LECT2 association with macrophage-mediated killing of Helicobacter pylori by activating NF-κB and nitric oxide production.

Helicobacter pylori employs unique methods to colonize the stomach, which induces chronic inflammation. It is also able to avoid eradication by macrophages and other immune cells. Leukocyte cell-derived chemotaxin 2 (LECT2), a multi-functional cytokine involved in many pathological conditions, has recently been shown to activate macrophages via the CD209a receptor. Therefore, we aimed to investigate the effects of LECT2 on H. pylori-infected macrophages. Macrophages were treated with recombinant LECT2, and both their ability to kill H. pylori and produce nitric oxide were analyzed. Western blot was performed to determine nuclear translocation and protein phosphorylation of p65, a subunit of nuclear factor (NF)-κB. Transfection experiments were performed to analyze the signaling pathway of LECT2 in macrophages. We found that treatment with LECT2 enhanced H. pylori killing and nitric oxide production in macrophages. In addition, DNA-binding activity and nuclear translocation of p65 were up-regulated by LECT2 treatment. Furthermore, we found that NF-κB activation by LECT2 was mediated by Raf-1 in macrophages, and Raf-1 phosphorylation was specifically altered in response to LECT2. Moreover, LECT2 induced Ser28 phosphorylation in the intracellular domain of CD209a. CD209a Ser28 phosphorylation was required for LECT2-induced Raf-1 and NF-κB activation in RAW264.7 macrophages. Our study showed that the effects of LECT2 on H. pylori killing and nitric oxide production were dependent on CD209a phosphorylation, Raf-1, and NF-κB activation. Together, these results demonstrate for the first time that exposure to LECT2 can modulate specific intracellular mechanisms downstream of CD209a to enhance H. pylori killing and nitric oxide production in macrophages.

15-Deoxy-prostaglandin J2 anti-inflammation in a rat model of chronic obstructive pulmonary disease and human bronchial epithelial cells via Nrf2 activation.

Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates antioxidant and anti-inflammatory genes, and it plays a crucial role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Moreover, 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) plays a protective role against oxidative stress and inflammation both in vivo and in vitro. In a previous study, we found that 15d-PGJ2 increased the expression of Nrf2 in a COPD rat model. This study aims to elucidate the role of 15d-PGJ2 in COPD pathogenesis and the relationship between Nrf2 and human bronchial epithelial (HBE) cells. Normal HBE (HBE) cells were cultured. Following cigarette smoke extract (CSE) stimulation, pre-incubation with or without small interfering RNA (siRNA) Nrf2, and stimulation with or without 15d-PGJ2, the expression levels of Nrf2, NF-κBp65, and IL-8 were detected by reverse transcription-polymerase chain reaction and western blot, respectively. The expression of NF-κBp65 and IL-8 in CSE-stimulated normal HBE cells was inhibited by 15d-PGJ2 at both the mRNA level and the protein level. Moreover, the expression of Nrf2 in normal HBE cells was improved by 15d-PGJ2 at both the mRNA level and the protein level. However, the inhibitory or improving effects of 15d-PGJ2 were disengaged by siRNA Nrf2 at both the mRNA level and the protein level. 15d-PGJ2 possesses anti-inflammatory properties in the pathogenesis of COPD, and HBE cells stimulated by CSE via Nrf2 activation.

Down-regulation of NF-κB signaling by Gordonia bronchialis prevents the activation of gut epithelial cells.

The immunomodulatory power of heat-killed Gordonia bronchialis was studied on gut epithelial cells activated with pro-inflammatory stimuli (flagellin, TNF-α or IL-1ß). Light emission of luciferase-transfected epithelial cells and mRNA expression of IL-1ß, TNF-α, IL-6, CCL20, IL-8 and MCP-1 were measured. NF-κB activation was assessed by immunofluorescence and immunoblotting, and induction of reactive oxygen species (ROS) was evaluated. In vivo inhibitory properties of G. bronchialis were studied with ligated intestinal loop assay and in a mouse model of food allergy. G. bronchialis promoted the down-regulation of the expression of CCL20 and IL-1ß on activated epithelial cells in a dose-dependent manner. A concomitant blocking of nuclear p65 translocation with increased production of ROS was found. In vivo experiments confirmed the inhibition of CCL20 expression and the suppression of IgE sensitization and hypersensitivity symptoms in the food allergy mouse model. In conclusion, heat-killed G. bronchialis inhibited the activation of NF-κB pathway in human epithelial cells, and suppressed the expression of CCL20. These results indicate that G. bronchialis may be used to modulate the initial steps of innate immune activation, which further suppress the allergic sensitization. This approach may be exploited as a therapy for intestinal inflammation.

Regulation of cell survival by resveratrol involves inhibition of NF kappa B-regulated gene expression in prostate cancer cells.

BACKGROUND: Polyphenols have been proposed as antitumoral agents. We have shown that resveratrol (RES) induced cell cycle arrest and promoted apoptosis in prostate cancer cells by inhibition of the PI3K pathway. The RES effects on NF kappaB activity in LNCaP cells (inducible NF kappaB), and PC-3 cells (constitutive NF kappaB) are reported. METHODS: Cells were treated with 1-150 microM of RES during 36 hr. NF kappaB subcellular localization was analyzed by western blot and immunofluorescence. I kappaB alpha was evaluated by immunoprecipitation followed by Western blot. Specific DNA binding of NF kappaB was determined by EMSA assays and NF kappaB-mediated transcriptional activity by transient transfection with a luciferase gene reporter system. RESULTS: RES induced a dose-dependent cytoplasmic retention of NF kappaB mediated by I kappaB alpha in PC-3 cells but not in LNCaP. RES-induced inhibition of NF kappaB specific binding to DNA was more significant in PC-3 cells. NF kappaB-mediated transcriptional activity induced by EGF and TNFalpha were inhibited by RES in both cell lines. LY294002 mimicked RES effects on NF kappaB activity. CONCLUSION: Antiproliferative and apoptotic effects of RES on human prostate cancer cells may be mediated by the inhibition of NF kappaB activity. This mechanism seems to be associated to RES-induced PI3K inhibition. RES could have therapeutic potential for prostate cancer treatment.