons as measured by a lower resting membrane potential and by the number of APs generated at 2× threshold. The post AP slow afterhyperpolarization (sAHP) was prolonged for GF compared with SPF and CONV-GF animals. Passive membrane characteristics were also altered in GF mice by a decrease in input resistance. CONCLUSIONS & INFERENCES: Here, we report the novel finding that commensal intestinal microbiota are necessary for normal excitability of gut sensory neurons and thus provide a potential mechanism for the transfer of information between the microbiota and nervous system.

The microbiota and the gut-brain axis: insights from the temporal and spatial mucosal alterations during colonisation of the germfree mouse intestine.

The influence of the gut microbiota on the nervous system, brain development and behaviour, in particular during microbial colonisation of the host, has recently been receiving profound interest. Our time-resolved mining of combined data analyses of the ex-germfree mouse intestine during a 30-day course of colonisation with conventional mouse faecal microbiota (conventionalisation), shed light on temporal altered expression of genes of which the products influenced functions of the nervous system. Plasma tryptophan and kynurenine levels reflected high indoleamine dioxygenase activity, which was supported by significant temporal induction of the encoding gene in all gut tissues. However, the majority of genes associated with neuronal development and function were reduced. Colonic substance P elevation in response to conventionalisation was higher only after 30-days. These results support a functional microbiota-neurohumoral relationship during conventionalisation and suggest a delayed neuronal response that is elicited only after the microbiota accommodating homeostasis has been accomplished.

Reduced anxiety-like behavior and central neurochemical change in germ-free mice.

BACKGROUND: There is increasing interest in the gut-brain axis and the role intestinal microbiota may play in communication between these two systems. Acquisition of intestinal microbiota in the immediate postnatal period has a defining impact on the development and function of the gastrointestinal, immune, neuroendocrine and metabolic systems. For example, the presence of gut microbiota regulates the set point for hypothalamic-pituitary-adrenal (HPA) axis activity. METHODS: We investigated basal behavior of adult germ-free (GF), Swiss Webster female mice in the elevated plus maze (EPM) and compared this to conventionally reared specific pathogen free (SPF) mice. Additionally, we measured brain mRNA expression of genes implicated in anxiety and stress-reactivity. KEY RESULTS: Germ-free mice, compared to SPF mice, exhibited basal behavior in the EPM that can be interpreted as anxiolytic. Altered GF behavior was accompanied by a decrease in the N-methyl-D-aspartate receptor subunit NR2B mRNA expression in the central amygdala, increased brain-derived neurotrophic factor expression and decreased serotonin receptor 1A (5HT1A) expression in the dentate granule layer of the hippocampus. CONCLUSIONS & INFERENCES: We conclude that the presence or absence of conventional intestinal microbiota influences the development of behavior, and is accompanied by neurochemical changes in the brain.

99th Dahlem conference on infection, inflammation and chronic inflammatory disorders: psycho-neuroimmunology and the intestinal microbiota: clinical observations and basic mechanisms.

This is a rapidly emerging field. The application of knowledge regarding the relationship between neural and immune systems in order to gain a better understanding of human conditions has been slow. In this discussion we describe how the brain and microbiota interact, and try to bring this into a context that is clinically relevant. We begin by describing established facts pertaining to the gut-brain axis and the role of gut bacteria. We then focus upon emerging data that will contribute to the generation of a new conceptual framework about the microbiota-gut-brain axis. In the final section we anticipate future directions of this field.

Bacterial strain-specific induction of Foxp3+ T regulatory cells is protective in murine allergy models.

BACKGROUND: The incidence of atopic disease has increased dramatically during recent decades and the potential immunoregulatory influence of the microbiota in these individuals is under investigation. OBJECTIVE: The aim of our study was to identify a bacterial strain that is protective in murine allergy models and to determine if microbial induction of T regulatory cells was associated with protection from allergic inflammation. METHODS: Three microbes (Bifidobacterium breve AH1205, B. longum AH1206 and Lactobacillus salivarius AH102) of human origin were fed to newborn, adult and germ-free animals. Induction of Foxp3(+) T regulatory cells was assessed by flow cytometry. Gene array analysis was performed on Peyer's patches. Strains were also examined for their protective effects in the ovalbumin (OVA) respiratory allergy model and the OVA-cholera toxin dietary allergy model. RESULTS: Bifidobacterium longum AH1206 consumption resulted in increased numbers of Foxp3(+) T regulatory cells in infant, adult and germ-free animals. B. breve AH1205 induced Foxp3(+) T regulatory cell expansion only in infant mice while L. salivarius AH102 did not alter T regulatory cell numbers in any animal model tested. B. longum AH1206 reduced the Peyer's patch gene expression associated with antigen presentation, TLR signalling and cytokine production while increasing the expression of genes associated with retinoic acid metabolism. B. longum AH1206 protected against airway inflammation in OVA-sensitized animals and B. longum AH1206 blocked the induction of IgE to orally administered OVA. Neither B. breve AH1205 nor L. salivarius AH102 had a protective effect in either model. CONCLUSION: Bacterial strain-specific induction of Foxp3(+) T regulatory cells in vivo is associated with protection from respiratory and oral allergy.

Lactobacillus reuteri ingestion and IK(Ca) channel blockade have similar effects on rat colon motility and myenteric neurones.

BACKGROUND: We have previously shown that ingestion of Lactobacillus reuteri may modulate colonic enteric neuron activity but with unknown effects on colon motility. The aim of the present report was to elucidate the neuronal mechanisms of action of the probiotic by comparing the effects on motility of L. reuteri ingestion with blockade of a specific ionic current in enteric neurons. METHODS: We have used intraluminal pressure recordings from ex vivo rat colon segments and whole cell patch clamp recordings from neurons of rat longitudinal muscle myenteric plexus preparations to investigate the effects of L. reuteri and TRAM-34 on colon motility and neurophysiology. The effects of daily feeding of 10(9) L. reuteri bacteria or acute application of TRAM-34 on threshold fluid filling pressure or pulse pressure was measured. KEY RESULTS: Lactobacillus reuteri increased intraluminal fluid filling pressure thresholds for evoking pressure pulses by 51% from 0.47 +/- 0.17 hPa; the probiotic also decreased the pulse pressure amplitudes, but not frequency, by 18% from 3.91 +/- 0.52 hPa. The intermediate conductance calcium-dependent potassium (IK(Ca)) channel blocker TRAM-34 (3 micromol L(-1)) increased filling threshold pressure by 43% from 0.52 +/- 0.22 hPa and reduced pulse pressure amplitude by 40% from 2.63 +/- 1.11 hPa; contraction frequency was unaltered. TRAM-34 (3 micromol L(-1)) reduced membrane polarization, leak conductance and the slow afterhyperpolarization current in 16/16 myenteric rat colon AH cells but 19/19 S cells were unaffected. CONCLUSIONS & INFERENCES: The present results are consistent with L. reuteri enhancing tonic inhibition of colon contractile activity by acting via the IK(Ca) channel current in AH cells.

Heightened central affective response to visceral sensations of pain and discomfort in IBS.

BACKGROUND Typically, conventional functional imaging methods involve repeated exposures to sensory stimulation. In rectal distension (RD) studies that involve multiple distensions, however, it is difficult to disambiguate the central response to RD from pathological alterations in peripheral neural responses associated with relaxation and accommodation of the rectum. METHODS This study addressed potential confounders found in previous imaging studies by collecting functional magnetic resonance imaging studies (fMRI) data during a single slow ramp-tonic distension paradigm and analysing fMRI signal changes using independent component analysis. KEY RESULTS Compared with controls, IBS participants showed increased activation of the anterior cingulate cortices, insula and ventral medial prefrontal regions suggesting heightened affective responses to painful visceral stimuli. In addition, the failure by IBS patients to down-regulate activity within ventral medial prefrontal and the posterior cingulate/precuneus regions was suggestive of reduced sensitivity to somatic changes and delayed shifts away from rest in ;default network' activity patterns. Controls showed heightened activation of the thalamus, striatal regions and dorsolateral prefrontal cortex suggesting greater arousal and salience-driven sustained attention reactions and greater modulation of affective responses to discomfort and pain. CONCLUSION&INFERENCES This work points to alterations in the central response to visceral pain and discomfort in IBS, highlighting diminished modulation and heightened internalization of affective reactions.

The D-alanine content of lipoteichoic acid is crucial for Lactobacillus plantarum-mediated protection from visceral pain perception in a rat colorectal distension model.

The mechanisms leading to positive effects of probiotics in irritable bowel syndrome and inflammatory bowel disease have not been clarified, but the possible involvement of cell wall components is widely discussed. Reduction of the D-alanine content of lipoteichoic acid (LTA) in Lactobacillus plantarum (Dlt(-) mutant) enhanced its anti-inflammatory properties in a mouse colitis model. Another lactobacillus species inhibited visceral pain perception in response to colorectal distension (CRD) in rats. Therefore, we investigated if LTA modification influences the constitutive intestinal pain perception in addition to modulation of cytokine release. Male Sprague-Dawley rats were gavaged with L. plantarum, L. plantarum Dlt(-) mutant or buffer control, respectively and the responses to CRD were tested in this non-inflammatory model. Tumour necrosis factor (TNF), interferon (IFN)-gamma and interleukin (IL)-10 release were measured in colon tissue homogenates and upon anti-CD3/CD28 activation of isolated splenocytes and mesenteric lymphocytes. Control animals showed significant bradycardia following noxious CRD, whereas only the L. plantarum Dlt(-) mutant inhibited the response. The mutant also decreased the activation-induced release of TNF and IFN-gamma from mesenteric T cells and the IL-10 concentration in colonic tissue, while increasing the activation-induced secretion of IL-10 in splenocytes and mesenteric lymphocytes and the baseline IL-10 release of splenocytes. In conclusion, d-alanine depletion of LTA in L. plantarum inhibited visceral pain perception in healthy, non-inflamed rats. Regardless of the non-inflammatory nature of the model decreased visceral pain perception was seen in parallel with anti-inflammatory properties.

Vagal dysfunction in irritable bowel syndrome assessed by rectal distension and baroreceptor sensitivity.

Autonomic nervous system dysfunction has been implicated in the pathophysiology of irritable bowel syndrome (IBS). This study characterized the autonomic response to rectal distension in IBS using baroreceptor sensitivity (BRS), a measure of autonomic function. Rectal bag pressure, discomfort, pain, ECG, blood pressure and BRS were continuously measured before, during and after rectal distension in 98 healthy volunteers (34 +/- 12 years old, 52 females) and 39 IBS patients (39 +/- 11 years old, 35 females). In comparison with the healthy volunteers, IBS patients experienced significantly more discomfort (69 +/- 2.2% vs 56 +/- 3.6%; P < 0.05), but not pain (9 +/- 1.4% vs 6 +/- 2.4%; ns) with rectal distension despite similar distension pressures (51 +/- 1.4 vs 54 +/- 2.4 mmHg; ns) and volumes (394 +/- 10.9 vs 398 +/- 21.5 mL; ns). With rectal distension, heart rate increased in both healthy volunteers (66 +/- 1 to 71 +/- 1 bpm; P < 0.05) and IBS patients (66 +/- 2 to 74 +/- 3 bpm; P < 0.05). Systolic blood pressure also increased in both healthy volunteers (121 +/- 2 to 143 +/- 2 mmHg; P < 0.05) and patients (126 +/- 3 to 153 +/- 4 mmHg (P < 0.05) as did diastolic blood pressure, 66 +/- 2 to 80 +/- 2 mmHg (P < 0.05), compared with 68 +/- 3 to 84 +/- 3 mmHg (P < 0.05) in IBS patients. The systolic blood pressure increase observed in IBS patients was greater than that seen in healthy volunteers and remained elevated in the post distension period (139 +/- 3 mmHg vs 129 +/- 2 mmHg; P < 0.05). IBS patients had lower BRS (7.85 +/- 0.4 ms mmHg(-1)) compared with healthy volunteers (9.4 +/- 0.3; P < 0.05) at rest and throughout rectal distension. Greater systolic blood pressure response to rectal distension and associated diminished BRS suggests a compromise of the autonomic nervous system in IBS patients.

Differential corticosterone responses to stress in the lung in two strains of Flinders rats.

BACKGROUND: Acute stress affects a variety of organs and cellular systems. These include the hypothalamic-pituitary-adrenal (HPA) axis, corticotropin-releasing factor (CRF), mast cells and nerves. Flinders-sensitive (FSL) rat strains have hypercholinergic responses and are more sensitive than Flinders-resistant rats (FRL) to anaphylaxis. OBJECTIVE: To investigate the effects of acute water avoidance stress (1 h) on FSL and FRL tracheal epithelial tissue. METHODS: We measured short circuit current (I(sc)) as a measure of tracheal response, and the effect of substance P (SP) on tracheal epithelium in Ussing chambers. Electron microscopy was performed to assess mast cell activation. RESULTS: Both strains showed increased I(sc) responses to stress, inhibited by prior injection of the CRF receptor 1 and 2 antagonist, alpha-helical CRF-(9-41). No increases in conductance were seen. Stress responses were accompanied by electron microscopic morphologic evidence for mast cell degranulation, which was not completely inhibited by alpha-helical CRF-(9-41) pre-treatment. Stress primed the epithelium for an enhanced response to SP in FSL, but this again was not inhibited by alpha-helical CRF-(9-41). FRL had 2.5 times the corticosterone response of FSL. CONCLUSION: Acute stress affects the tracheal epithelium, not accompanied by changes in ion permeability, but associated with mast cell degranulation. Because blunted HPA axis responses are associated with vulnerability to inflammation, this may partially explain the findings. These stress effects on the lung have a genetic basis associated with relative corticosterone responses, are complex and only in part mediated by CRF.

Neurotrophin-3, but not nerve growth factor, promotes survival of human intestinal mast cells.

Neurotrophins are potent regulators of neuronal cell survival and function. Nerve growth factor (NGF) was shown to reduce apoptosis in cord blood-derived mast cells. Here, we examined the effect of the neurotrophins NGF and neurotrophin (NT)-3 on survival and mediator release of human intestinal mast cells. Mast cells isolated from normal intestinal tissue were cultured in the presence of NGF, NT-3, or stem cell factor (SCF) alone or in the presence of SCF together with each neurotrophin. NGF or NT-3 alone did not promote mast cell survival. In contrast, mast cell recovery was increased twofold when mast cells were cultured with NT-3 in addition to SCF for 14 days compared with control. Mast cell recovery was further increased following a combined addition of NT-3, SCF and IL-4. NT-3 mediated mast cell growth was dependent on the primary receptor for NT-3 TrkC. NGF in combination with SCF or with SCF and IL-4 showed no effect on mast cell survival. Histamine release and histamine content per mast cell remained unchanged, whereas leukotriene C4 release decreased if mast cells were cultured with NGF or NT-3 in addition to SCF. In summary, NT-3 affects mature human mast cells by promoting mast cell survival, whereas NGF does not.

Inhibitory effects of Lactobacillus reuteri on visceral pain induced by colorectal distension in Sprague-Dawley rats.

BACKGROUND AND AIMS: Probiotic bacteria are being investigated as possible treatments for many intestinal disorders. The present study aimed to explore the effects of live, heat killed, or gamma irradiated Lactobacillus reuteri on cardio-autonomic response and single fibre unit discharge in dorsal root ganglia to colorectal distension in healthy Sprague-Dawley rats housed under conventional conditions. The effects of this treatment on somatic pain were also examined. METHODS: 1x10(9) bacteria were given by gavage for nine days. Colorectal distension occurred under anaesthesia. Heart rate was measured through continuous electrocardiography. Single fibre unit discharge was recorded from the 6th left lumbar dorsal root ganglion. Somatic pain was evaluated by the tail flick and paw pressure tests. RESULTS: Colorectal distension caused a pressure dependent bradycardia in the control (native medium) group. Treatment with live, heat killed, or gamma irradiated bacteria as well as their products (conditioned medium) prevented the pain response even during the maximum distension pressure (80 mm Hg). Both viable and non-viable bacteria significantly decreased dorsal root ganglion single unit activity to distension. No effects on somatic pain were seen with any treatment. CONCLUSIONS: Oral administration of either live or killed probiotic bacteria or conditioned medium inhibited the constitutive cardio-autonomic response to colorectal distension in rats through effects on enteric nerves. These data may provide a novel explanation for beneficial probiotic effects on visceral pain.

What is the physiological function of mast cells?

Under physiological conditions, skin mast cells preferentially localize around nerves, blood vessels and hair follicles. This observation, which dates back to Paul Ehrlich, intuitively suggests that these enigmatic, multifacetted protagonists of natural immunity are functionally relevant to many more aspects of tissue physiology than just to the generation of inflammatory and vasodilatory responses to IgE-dependent environmental antigens. And yet, for decades, mainstream-mast cell research has been dominated by a focus on the -undisputedly prominent and important - mast cell functions in type I immune responses and in the pathogenesis and management of allergic diseases. Certainly, it is hard to believe that the very large and rather selectively distributed number of mast cells in normal, uninflamed, non-infected, non-traumatized mammalian skin or mucosal tissue simply hanging around there lazily day and night, just wait for the odd allergen or parasite-associated antigen to come by so the mast cell can finally swing into action. Indeed, the past decade has witnessed a renaissance of mast cell research 'beyond allergy', along with a more systematic exploration of the surprisingly wide range of physiological functions that mast cells may be involved in. The current debate sketches many exciting horizons that have recently come into our vision during this intriguing, ongoing search.

Nerve-mast cell (RBL) interaction: RBL membrane ruffling occurs at the contact site with an activated neurite.

Mast cell-neurite interaction serves as a model for neuroimmune interaction. We have shown that neurite-mast cell communication can occur via substance P interacting with neurokinin (NK)-1 receptors on the mucosal mast cell-like cell, the rat basophilic leukemia (RBL) cell. Neurite (murine superior cervical ganglia) and RBL cell [expressing the granule-associated antigen CD63-green fluorescent protein (GFP) conjugate] cocultures were established and stimulated with bradykinin (BK; 10 nM) or scorpion venom (SV; 10 pg/ml), both of which activate only neurites. Cell activation was assessed by confocal imaging of Ca2+ (cells preloaded with fluo 3), and analyses of RBL CD63-GFP+ granule movement were conducted. Neurite activation by BK or SV was followed by RBL Ca2+ mobilization, which was inhibited by an NK-1 receptor antagonist (NK-1 RA). Moreover, membrane ruffling was observed on RBL pseudopodial extensions in contact with the activated neurite, but not on noncontacting pseudopodia. RBL membrane ruffling was inhibited by NK-1 RA, but not NK-2 RA, and was accompanied by a significant increase in granule movement (0.13 +/- 0.04 vs. 0.05 +/- 0.01 microm/s) that was most evident at the point of neurite contact: many of the granules moved toward the plasmalemma. This is the first documentation of such precise (restricted to the membrane's contact site) transfer of information between nerves and mast cells that could allow for very subtle in vivo communication between these two cell types.