Properties of myenteric neurones and mucosal functions in the distal colon of diet-induced obese mice.

Colonic transit and mucosal integrity are believed to be impaired in obesity. However, a comprehensive assessment of altered colonic functions, inflammatory changes and neuronal signalling of obese animals is missing. In mice, we studied the impact of diet-induced obesity (DIO) on: (i) in vivo colonic transit; (ii) signalling in the myenteric plexus by recording responses to nicotine and 2-methyl-5-hydroxytryptamine (2-methyl-5-HT), together with the expression of tryptophan hydroxylase (TPH) 1 and 2, serotonin reuptake transporter, choline acetyltransferase and the paired box gene 4; and (iii) expression of proinflammatory cytokines, epithelial permeability and density of macrophages, mast cells and enterochromaffin cells. Compared with controls, colon transit and neuronal sensitivity to nicotine and 2-methyl-5-HT were enhanced in DIO mice fed for 12 weeks. This was associated with increased tissue acetylcholine and 5-hydroxytryptamine (5-HT) content, and increased expression of TPH1 and TPH2. In DIO mice, upregulation of proinflammatory cytokines was found in fat tissue, but not in the gut wall. Accordingly, mucosal permeability or integrity was unaltered without signs of immune cell infiltration in the gut wall. Body weight showed positive correlations with adipocyte markers, tissue levels of 5-HT and acetylcholine, and the degree of neuronal sensitization. DIO mice fed for 4 weeks showed no neuronal sensitization, had no signs of gut wall inflammation and showed a smaller increase in leptin, interleukin-6 and monocyte chemoattractant protein 1 expression in fat tissue. DIO is associated with faster colonic transit and impacts on acetylcholine and 5-HT metabolism with enhanced responsiveness of enteric neurones to both mediators after 12 weeks of feeding. Our study demonstrates neuronal plasticity in DIO prior to the development of a pathological histology or abnormal mucosal functions. This questions the common assumption that increased mucosal inflammation and permeability initiate functional disorders in obesity.

Diet-induced obesity has neuroprotective effects in murine gastric enteric nervous system: involvement of leptin and glial cell line-derived neurotrophic factor.

Nutritional factors can induce profound neuroplastic changes in the enteric nervous system (ENS), responsible for changes in gastrointestinal (GI) motility. However, long-term effects of a nutritional imbalance leading to obesity, such as Western diet (WD), upon ENS phenotype and control of GI motility remain unknown. Therefore, we investigated the effects of WD-induced obesity (DIO) on ENS phenotype and function as well as factors involved in functional plasticity. Mice were fed with normal diet (ND) or WD for 12 weeks. GI motility was assessed in vivo and ex vivo. Myenteric neurons and glia were analysed with immunohistochemical methods using antibodies against Hu, neuronal nitric oxide synthase (nNOS), Sox-10 and with calcium imaging techniques. Leptin and glial cell line-derived neurotrophic factor (GDNF) were studied using immunohistochemical, biochemical or PCR methods in mice and primary culture of ENS. DIO prevented the age-associated decrease in antral nitrergic neurons observed in ND mice. Nerve stimulation evoked a stronger neuronal Ca(2+) response in WD compared to ND mice. DIO induced an NO-dependent increase in gastric emptying and neuromuscular transmission in the antrum without any change in small intestinal transit. During WD but not ND, a time-dependent increase in leptin and GDNF occurred in the antrum. Finally, we showed that leptin increased GDNF production in the ENS and induced neuroprotective effects mediated in part by GDNF. These results demonstrate that DIO induces neuroplastic changes in the antrum leading to an NO-dependent acceleration of gastric emptying. In addition, DIO induced neuroplasticity in the ENS is likely to involve leptin and GDNF.

Diet-induced obesity in young mice: Consequences on the pancreatic intrinsic nervous system control of insulin secretion.

INTRODUCTION: Obesity has become a pandaemic even in children. We aimed to investigate the impact of obesity in youth on later pancreatic intrinsic nervous system (PINS) phenotype and control of insulin secretion. METHODS: Young mice (5-week-old, T0 group) were fed either a normal diet (ND group) or a Western diet (WD group) for 12 weeks. Pancreas nervous system density, PINS phenotype and pancreas anatomy were analysed by immunohistochemistry at T0 and in adulthood (ND and WD groups). Insulin secretion was also studied in these 3 groups using a new model of ex vivo pancreatic culture, where PINS was stimulated by nicotinic and nitrergic agonists with and without antagonists. Insulin was assayed in supernatants by ELISA. RESULTS: Pancreas nervous system density decreased with age in ND (P < .01) but not in WD mice (P = .08). Western diet decreased the PINS nitrergic component as compared to normal diet (P < .01) but it did not modify its cholinergic component (P = .50). Nicotinic PINS stimulation induced greater insulin secretion in ND compared to WD mice (P < .001) whereas nitrergic stimulation significantly decreased insulin secretion in ND mice (P < .001) and tended to increase insulin secretion in WD mice (P = .08). Endocrine pancreas anatomy was not modified by the Western diet as compared to the normal diet (P = .93). CONCLUSIONS: Early Western diet induced neuronal density and phenotype changes in PINS that might be involved in the pancreas insulin secretion dysfunctions associated with obesity.

The Infant-Derived Bifidobacterium bifidum Strain CNCM I-4319 Strengthens Gut Functionality.

Bifidobacteria are among the first colonisers of the gastrointestinal tract of breast-fed newborns due to, among other things, their ability to metabolise oligosaccharides naturally occurring in human milk. The presence of bifidobacteria in the infant gut has been shown to promote intestinal health and homeostasis as well as to preserve a functional gut barrier, thus positively influencing host health and well-being. Among human-associated gut commensals, Bifidobacterium bifidum has been described as the only species capable of the extracellular degradation of both mucin-type glycans and HMOs, thereby giving this species a special role as a commensal gut forager of both host and diet-derived glycans. In the present study, we assess the possible beneficial properties and probiotic potential of B. bifidum strain CNCM I-4319. In silico genome analysis and growth experiments confirmed the expected ability of this strain to consume HMOs and mucin. By employing various animal models, we were also able to assess the ability of B. bifidum CNCM I-4319 to preserve gut integrity and functionality from stress-induced and inflammatory damage, thereby enforcing its potential as an effective probiotic strain.

Neuroplasticity and neuroprotection in enteric neurons: role of epithelial cells.

Neurons of enteric nervous system (ENS) regulate intestinal epithelial cells (IEC) functions but whether IEC can impact upon the neurochemical coding and survival of enteric neurons remain unknown. Neuro-epithelial interactions were studied using a coculture model composed of IEC lines and primary culture of rat ENS or human neuroblastoma cells (SH-SY5Y). Neurochemical coding of enteric neurons was analysed by immunohistochemistry and quantitative PCR. Neuroprotective effects of IEC were tested by measuring neuron specific enolase (NSE) release or cell permeability to 7-amino-actinomycin D (7-AAD). Following coculture with IEC, the percentage of VIP-immunoreactive (IR) neurons but not NOS-IR and VIP mRNA expression were significantly increased. IEC significantly reduced dopamine-induced NSE release and 7-AAD permeability in culture of ENS and SH-SY5Y, respectively. Finally, we showed that NGF had neuroprotective effects but reduced VIP expression in enteric neurons. In conclusion, our study identified a novel role for IEC in the regulation of enteric neuronal properties.

Inducible mouse model of chronic intestinal pseudo-obstruction by smooth muscle-specific inactivation of the SRF gene.

BACKGROUND & AIMS: Serum response factor (SRF) regulates the expression of muscle genes and immediate early genes. We investigated the consequences of inactivating this transcription factor SRF in adult gastrointestinal smooth muscle cells. METHODS: SRF-floxed mice were crossed with SM-CreER(T2)(ki) mice expressing a tamoxifen-inducible recombinase in smooth muscle cells. Tamoxifen was injected into 12-week-old animals to activate the CreER(T2) and excise the SRF gene. RESULTS: SRF was down-regulated in the smooth muscle cells of the gastrointestinal tract, urinary bladder, and aorta. The mutant mice developed severe dilation of the intestinal tract associated with food stasis and air-fluid levels in the lumen 13 days after tamoxifen treatment. Mutant mice displayed cachexia and died between days 13 and 22. The dilation was associated with a thinning of the muscularis propria and was also observed in the urinary bladder. Ex vivo colonic contraction induced by electric field stimulation and carbachol was impaired in the mutant mice before the occurrence of the dilation phenotype. The expression of several genes, including those encoding smooth muscle actin, the heavy chain of smooth muscle myosin, and smoothelin, was 60% to 70% lower in mutants than in controls, and mutants also had a lower F/G actin ratio. CONCLUSIONS: SRF plays a central role in maintaining visceral smooth muscle contractile function in adults. Mice with a smooth muscle cell-specific SRF mutation develop a severe motility disorder resembling chronic intestinal pseudo-obstruction in humans and may be used as an inducible model of this disorder.

Mucosal mast cell proteases are involved in colonic permeability alterations and subsequent bacterial translocation in endotoxemic rats.

LPS-induced endotoxemia is associated with gut immune stimulation, mucosal inflammation, colonic paracellular permeability (CPP) alteration, and it promotes bacterial translocation (BT). Gut permeability increase linked to LPS promotes mucosal barrier dysfunction resulting to BT. However, the mechanisms involved in these alterations remain unknown. We aimed to evaluate the role of colonic mucosal mast cells and luminal serine protease activity (PA) in the alterations of CPP and BT induced by LPS. Rats receiving doxantrazole, a mast cell stabilizer, combined or not with LPS from Escherichia coli and CPP as well as BT were evaluated after each treatment. Mucosal mast cell activation was assessed by histological methods and by rat mast cell protease 2 level measurement in colonic content. Colonic luminal PA and mucosal inflammation (myeloperoxidase activity) were biochemically determined. In addition, the ability of luminal contents to act on CPP was evaluated in vitro in Ussing chambers. Peripheral administration of LPS promoted mast cell degranulation and increased CPP, BT, mucosal myeloperoxidase activity as well as rat mast cell protease 2 levels, and PA in colonic content. LPS-induced CPP increase and BT were prevented by doxantrazole. In vitro, exposure of the apical side of colonic tissues with supernatants from colonic contents of LPS-treated rats increased CPP. This effect was blocked by the serine protease inhibitor soybean trypsin inhibitor. Our data bring evidence of a key role of mucosal mast cells in LPS-induced increase of CPP and BT through the release of serine proteases into the colonic lumen.

Melanoma cells treated with GGTI and IFN-gamma allow murine vaccination and enhance cytotoxic response against human melanoma cells.

BACKGROUND: Suboptimal activation of T lymphocytes by melanoma cells is often due to the defective expression of class I major histocompatibility antigens (MHC-I) and costimulatory molecules. We have previously shown that geranylgeranyl transferase inhibition (done with GGTI-298) stimulates anti-melanoma immune response through MHC-I and costimulatory molecule expression in the B16F10 murine model [1]. METHODOLOGY/PRINCIPAL FINDINGS: In this study, it is shown that vaccination with mIFN-gand GGTI-298 pretreated B16F10 cells induces a protection against untreated tumor growth and pulmonary metastases implantation. Furthermore, using a human melanoma model (LB1319-MEL), we demonstrated that in vitro treatment with hIFN-gamma and GGTI-298 led to the up regulation of MHC-I and a costimulatory molecule CD86 and down regulation of an inhibitory molecule PD-1L. Co-culture experiments with peripheral blood mononuclear cells (PBMC) revealed that modifications induced by hIFN-gamma and GGTI-298 on the selected melanoma cells, enables the stimulation of lymphocytes from HLA compatible healthy donors. Indeed, as compared with untreated melanoma cells, pretreatment with hIFN-gamma and GGTI-298 together rendered the melanoma cells more efficient at inducing the: i) activation of CD8 T lymphocytes (CD8+/CD69+); ii) proliferation of tumor-specific CD8 T cells (MelanA-MART1/TCR+); iii) secretion of hIFN-gamma; and iv) anti-melanoma specific cytotoxic cells. CONCLUSIONS/SIGNIFICANCE: These data indicate that pharmacological treatment of melanoma cell lines with IFN-gamma and GGTI-298 stimulates their immunogenicity and could be a novel approach to produce tumor cells suitable for vaccination and for stimulation of anti-melanoma effector cells.

Pathways involved in gut mucosal barrier dysfunction induced in adult rats by maternal deprivation: corticotrophin-releasing factor and nerve growth factor interplay.

Neonatal maternal deprivation (NMD) increases gut paracellular permeability (GPP) through mast cells and nerve growth factor (NGF), and modifies corticotrophin-releasing factor (CRF) and corticosterone levels. CRF, corticosterone and mast cells are involved in stress-induced mucosal barrier impairment. Consequently, this study aimed to specify whether corticosteronaemia and colonic expression of both preproCRF and CRF are modified by NMD, and to determine if altered expression may participate in the elevated GPP in connection with NGF and mast cells. Male Wistar rat pups were either separated from postnatal days 2-14, or left undisturbed with their dam. At 12 weeks of age, adult rats were treated with mifepristone (an antagonist of corticoid receptors), alpha-helical CRF((9-41)) (a non-specific CRF receptor antagonist), or SSR-125543 (CRF-R(1) receptor antagonist). We also determined corticosteronaemia and both colonic preproCRF and CRF expression. Then, control rats were treated by CRF, doxantrazole (mast cell stabilizer), BRX-537A (a mast cell activator) and anti-NGF antibody. NMD did not modify colonic CRF level but increased colonic preproCRF expression and corticosteronaemia. Peripheral CRF, via CRF-R(1) receptor, but not corticosterone, was involved in the elevated GPP observed in these rats, through a mast-cell-mediated mechanism, since the increase of GPP induced by exogenous CRF was abolished by doxantrazole. Anti-NGF antibody treatment also reduced the elevated GPP induced by CRF or BRX-537A. CRF acts through CRF-R(1) receptors to stimulate NGF release from mast cells, which participates in the elevated GPP observed in NMD adult rats. This suggests that early traumatic experience induced neuro-endocrine dysfunction, involved in alterations of gut mucosal barrier.

Sex steroid regulation of macrophage migration inhibitory factor in normal and inflamed colon in the female rat.

BACKGROUND AND AIMS: Sex steroids influence IBD symptoms. Macrophage migration inhibitory factor (MIF), a target of sex steroids in other inflammatory models, promotes interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha release in colitis. We investigated whether estradiol and progesterone influence MIF, IL-1beta, and TNF-alpha production in experimental colitis. METHODS: Colonic MIF, IL-1beta, and TNF-alpha levels were measured in cyclic and ovariectomized rats, with or without estradiol benzoate (EB) or progesterone (P) replacement. MIF distribution was assessed by immunohistochemistry. Cytokines, myeloperoxidase activity, macroscopic damage, and plasma corticosterone were assessed 24 hours after intrarectal trinitrobenzene sulfonic acid (TNBS), with and without neutralizing anti-MIF antibody. Effects of EB and P on myeloperoxidase activity and MIF concentration were also assessed at 7 days in dextran sulfate sodium-induced colitis. RESULTS: Basal IL-1beta and TNF-alpha contents did not fluctuate during the estrous cycle, while MIF concentrations increased from estrus (estrogen dominance) to metestrus (P dominance; P < .05). EB and P treatment mimicked these effects in ovariectomized rats, and similarly altered MIF immunostaining. Progesterone dominance aggravated TNBS colitis in comparison with estrogen. Progesterone enhanced TNBS-induced MIF (P < .001) and TNF-alpha (P < .01) production, while EB decreased MIF (P < .01) and IL-beta levels (P < .01). Anti-MIF antibody prevented P-mediated up-regulation of TNF-alpha, improved TNBS colitis, and enhanced plasma corticosterone. At 7 days after dextran sulfate sodium, EB decreased myeloperoxidase activity and MIF concentration, while P had no effect. CONCLUSIONS: Estrogen decreases while progesterone increases MIF production in the female rat colon. Changes in basal MIF contents may affect colon susceptibility to inflammation, by modulating TNF-alpha and IL-1beta production during early stages of colitis.

Myosin light chain kinase is involved in lipopolysaccharide-induced disruption of colonic epithelial barrier and bacterial translocation in rats.

Sepsis is associated with bacterial translocation (BT) and changes in colonic paracellular permeability (CPP), but the link between these effects is unknown. The present study aimed to identify whether changes in CPP after lipopolysaccharide (LPS) administration triggers BT, colonic inflammation, visceral pain, and sickness behavior and to evaluate the role of myosin light chain kinase (MLCK) in colonocyte cytoskeleton contraction. Rats received the MLCK inhibitor ML-7 alone or combined with LPS. CPP was measured for 6 hours after administration. Visceral pain, food intake, BT, electron microscopy of tight junctions of colonocytes, cytokine levels, and Western blotting of phosphorylated MLC from colonic mucosa were assessed in a time range of 0 to 3 hours after treatment. Sepsis increased CPP at 0 to 6 hours after LPS and associated with tight junction morphological changes, increased MLC phosphorylation, and mucosal release of proinflammatory cytokines. Massive BT, visceral hyperalgesia, and reduced food intake were also observed. Addition of ML-7 prevented all LPS-induced effects, except for changes in food intake. In conclusion, LPS-mediated effects on CPP include gut inflammation, BT, and visceral hyperalgesia. Inhibition of MLCK-dependent colonocyte cytoskeleton contraction by ML-7 prevents the LPS-induced alterations of CPP and its subsequent effects.