Attenuation of Oxytocin and Serotonin 2A Receptor Signaling through Novel Heteroreceptor Formation.

The oxytocin receptor (OTR) and the 5-hydroxytryptamine 2A receptor (5-HTR2A) are expressed in similar brain regions modulating central pathways critical for social and cognition-related behaviors. Signaling crosstalk between their endogenous ligands, oxytocin (OT) and serotonin (5-hydroxytryptamine, 5-HT), highlights the complex interplay between these two neurotransmitter systems and may be indicative of the formation of heteroreceptor complexes with subsequent downstream signaling changes. In this study, we assess the possible formation of OTR-5HTR2A heteromers in living cells and the functional downstream consequences of this receptor-receptor interaction. First, we demonstrated the existence of a physical interaction between the OTR and 5-HTR2Ain vitro, using a flow cytometry-based FRET approach and confocal microscopy. Furthermore, we investigated the formation of this specific heteroreceptor complex ex vivo in the brain sections using the Proximity Ligation Assay (PLA). The OTR-5HTR2A heteroreceptor complexes were identified in limbic regions (including hippocampus, cingulate cortex, and nucleus accumbens), key regions associated with cognition and social-related behaviors. Next, functional cellular-based assays to assess the OTR-5HTR2A downstream signaling crosstalk showed a reduction in potency and efficacy of OT and OTR synthetic agonists, carbetocin and WAY267464, on OTR-mediated Gαq signaling. Similarly, the activation of 5-HTR2A by the endogenous agonist, 5-HT, also revealed attenuation in Gαq-mediated signaling. Finally, altered receptor trafficking within the cell was demonstrated, indicative of cotrafficking of the OTR/5-HTR2A pair. Overall, these results constitute a novel mechanism of specific interaction between the OT and 5-HT neurotransmitters via OTR-5HTR2A heteroreceptor formation and provide potential new therapeutic strategies in the treatment of social and cognition-related diseases.

Natural killer cells protect against mucosal and systemic infection with the enteric pathogen Citrobacter rodentium.

Natural killer (NK) cells are traditionally considered in the context of tumor surveillance and viral defense, but their role in bacterial infections, particularly those caused by enteric pathogens, is less clear. C57BL/6 mice were orally gavaged with Citrobacter rodentium, a murine pathogen related to human diarrheagenic Escherichia coli. We used polyclonal anti-asialo GM1 antibody to actively deplete NK cells in vivo. Bioluminescent imaging and direct counts were used to follow infection. Flow cytometry and immunofluorescence microscopy were used to analyze immune responses. During C. rodentium infection, NK cells were recruited to mucosal tissues, where they expressed a diversity of immune-modulatory factors. Depletion of NK cells led to higher bacterial loads but less severe colonic inflammation, associated with reduced immune cell recruitment and lower cytokine levels. NK cell-depleted mice also developed disseminated systemic infection, unlike control infected mice. NK cells were also cytotoxic to C. rodentium in vitro.

Mechanism of protection of transepithelial barrier function by Lactobacillus salivarius: strain dependence and attenuation by bacteriocin production.

Enhanced barrier function is one mechanism whereby commensals and probiotic bacteria limit translocation of foreign antigens or pathogens in the gut. However, barrier protection is not exhibited by all probiotic or commensals and the strain-specific molecules involved remain to be clarified. We evaluated the effects of 33 individual Lactobacillus salivarius strains on the hydrogen peroxide (H(2)O(2))-induced barrier impairment in human epithelial Caco-2 cells. These strains showed markedly different effects on H(2)O(2)-induced reduction in transepithelial resistance (TER). The effective strains such as UCC118 and CCUG38008 attenuated H(2)O(2)-induced disassembly and relocalization of tight junction proteins, but the ineffective strain AH43324 did not. Strains UCC118 and CCUG38008 induced phosphorylation of extracellular signal-regulated kinase (ERK) in Caco-2 cells, and the ERK inhibitor U0126 attenuated the barrier-protecting effect of these strains. In contrast, the AH43324 strain induced phosphorylation of Akt and p38, which was associated with an absence of a protective effect. Global transcriptome analysis of UCC118 and AH43324 revealed that some genes in a bacteriocin gene cluster were upregulated in AH43324 under TER assay conditions. A bacteriocin-negative UCC118 mutant displayed significantly greater suppressive effect on H(2)O(2)-induced reduction in TER compared with wild-type UCC118. The wild-type strain augmented H(2)O(2)-induced phosphorylation of Akt and p38, whereas a bacteriocin-negative UCC118 mutant did not. These observations indicate that L. salivarius strains are widely divergent in their capacity for barrier protection, and this is underpinned by differences in the activation of intracellular signaling pathways. Furthermore, bacteriocin production appears to have an attenuating influence on lactobacillus-mediated barrier protection.

Characterization of pro-inflammatory flagellin proteins produced by Lactobacillus ruminis and related motile Lactobacilli.

Lactobacillus ruminis is one of at least twelve motile but poorly characterized species found in the genus Lactobacillus. Of these, only L. ruminis has been isolated from mammals, and this species may be considered as an autochthonous member of the gastrointestinal microbiota of humans, pigs and cows. Nine L. ruminis strains were investigated here to elucidate the biochemistry and genetics of Lactobacillus motility. Six strains isolated from humans were non-motile while three bovine isolates were motile. A complete set of flagellum biogenesis genes was annotated in the sequenced genomes of two strains, ATCC25644 (human isolate) and ATCC27782 (bovine isolate), but only the latter strain produced flagella. Comparison of the L. ruminis and L. mali DSM20444(T) motility loci showed that their genetic content and gene-order were broadly similar, although the L. mali motility locus was interrupted by an 11.8 Kb region encoding rhamnose utilization genes that is absent from the L. ruminis motility locus. Phylogenetic analysis of 39 motile bacteria indicated that Lactobacillus motility genes were most closely related to those of motile carnobacteria and enterococci. Transcriptome analysis revealed that motility genes were transcribed at a significantly higher level in motile L. ruminis ATCC27782 than in non-motile ATCC25644. Flagellin proteins were isolated from L. ruminis ATCC27782 and from three other Lactobacillus species, while recombinant flagellin of aflagellate L. ruminis ATCC25644 was expressed and purified from E. coli. These native and recombinant Lactobacillus flagellins, and also flagellate L. ruminis cells, triggered interleukin-8 production in cultured human intestinal epithelial cells in a manner suppressed by short interfering RNA directed against Toll-Like Receptor 5. This study provides genetic, transcriptomic, phylogenetic and immunological insights into the trait of flagellum-mediated motility in the lactobacilli.

The sphingosine-1-phosphate analogue FTY720 impairs mucosal immunity and clearance of the enteric pathogen Citrobacter rodentium.

The sphingosine-1-phosphate (S1P) analogue FTY720 is therapeutically efficacious in multiple sclerosis and in the prevention of transplant rejection. It prevents the migration of lymphocytes to sites of pathology by trapping them within the peripheral lymph nodes, mesenteric lymph nodes (MLNs), and Peyer's patches. However, evidence suggests that its clinical use may increase the risk of mucosal infections. We investigated the impact of FTY720 treatment on susceptibility to gastrointestinal infection with the mouse enteric pathogen Citrobacter rodentium. This attaching and effacing bacterium induces a transient bacterial colitis in immunocompetent mice that resembles human infection with pathogenic Escherichia coli. FTY720 treatment induced peripheral blood lymphopenia, trapped lymphocytes in the MLNs, and prevented the clearance of bacteria when mice were infected with luciferase-tagged C. rodentium. FTY720-treated C. rodentium-infected mice had enhanced colonic inflammation, with significantly higher colon mass, colon histopathology, and neutrophil infiltration than vehicle-infected animals. In addition, FTY720-treated infected mice had significantly lower numbers of colonic dendritic cells, macrophages, and T cells. Gene expression analysis demonstrated that FTY720-treated infected mice had an impaired innate immune response and a blunted mucosal adaptive immune response, including Th1 cytokines. The data demonstrate that the S1P analogue FTY720 adversely affects the immune response to and clearance of C. rodentium.

Influence of adhesion and bacteriocin production by Lactobacillus salivarius on the intestinal epithelial cell transcriptional response.

Lactobacillus salivarius strain UCC118 is a human intestinal isolate that has been extensively studied for its potential probiotic effects in human and animal models. The objective of this study was to determine the effect of L. salivarius UCC118 on gene expression responses in the Caco-2 cell line to improve understanding of how the strain might modulate intestinal epithelial cell phenotypes. Exposure of Caco-2 cells to UCC118 led to the induction of several human genes (TNFAIP3, NFKBIA, and BIRC3) that are negative regulators of inflammatory signaling pathways. Induction of chemokines (CCL20, CXCL-1, and CXCL-2) with antimicrobial functions was also observed. Disruption of the UCC118 sortase gene srtA causes reduced bacterial adhesion to epithelial cells. Transcription of three mucin genes was reduced significantly when Caco-2 cells were stimulated with the ΔsrtA derivative of UCC118 compared to cells stimulated with the wild type, but there was no significant change in the transcription levels of the anti-inflammatory genes. UCC118 genes that were significantly upregulated upon exposure to Caco-2 cells were identified by bacterial genome microarray and consisted primarily of two groups of genes connected with purine metabolism and the operon for synthesis of the Abp118 bacteriocin. Following incubation with Caco-2 cells, the bacteriocin synthesis genes were transcribed at higher levels in the wild type than in the ΔsrtA derivative. These data indicate that L. salivarius UCC118 influences epithelial cells both through modulation of the inflammatory response and by modulation of intestinal cell mucin production. Sortase-anchored cell surface proteins of L. salivarius UCC118 have a central role in promoting the interaction between the bacterium and epithelial cells.

Differential intestinal M-cell gene expression response to gut commensals.

Different rates of bacterial translocation across the gut mucosa have been reported but few studies have examined translocation of commensals at the level of the gut epithelial microfold (M) cell. We used an in vitro M-cell model to quantify translocation and determine the transcriptional response of M cells to various commensal bacteria. The transport kinetics and gene expression profile of M cells in response to different bacterial strains, namely Lactobacillus salivarius, Escherichia coli and Bacteroides fragilis, was assessed. Bacterial strains translocated across M cells with different efficiencies; E. coli and B. fragilis translocated with equal efficiency whereas L. salivarius translocated with less efficiency. Microarray analysis of the M cell response showed both common and differential gene expression changes between the bacterial strains. In the presence of bacteria, but not control beads, up-regulated genes were mainly involved in transcription regulation whereas pro-inflammatory and stress response genes were primarily up-regulated by E. coli and B. fragilis, but not L. salivarius nor beads. Translocation of bacteria and M-cell gene expression responses were confirmed in murine M cells following bacterial challenge in vivo. These results demonstrate that M cells have the ability to discriminate between different commensal bacteria and modify subsequent immune responses.

Bifidobacterial surface-exopolysaccharide facilitates commensal-host interaction through immune modulation and pathogen protection.

Bifidobacteria comprise a significant proportion of the human gut microbiota. Several bifidobacterial strains are currently used as therapeutic interventions, claiming various health benefits by acting as probiotics. However, the precise mechanisms by which they maintain habitation within their host and consequently provide these benefits are not fully understood. Here we show that Bifidobacterium breve UCC2003 produces a cell surface-associated exopolysaccharide (EPS), the biosynthesis of which is directed by either half of a bidirectional gene cluster, thus leading to production of one of two possible EPSs. Alternate transcription of the two opposing halves of this cluster appears to be the result of promoter reorientation. Surface EPS provided stress tolerance and promoted in vivo persistence, but not initial colonization. Marked differences were observed in host immune response: strains producing surface EPS (EPS(+)) failed to elicit a strong immune response compared with EPS-deficient variants. Specifically, EPS production was shown to be linked to the evasion of adaptive B-cell responses. Furthermore, presence of EPS(+) B. breve reduced colonization levels of the gut pathogen Citrobacter rodentium. Our data thus assigns a pivotal and beneficial role for EPS in modulating various aspects of bifidobacterial-host interaction, including the ability of commensal bacteria to remain immunologically silent and in turn provide pathogen protection. This finding enforces the probiotic concept and provides mechanistic insights into health-promoting benefits for both animal and human hosts.

Shining a light on intestinal traffic.

Inflammatory bowel disease (IBD), encompassing Crohn's disease and ulcerative colitis, is associated with enhanced leukocyte infiltration to the gut, which is directly linked to the clinical aspects of these disorders. Thus, leukocyte trafficking is a major target for IBD therapy. Past and emerging techniques to study leukocyte trafficking both in vitro and in vivo have expanded our knowledge of the leukocyte migration process and the role of inhibitors. Various strategies have been employed to target chemokine- and integrin-ligand interactions within the multistep adhesion cascade and the S1P/S1PR1 axis in leukocyte migration. Though there is an abundance of preclinical data demonstrating efficacy of leukocyte trafficking inhibitors, many have yet to be confirmed in clinical studies. Vigilance for toxicity and further research is required into this complex and emerging area of IBD therapy.

Induction and activation of adaptive immune populations during acute and chronic phases of a murine model of experimental colitis.

BACKGROUND: Dextran sodium sulphate (DSS) is commonly used to induce intestinal inflammation in rodents. Despite its continuing importance as a model system for examining IBD pathogenesis, the mucosal and systemic immune responses have not been comprehensively documented. AIMS: The purpose of this study was to dissect functional and phenotypic changes in both immune compartments associated with acute and chronic DSS-induced colitis. METHODS: C57BL/6 mice were exposed to 3% DSS for 6 days followed by 20 days of water, and organs (spleens, MLN and colons) were harvested during both acute and chronic phases of colitis to examine innate and adaptive cell populations. RESULTS: As early as 1 day post DSS, significant changes in the percentage, distribution and activation status of all innate cell populations examined were noted. These striking differences continued in systemic and mucosal lymphoid tissues throughout the acute phase (days 5-12). Significantly, during the late acute and chronic phases T and B cells accumulated in the colon. In contrast, in the spleens of chronically inflamed mice T and B cells were significantly decreased whereas neutrophils, macrophages, and IL-6 and IL-17 positive cells were increased. CONCLUSIONS: Our data provides important insights into the mucosal and systemic immune responses induced by DSS administration. Notably, we show that adaptive immune responses are induced during both acute and chronic colitis. This will facilitate a more informed and sophisticated use of this model both for investigating basic mechanisms of intestinal inflammation and for the evaluation of potential new therapeutic agents for IBD.

Technical Advance: Function and efficacy of an {alpha}4-integrin antagonist using bioluminescence imaging to detect leukocyte trafficking in murine experimental colitis.

Leukocyte trafficking is a therapeutic target in IBD. The integrins α4ß and α4ß1 regulate leukocyte migration into tissues and lymphoid organs. Current strategies rely on biologics, such as mAb, to inhibit leukocyte recruitment. Here we show the in vivo therapeutic effects of a small molecule α4-integrin antagonist (GSK223618A) in a leukocyte-trafficking model and a murine model of colitis. Leukocytes isolated from MLNs of transgenic ß-actin-luc+ mice were injected i.v. into recipients with DSS-induced colitis. Recipient mice were orally gavaged with vehicle or an α4-integrin antagonist 1 h pre-adoptive transfer, followed by bioluminescence whole body and ex vivo organ imaging 4 h post-transfer. To confirm its therapeutic effect, the α4-integrin antagonist was given orally twice daily for 6 days to mice with DSS-induced colitis, starting on Day 3. Clinical, macroscopic, and histological signs of inflammation were assessed and gene-expression profiles analyzed. Using bioluminescence imaging, we tracked and quantified leukocyte migration to the inflamed gut and demonstrated its inhibition by a small molecule α4-integrin antagonist. Additionally, the therapeutic effect of the antagonist was confirmed in DSS-induced colitis in terms of clinical, macroscopic, and histological signs of inflammation. Gene expression analysis suggested enhancement of tissue healing in compound-treated animals. Inhibition of leukocyte trafficking using small molecule integrin antagonists is a promising alternative to large molecule biologics. Furthermore, in vivo bioluminescence imaging is a valuable strategy for preclinical evaluation of potential therapeutics that target leukocyte trafficking in inflammatory diseases.

Type I insulin-like growth factor receptor expression on colorectal adenocarcinoma cell lines is decreased in response to the chemopreventive agent N-acetyl-l-cysteine.

Increased expression of the type I insulin-like growth factor receptor (IGF-1R) is associated with colon cancer, while the antioxidant N-acetyl-l-cysteine (NAC) is known to suppress colonic proliferation. We demonstrate that NAC down-regulates the expression of IGF-1R on three colorectal adenocarcinoma cell lines (HT29, SW480, and LoVo). NAC also abrogates the proliferative effect of IGF-I on HT29 cells. This indicates a novel mechanism for the therapeutic effects of NAC.