Loss of vagal integrity disrupts immune components of the microbiota-gut-brain axis and inhibits the effect of Lactobacillus rhamnosus on behavior and the corticosterone stress response.

The vagus nerve is one of the major signalling components between the gut microbiota and brain. However, the exact relationship between gut-brain signaling along the vagus and the effects of gut microbes on brain function and behaviour is unclear. In particular, the relationship between the vagus nerve and immune signaling, that also appears to play a critical role in microbiota-gut-brain communication, has not been delineated. The aim of the present study was to determine the effect of subdiaphragmatic vagotomy on peripheral and central immune changes associated with the anxiolytic actions of L.rhamnosus. Male mice underwent vagotomy or sham surgery, followed by administration of L.rhamnosus for 14 days. L.rhamnosus administration following sham surgery resulted in reduced anxiety-like behaviour, and an attenuation of the hypothalamic-pituitary-adrenal axis (HPA axis), as indicated by reduced plasma corticosterone after acute restraint stress. These effects were associated with an increase in splenic T regulatory cells and a decrease in activated microglia in the hippocampus. The anxiolytic effects, HPA modulation and increase in T regulatory cells were prevented by vagotomy, whereas vagotomy alone led to a significant increase in activated microglia in the hippocampus that was not altered with L.rhamnosus treatment. Thus, both microbe induced and constitutive vagal signaling influences critical immune components of the microbiota-gut-brain axis. These findings suggest that, rather than acting as a direct neural link to the central nervous system, the role of the vagus nerve in gut-microbe to brain signalling is as an integral component of a bi-directional neuroimmunoendocrine pathway.

CD4+CD25+ T Cells are Essential for Behavioral Effects of Lactobacillus rhamnosus JB-1 in Male BALB/c mice.

Over the past decade there has been increasing interest in the involvement of the microbiota-gut-brain axis in mental health. However, there are major gaps in our knowledge regarding the complex signaling systems through which gut microbes modulate the CNS. The immune system is a recognized mediator in the bidirectional communication continuously occurring between gut and brain. We previously demonstrated that Lactobacillus rhamnosus JB-1 (JB-1), a bacterial strain that has anxiolytic- and antidepressant-like effects in mice, modulates the immune system through induction of immunosuppressive T regulatory cells. Here we examined a potential causal relationship between JB-1 induced regulatory T cells and the observed effects on behaviour. We found that depletion of regulatory T cells, via treatment with monoclonal antibody against CD25, inhibited the antidepressant- and anxiolytic-like effects induced by 4-week oral administration of JB-1 in mice. Ly6Chi monocytes were found to be decreased in JB-1 fed mice with intact regulatory T cells, but not in JB-1 fed mice following depletion. Furthermore, adoptive transfer of CD4+CD25+ cells, from JB-1 treated donor mice, but not from controls, induced antidepressant- and anxiolytic-like effects in recipient mice. Ly6Chi monocytes were also significantly decreased in mice receiving CD4+CD25+ cells from JB1 fed donors. This study identifies cells within the CD4+CD25+ population, most likely regulatory T cells, as both necessary and sufficient in JB-1-induced antidepressant- and anxiolytic-like effects in mice, providing novel mechanistic insight into microbiota-gut-brain communication in addition to highlighting the potential for immunotherapy in psychiatric disorders.

Human Milk Oligosaccharides Attenuate Antigen-Antibody Complex Induced Chemokine Release from Human Intestinal Epithelial Cell Lines.

There has been increased interest in the use of dietary ingredients, including prebiotics such as human-milk oligosaccharides (HMOs), as therapeutic strategies for food allergy. Understanding the mechanisms underlying the beneficial effects of HMOs is important to realizing their therapeutic potential. Here we demonstrate that the HMO, 6'-sialyllactose (6'SL) inhibited chemokine (IL-8 and CCL20) release from T-84 and HT-29 cells stimulated with antigen-antibody complex, TNFα or PGE2 ; an effect that was PPARγ dependent and associated with decreased activity of the transcription factors AP-1 and NFκB. In contrast, 2'-fucosyllactose (2'FL) selectively inhibited CCL20 release in response to antigen antibody complex in a PPARγ independent manner. This study reinforces the concept that structurally different oligosaccharides have distinct biological activities and identifies, for the first time, that the HMOs, 6'SL, and 2'FL, modulate human epithelial cell responses related to allergic disease. These findings encourage further investigation of the therapeutic potential of specific HMOs in food allergy. PRACTICAL APPLICATION: This study provides evidence for direct effects of HMOs in addition to their prebiotic role and demonstrates, for the first time, modulation of Ag-IgE complex activation of human epithelial cells that may have important implications for food-allergy. The study also reinforces the concept that structurally different oligosaccharides have distinct biological activities. In determining the composition of infant formula, addition of oligosaccharides with specific structures may provide direct modulation of immune responses and potentially attenuate symptoms or development of food allergy.

The bacterial quorum-sensing molecule, N-3-oxo-dodecanoyl-L-homoserine lactone, inhibits mediator release and chemotaxis of murine mast cells.

OBJECTIVE: Bacterial colonization relies on communication between bacteria via so-called "quorum-sensing molecules", which include the acyl-homoserine lactone group. Certain acyl-homoserine lactones can modulate mammalian cell function and are thought to contribute to bacterial pathogenicity. Given the role of mast cells in host defense, we investigated the ability of acyl-homoserine lactones to modulate mast cell function. METHODS: We utilized murine primary mast cell cultures to assess the effect of acyl-homoserine lactones on degranulation and cytokine release in response to different stimuli. We also assessed cell migration in response to chemoattractants. The effect of acyl-homoserine lactones in vivo was tested using a passive cutaneous anaphylaxis model. RESULTS: Two of the tested quorum-sensing molecules, N-3-oxo-dodecanoyl-L-homoserine lactone and N-Dodecanoyl-L-homoserine lactone, inhibited IgE dependent and independent degranulation and mediator release from primary mast cells. Further testing of N-3-oxo-dodecanoyl-L-homoserine lactone, the most potent inhibitor and a product of Pseudomonas aeruginosa, revealed that it also attenuated chemotaxis and LPS induced cytokine production. In vivo, N-3-oxo-dodecanoyl-L-homoserine lactone inhibited the passive cutaneous anaphylaxis response in mice. CONCLUSION: The ability of N-3-oxo-dodecanoyl-L-homoserine lactone to stabilize mast cells may contribute to the pathogenicity of P. aeruginosa but could potentially be exploited therapeutically in allergic disease.

Superior Cervical Ganglia Neurons Induce Foxp3+ Regulatory T Cells via Calcitonin Gene-Related Peptide.

The nervous and immune systems communicate bidirectionally, utilizing diverse molecular signals including cytokines and neurotransmitters to provide an integrated response to changes in the body's internal and external environment. Although, neuro-immune interactions are becoming better understood under inflammatory circumstances and it has been evidenced that interaction between neurons and T cells results in the conversion of encephalitogenic T cells to T regulatory cells, relatively little is known about the communication between neurons and naïve T cells. Here, we demonstrate that following co-culture of naïve CD4+ T cells with superior cervical ganglion neurons, the percentage of Foxp3 expressing CD4+CD25+ cells significantly increased. This was mediated in part by immune-regulatory cytokines TGF-ß and IL-10, as well as the neuropeptide calcitonin gene-related peptide while vasoactive intestinal peptide was shown to play no role in generation of T regulatory cells. Additionally, T cells co-cultured with neurons showed a decrease in the levels of pro-inflammatory cytokine IFN-γ released upon in vitro stimulation. These findings suggest that the generation of Tregs may be promoted by naïve CD4+ T cell: neuron interaction through the release of neuropeptide CGRP.

Gut commensal microvesicles reproduce parent bacterial signals to host immune and enteric nervous systems.

Ingestion of a commensal bacteria, Lactobacillus rhamnosus JB-1, has potent immunoregulatory effects, and changes nerve-dependent colon migrating motor complexes (MMCs), enteric nerve function, and behavior. How these alterations occur is unknown. JB-1 microvesicles (MVs) are enriched for heat shock protein components such as chaperonin 60 heat-shock protein isolated from Escherichia coli (GroEL) and reproduce regulatory and neuronal effects in vitro and in vivo. Ingested labeled MVs were detected in murine Peyer's patch (PP) dendritic cells (DCs) within 18 h. After 3 d, PP and mesenteric lymph node DCs assumed a regulatory phenotype and increased functional regulatory CD4(+)25(+)Foxp3+ T cells. JB-1, MVs, and GroEL similarly induced phenotypic change in cocultured DCs via multiple pathways including C-type lectin receptors specific intercellular adhesion molecule-3 grabbing non-integrin-related 1 and Dectin-1, as well as TLR-2 and -9. JB-1 and MVs also decreased the amplitude of neuronally dependent MMCs in an ex vivo model of peristalsis. Gut epithelial, but not direct neuronal application of, MVs, replicated functional effects of JB-1 on in situ patch-clamped enteric neurons. GroEL and anti-TLR-2 were without effect in this system, suggesting the importance of epithelium neuron signaling and discrimination between pathways for bacteria-neuron and -immune communication. Together these results offer a mechanistic explanation of how Gram-positive commensals and probiotics may influence the host's immune and nervous systems.

The parasympathetic nervous system as a regulator of mast cell function.

Often considered as the archetype of neuroimmune communication, much of our understanding of the bidirectional relationship between the nervous and immune systems has come from the study of mast cell-nerve interaction. Mast cells play a role in resistance to infection and are extensively involved in inflammation and subsequent tissue repair. Thus, the relationship between mast cells and neurons enables the involvement of peripheral and central nervous systems in the regulation of host defense mechanisms and inflammation. Recently, with the identification of the cholinergic anti-inflammatory pathway, there has been increased interest in the role of the parasympathetic nervous system in regulating immune responses. Classical neurotransmitters and neuropeptides released from cholinergic and inhibitory NANC neurons can modulate mast cell activity, and there is good evidence for the existence of parasympathetic nerve-mast cell functional units in the skin, lung, and intestine that have the potential to regulate a range of physiological processes.

The mast cell-nerve functional unit: a key component of physiologic and pathophysiologic responses.

A key characteristic of mast cells appears to be an ability to span the division between nervous and immune system. Indeed, much of our understanding of the bi-directional relationship between the nervous and immune systems has come from the study of mast cell-nerve interaction. Although differences in species have been reported, morphologic as well as functional associations between mast cell and nerves are found in most tissues in many mammalian species, including humans. These interactions are involved in the regulation of physiologic homeostatic processes as well as in disease mechanisms. Here we discuss the influence of cholinergic and sensory neurons on mast cells as well as the importance of mast cell nerve interactions at specific tissue sites, including the brain.

The vagus nerve modulates CD4+ T cell activity.

The vagus nerve has a counter-inflammatory role in a number of model systems. While the majority of these anti-inflammatory effects have been ascribed to the activation of nicotinic receptors on macrophages, little is known about the role of the vagus in modulating the activity of other cells involved in inflammatory responses. Here, we demonstrate that following subdiaphragmatic vagotomy of mice CD4(+) T cells from the spleen proliferated at a higher rate and produced more pro-inflammatory cytokines, including TNF and IFN-gamma, upon in vitro stimulation. Cell responses were restored to control levels following the administration of nicotine and the treatment of non-vagotomized animals with a nicotinic receptor antagonist could mimic the effect of vagotomy. Our results suggest that vagal input constitutively down-regulates T cell function through action at nicotinic receptors and the role of the vagus in regulating immune responses is more extensive than previously demonstrated.

Oral treatment with live Lactobacillus reuteri inhibits the allergic airway response in mice.

RATIONALE: Clinical trials have demonstrated that probiotics may be effective in the treatment and prevention of atopic disease in children but there have been few reports of therapeutic effects of oral probiotics outside the gastrointestinal tract. OBJECTIVES: We investigated the effect of two probiotic organisms on the response to antigen challenge in a mouse model of allergic airway inflammation. METHODS: We used an ovalbumin-sensitized asthma model in BALB/c and Toll-like receptor 9-deficient mice. Animals were treated with probiotic organisms via gavaging needle before antigen challenge. After antigen challenge, airway responsiveness to methacholine, influx of inflammatory cells to the lung, and cytokine levels in bronchoalveolar lavage fluid were assessed. RESULTS: Oral treatment with live Lactobacillus reuteri but not Lactobacillus salivarius significantly attenuated the influx of eosinophils to the airway lumen and parenchyma and reduced the levels of tumor necrosis factor, monocyte chemoattractant protein-1, IL-5, and IL-13 in bronchoalveolar lavage fluid of antigen-challenged animals, but there was no change in eotaxin or IL-10. L. reuteri but not L. salivarius also decreased allergen-induced airway hyperresponsiveness. These responses were dependent on Toll-like receptor 9 and were associated with increased activity of indoleamine 2,3-dioxygenase. Killed organisms did not mimic the ability of the live L. reuteri to attenuate inflammation or airway hyperresponsiveness. CONCLUSION: Oral treatment with live L. reuteri can attenuate major characteristics of an asthmatic response in a mouse model of allergic airway inflammation. These results suggest that oral treatment with specific live probiotic strains may have therapeutic potential in the treatment of allergic airway disease.

Proteinase-activated receptor 2 activation in the airways enhances antigen-mediated airway inflammation and airway hyperresponsiveness through different pathways.

BACKGROUND: Serine proteinases such as mast cell tryptase, trypsin-like enzymes, and certain allergens are important in the pathogenesis of asthma. These proteinases can activate the proteinase-activated receptor (PAR)-2, which has been shown to be upregulated in the airways of patients with asthma. OBJECTIVE: The purpose of this study was to investigate PAR-2 activation in the airways during allergen challenge and its effects on the 2 principle features of asthma, airway inflammation and airway hyperresponsiveness (AHR). METHODS: Proteinase-activated receptor 2 activating peptide SLIGRL-NH2 (PAR-2 activating peptide [ap]) or control peptide LSIGRL-NH2 (PAR-2 control peptide [cp]) was administered alone or in conjunction with ovalbumin intranasally to mice, and AHR and airway inflammation were evaluated. RESULTS: PAR2ap did not induce AHR or airway inflammation in ovalbumin-sensitized mice that had not been challenged with ovalbumin. When administered with ovalbumin, PAR-2ap enhanced AHR and airway inflammation compared with ovalbumin administered alone or with PAR-2cp. The enhanced AHR persisted for 5 days, whereas the enhancement to airway inflammation dissipated. Mice administered PAR-2ap alone during the 5 days after the final antigen challenge demonstrated an additional enhancement to airway inflammation compared with the control animals. PAR-2ap administered with allergen increased TNF and IL-5 mRNA in lung tissue and IL-13 and TNF in bronchoalveolar lavage fluid. CONCLUSION: Exogenous PAR-2 activation in parallel with allergen challenge enhances allergen-mediated AHR and airway inflammation through distinct mechanisms. PAR-2 activation can also enhance established airway inflammation even when dissociated from exposure to allergen. Therefore, PAR-2 activation may play a pathogenic role in the development of AHR and airway inflammation.

Live Lactobacillus rhamnosus [corrected] is essential for the inhibitory effect on tumor necrosis factor alpha-induced interleukin-8 expression.

The mechanism of the apparent anti-inflammatory action of probiotic organisms is unclear. Lactobacillus reuteri is effective in inhibiting colitis in interleukin-10 (IL-10)-deficient mice. Nerve growth factor (NGF), in addition to its activity on neuronal cell growth, has significant anti-inflammatory effects in several experimental systems in vitro and in vivo, including a model of colitis. Our experiments were designed to explore the mechanism of effect of L. reuteri in the human epithelial cell lines T84 and HT29 on cytokine and NGF synthesis and IL-8 response to tumor necrosis factor alpha (TNF-alpha). Epithelial cells were cultured for various times with live and killed L. reuteri and examined by reverse transcription-PCR for NGF, IL-10, and TNF-alpha-induced IL-8 expression. An enzyme-linked immunosorbent assay was used to quantitate intracellular IL-8 and secreted product. Western blotting and confocal microscopy were used to determine the effects on IkappaB and NF-kappaB, respectively. Live but not heat-killed or gamma-irradiated L. reuteri upregulated NGF and dose dependently inhibited constitutive synthesis by T84 and HT29 cells of IL-8 and that induced by TNF-alpha in terms of mRNA and intracellular and secreted protein. Similarly, L. reuteri inhibited IL-8 synthesis induced by Salmonella enterica serovar Typhimurium. L. reuteri required preincubation and adherence for effect, inhibited translocation of NF-kappaB to the nuclei of HeLa cells, and prevented degradation of IkappaB. Neither cellular lysates nor media supernatants had any effect on TNF-alpha-induced IL-8. The conclusion is that L. reuteri has potent direct anti-inflammatory activity on human epithelial cells, which is likely to be related to the activity of ingested probiotics. L. reuteri also upregulates an unusual anti-inflammatory molecule, NGF, and inhibits NF-kappaB translocation to the nucleus.

Inhibition of calpain is a component of nitric oxide-induced down-regulation of human mast cell adhesion.

Nitric oxide is an important messenger that regulates mast cell activity by modifications to gene expression and intracellular pathways associated with exocytosis and adhesion. Integrin interactions with extracellular matrix components modulate an array of cell activities, including mediator production and secretion. To investigate the molecular mechanisms underlying NO regulation of mast cell function, we studied its effects on adhesion of a human mast cell line (HMC-1) to fibronectin (FN). The NO donors S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine strongly down-regulated the adhesion of HMC-1 to FN. Inhibitors of soluble guanylate cyclase and protein kinase G did not alter the response of cells to NO. A peroxynitrite scavenger did not affect modulation of adhesion by NO, nor could the effect of NO be mimicked by the peroxynitrite-producing compound 3-morpholinosydnonimine. NO donors inhibited the cysteine protease, calpain, while calpain inhibitors mimicked the effect of NO and led to a decrease in the ability of HMC-1 cells to adhere to FN. Thus, NO is an effective down-regulator of human mast cell adhesion. The mechanism for this action does not involve peroxynitrite or activation of soluble guanylate cyclase. Instead, a portion of NO-induced down-regulation of adhesion may be attributed to inhibition of the cysteine protease, calpain, an enzyme that has been associated with control of integrin activation in other cell types. The inhibition of calpain is most likely mediated via nitrosylation of its active site thiol group. Calpain may represent a novel therapeutic target for the regulation of mast cell activity in inflammatory disorders.