Posttranslational Inhibition of Proinflammatory Chemokine Secretion in Intestinal Epithelial Cells Implications for Specific IBD Indications

Background and Aim: Inflammatory bowel diseases (IBD) are immune-mediated chronic diseases that are characterized by an overreaction of the intestinal immune system to the intestinal microbiota. VSL#3, a mixture of 8 different lactic acid bacteria, is a clinically relevant probiotic compound in the context of IBD, but the bacterial structures and molecular mechanisms underlying the observed protective effects are largely unknown. The intestinal epithelium plays a very important role in the maintenance of the intestinal homeostasis, as the intestinal epithelial cells (IEC) are capable of sensing, processing, and reacting upon signals from the luminal microbiota and the intestinal immune system. This immune regulatory function of the IEC is lost in IBD owing to dysregulated activation of the IEC. Thus, the aim of this study was to reveal protective mechanisms of VSL#3 on IEC function.

Results: In vitro, VSL#3 was found to selectively inhibit activation-induced secretion of the T-cell chemokine interferon-inducible protein (IP)-10 in IEC. Cell wall-associated proteins of VSL#3-derived Lactobacillus casei (L. casei) were identified to be the active anti-inflammatory component of VSL#3. Mechanistically, L. casei did not impair initial IP-10 protein production, but induced posttranslational degradation of IP-10 in IEC. Feeding studies in tumor necrosis factor (TNF)(Delta ARE/+) mice, a mouse model for experimental ileitis, revealed that neither VSL#3 nor L. casei is capable of reducing ileal inflammation. Even preweaning feeding of VSL#3 did not prevent the development of severe ileitis in TNF Delta ARE/+ mice. In contrast, VSL#3 feeding studies in IL-10-/- mice, a model for experimental colitis, revealed that VSL#3 has local, intestinal compartment-specific protective effects on the development of inflammation. Reduced histopathologic inflammation in the cecum of IL-10-/- mice after VSL#3 treatment was found to correlate with reduced levels of IP-10 protein in primary cecal epithelial cells.

Conclusion and Outlook: These results suggest that the inhibitory effect of VSL#3-derived L. casei on IP-10 secretion in IEC is an important probiotic mechanism that contributes to the anti-inflammatory effects of VSL#3 in specific subsets of patients with IBD. An important future aim is the identification of the active probiotic protein, which could serve as a basis for the development of new efficient therapies in the context of IBD.

Post-Translational Inhibition of IP-10 Secretion in IEC by Probiotic Bacteria: Impact on Chronic Inflammation

Background: Clinical and experimental studies suggest that the probiotic mixture VSL#3 has protective activities in the context of inflammatory bowel disease (IBD). The aim of the study was to reveal bacterial strain-specific molecular mechanisms underlying the anti-inflammatory potential of VSL#3 in intestinal epithelial cells (IEC).

Methodology/Principal Findings: VSL#3 inhibited TNF-induced secretion of the T-cell chemokine interferon-inducible protein (IP-10) in Mode-K cells. Lactobacillus casei (L. casei) cell surface proteins were identified as active anti-inflammatory components of VSL#3. Interestingly, L. casei failed to block TNF-induced IP-10 promoter activity or IP-10 gene transcription at the mRNA expression level but completely inhibited IP-10 protein secretion as well as IP-10-mediated T-cell transmigration. Kinetic studies, pulse-chase experiments and the use of a pharmacological inhibitor for the export machinery (brefeldin A) showed that L. casei did not impair initial IP-10 production but decreased intracellular IP-10 protein stability as a result of blocked IP-10 secretion. Although L. casei induced IP-10 ubiquitination, the inhibition of proteasomal or lysosomal degradation did not prevent the loss of intracellular IP-10. Most important for the mechanistic understanding, the inhibition of vesicular trafficking by 3-methyladenine (3-MA) inhibited IP-10 but not IL-6 expression, mimicking the inhibitory effects of L. casei. These findings suggest that L. casei impairs vesicular pathways important for the secretion of IP-10, followed by subsequent degradation of the proinflammatory chemokine. Feeding studies in TNF Delta ARE and IL-10(-/-) mice revealed a compartimentalized protection of VSL#3 on the development of cecal but not on ileal or colonic inflammation. Consistent with reduced tissue pathology in IL-10(-/-) mice, IP-10 protein expression was reduced in primary epithelial cells.

Conclusions/Significance: We demonstrate segment specific effects of probiotic intervention that correlate with reduced IP-10 protein expression in the native epithelium. Furthermore, we revealed post-translational degradation of IP-10 protein in IEC to be the molecular mechanism underlying the anti-inflammatory effect.

Effect of tea polyphenols and tea pigments on the inhibition of precancerous liver lesions in rats

The objective of this study was to investigate the inhibitory effect of tea components, tea polyphenols and tea pigments, on precancerous liver lesions in rats. A rat liver precancerous lesion model was established by multiple low-dosage N-nitrosodiethylamine (NDEA) injections, followed by intraperitoneal CCl4 injection and partial hepatectomy (PH). Tea pigments (0.1%) or tea polyphenols (0.1%) were given to Wistar rats in drinking water during the eight weeks of the experiment. The number and area of glutathione S-transferase Pi-positive foci in the rat liver were used as biomarkers of precancerous liver lesions. Western and Northern blot techniques were used to detect rat liver GST-Pi expression at the protein and mRNA levels. At the end of the experiment tea polyphenols and tea pigments significantly decreased the number and area of GST-Pi-positive foci that were overexpressed in the NDEA-CCl4-PH-treated rats compared with the positive control group. The results also showed that GST-Pi mRNA and protein expression increased significantly in the NDEA-CCl4-PH-treated group, which is consistent with the changing of GST-Pi-positive foci. Tea pigments and tea polyphenols had an inhibitory effect on the overexpression of GST-Pi mRNA and protein in NDEA-CCl4-PH-treated rats. These results suggest that tea pigments and tea polyphenols are effective in preventing the occurrence and progression of precancerous liver lesions in rats.

α-Adrenergic agonism enhances the growth hormone (GH) response to GH-releasing hormone through an inhibition of hypothalamic somatostatin release in normal men

The purpose of this study was to investigate the precise mechanism by which central a-adrenergic pathways modulate GH secretion in humans. In 10 normal subjects we compared the pattern of clonidine-induced GH release to that elicited by GH-releasing hormone (GHRH) given at a time of presumably similar responsiveness of the somatotrope. We also evaluated the effect of stimulation by GHRH (either endogenous, by administration of clonidine, or exogenous) on the GH response to a further exogenous GHRH stimulation. In 2 experiments the administration of clonidine (0.150 mg, orally) at 0 or 60 min was followed by a GHRH [GRF-(1-29); 1 µg/kg, iv] challenge at 180 min. In other experiments subjects received on separate occasions placebo or clonidine at 0 min, followed by GHRH at 60 min and again at 180 min. In a further experiment the administration of clonidine at 0 min was followed by 2 GHRH challenges (60 and 180 min later). The administration of clonidine 60 or 120 min, but not 180 min, before the GHRH bolus significantly (P <0.01) increased the GH responses to this challenge compared to those elicited by GHRH when given after placebo in a period of a similar somatotrope responsiveness. These, in turn, were significantly (P <0.05) higher than those elicited by clonidine alone. The close relationship between pre-GHRH plasma GH values and GHRH-elicited GH peaks, not observed for clonidine, was lost after pretreatment with this drug. These data indicate that clonidine was able to disrupt the intrinsic hypothalamic-somatotroph rhythm, suggesting that a-adrenergic pathways have a major inhibitory effect on somatostatin release. Our data also indicate that GH responses to a GHRH bolus administered 120 min after a prior GHRH challenge are dependent on two parameters: the intrinsic hypothalamic-somatotroph rhythm at the time of the second GHRH bolus, and the magnitude of GH secretion elicited by the previous somatotroph stimulation. In summary, a-adrenergic agonism appears to act primarily in GH control by inhibiting the hypothalamic release of somatostatin, rather than by stimulating GHRH secretion.

Glycation, glycoxidation, and cross-linking of collagen by glucose. Kinetics, mechanisms, and inhibition of late stages of the Maillard reaction

The Maillard or browning reaction between sugar and protein contributes to the increased chemical modification and cross-linking of long-lived tissue proteins in diabetes. To evaluate the role of glycation and oxidation in these reactions, we have studied the effects of oxidative and antioxidative conditions and various types of inhibitors on the reaction of glucose with rat tail tendon collagen in phosphate buffer at physiological pH and temperature. The chemical modifications of collagen that were measured included fructoselysine, the glycoxidation products N epsilon-(carboxymethyl)lysine and pentosidine and fluorescence. Collagen cross-linking was evaluated by analysis of cyanogen bromide peptides using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by changes in collagen solubilization on treatment with pepsin or sodium dodecylsulfate. Although glycation was unaffected, formation of glycoxidation products and cross-linking of collagen were inhibited by antioxidative conditions. The kinetics of formation of glycoxidation products proceeded with a short lag phase and were independent of the amount of Amadori adduct on the protein, suggesting that autoxidative degradation of glucose was a major contributor to glycoxidation and cross-linking reactions. Chelators, sulfhydryl compounds, antioxidants, and aminoguanidine also inhibited formation of glycoxidation products, generation of fluorescence, and cross-linking of collagen without significant effect on the extent of glycation of the protein. We conclude that autoxidation of glucose or Amadori compounds on protein plays a major role in the formation of glycoxidation products and cross-liking of collagen by glucose in vitro and that chelators, sulfhydryl compounds, antioxidants, and aminoguanidine act as uncouplers of glycation from subsequent glycoxidation and cross-linking reactions.

SAHA overcomes FLIP-mediated inhibition of SMAC mimetic-induced apoptosis in mesothelioma

Malignant pleural mesothelioma (MPM) is a highly pro-inflammatory malignancy that is rapidly fatal and increasing in incidence. Cytokine signaling within the pro-inflammatory tumor microenvironment makes a critical contribution to the development of MPM and its resistance to conventional chemotherapy approaches. SMAC mimetic compounds (SMCs) are a promising class of anticancer drug that are dependent on tumor necrosis factor alpha (TNFa) signaling for their activity. As circulating TNFa expression is significantly elevated in MPM patients, we examined the sensitivity of MPM cell line models to SMCs. Surprisingly, all MPM cell lines assessed were highly resistant to SMCs either alone or when incubated in the presence of clinically relevant levels of TNFa. Further analyses revealed that MPM cells were sensitized to SMC-induced apoptosis by siRNA-mediated downregulation of the caspase 8 inhibitor FLIP, an antiapoptotic protein overexpressed in several cancer types including MPM. We have previously reported that FLIP expression is potently downregulated in MPM cells in response to the histone deacetylase inhibitor (HDACi) Vorinostat (SAHA). In this study, we demonstrate that SAHA sensitizes MPM cells to SMCs in a manner dependent on its ability to downregulate FLIP. Although treatment with SMC in the presence of TNFa promoted interaction between caspase 8 and the necrosis-promoting RIPK1, the cell death induced by combined treatment with SAHA and SMC was apoptotic and mediated by caspase 8. These results indicate that FLIP is a major inhibitor of SMC-mediated apoptosis in MPM, but that this inhibition can be overcome by the HDACi SAHA. © 2013 Macmillan Publishers Limited All rights reserved.

Histamine H3 receptor-mediated inhibition of sympathetically evoked mydriasis in rats

This study was designed to determine if the histamine H3 receptor agonist R-alpha-methylhistamine would play a role in modulation of sympathetically evoked mydriasis in anesthetized rats, and if so, to ascertain the specific receptor subtype(s) involved. Reproducible frequency-response curves of pupillary dilation were generated by stimulation of the cervical preganglionic sympathetic nerve (1-32 Hz). Systemic administration of R-alpha-methylhistamine (0.3-3.0 mg kg(-1)) produced a dose-related inhibition of the evoked mydriasis. The greatest inhibition was seen at lower frequency levels, with about 43% depression observed at 2 Hz. The specific histamine H3 receptor antagonist, clobenpropit (3.0 mg kg(-1), i.v.), blocked the inhibitory effect of R-alpha-methylhistamine, whereas neither the histamine H2 receptor antagonist, cimetidine (5.0 mg kg(-1), i.v.), nor the histamine H1 receptor antagonist, chlorpheniramine (0.5 mg kg(-1), i.v.), was effective. The histamine H2 receptor agonist, dimaprit (10 mg kg(-1), i.v.), was also without effect on the evoked mydriasis. R-alpha-methylhistamine (3.0 mg kg(-1)) did not inhibit phenylephrine-induced mydriasis. These results support the conclusion that R-alpha-methylhistamine produces inhibition of sympathetically evoked mydriasis via histamine H3 receptor stimulation, presumably by an action on presynaptic histamine H3 receptors.