Probiotic Lactobacillus reuteri promotes TNF-induced apoptosis in human myeloid leukemia-derived cells by modulation of NF-kappaB and MAPK signalling

The molecular mechanisms of pro-apoptotic effects of human-derived Lactobacillus reuteri ATCC PTA 6475 were investigated in this study. L. reuteri secretes factors that potentiate apoptosis in myeloid leukemia-derived cells induced by tumour necrosis factor (TNF), as indicated by intracellular esterase activity, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling assays and poly (ADP-ribose) polymerase cleavage. L. reuteri downregulated nuclear factor-κB (NF-κB)-dependent gene products that mediate cell proliferation (Cox-2, cyclin D1) and cell survival (Bcl-2, Bcl-xL). L. reuteri suppressed TNF-induced NF-κB activation, including NF-κB-dependent reporter gene expression in a dose-and time-dependent manner. L. reuteri stabilized degradation of IκBα and inhibited nuclear translocation of p65 (RelA). Although phosphorylation of IκBα was not affected, subsequent polyubiquitination necessary for regulated IκBα degradation was abrogated by L. reuteri . In addition, L. reuteri promoted apoptosis by enhancing mitogen-activated protein kinase (MAPK) activities including c-Jun N-terminal kinase and p38 MAPK. In contrast, L. reuteri suppressed extracellular signal-regulated kinases 1/2 in TNF-activated myeloid cells. L. reuteri may regulate cell proliferation by promoting apoptosis of activated immune cells via inhibition of IκBα ubiquitination and enhancing pro-apoptotic MAPK signalling. An improved understanding of L. reuteri- mediated effects on apoptotic signalling pathways may facilitate development of future probiotics-based regimens for prevention of colorectal cancer and inflammatory bowel disease.     

Signal transduction of MCP-1 expression induced by pancreatitis-associated ascitic fluid in pancreatic acinar cells

Pancreatitis-associated ascitic fluid (PAAF) is known to contribute to the progression of acute pancreatitis (AP). We have investigated the capability of PAAF to activate the expression of MCP-1 in pancreatic acinar cells and the involvement of MAPK, NF-κB and STAT3 as downstream signalling transduction pathways. The actions of dexamethasone (Dx) and N-acetylcysteine (NAC) on the PAAF's acinar effects have also been evaluated. Acinar cells were incubated for 1 hr with PAAF collected from rats with severe AP induced by sodium taurocholate in the absence or presence of Dx (10⁻⁷ M) or NAC (30 mM). MCP-1 mRNA expression, phospho-p38-MAPK, IκBα, nuclear p65 levels and nuclear translocation of STAT3 were analysed. In response to PAAF, overexpression of MCP-1, phosphorylation of p38-MAPK, degradation of IκBα and increases in p65 nuclear levels and STAT3 activity were found in acinar cells. PAAF-mediated MCP-1 up-regulation was completely suppressed by Dx and NAC. MAPK activation was only inhibited by NAC, NF-κB activation was repressed by Dx and NAC, and STAT3 pathway was strongly blocked by Dx and significantly reduced by NAC. In conclusion, acinar cells were activated by PAAF to produce MCP-1, mainly via NF-κB and STAT3 pathways. Both downstream pathways were targeted by Dx and NAC to repress the PAAF-mediated acinar MCP-1 up-regulation.     

Modulation of host immune responses, induction of apoptosis and inhibition of NF-kappaB activation by the Bordetella type III secretion system

Bordetella bronchiseptica establishes respiratory tract infections in laboratory animals with high efficiency. Colonization persists for the life of the animal and infection is usually asymptomatic in immunocompetent hosts. We hypothesize that this reflects a balance between immunostimulatory events associated with infection and immunomodulatory events mediated by the bacteria. We have identified 15 loci that are part of a type III secretion apparatus in B. bronchiseptica and three secreted proteins. The functions of the type III secretion system were investigated by comparing the phenotypes of wild-type bacteria with two strains that are defective in type III secretion using in vivo and in vitro infection models. Type III secretion mutants were defective in long-term colonization of the trachea in immunocompetent mice. The mutants also elicited higher titres of anti- Bordetella antibodies upon infection compared with wild-type bacteria. Type III secretion mutants also showed increased lethal virulence in immunodeficient SCID-beige mice. These observations suggest that type III-secreted products of B. bronchiseptica interact with components of both innate and adaptive immune systems of the host. B. bronchiseptica induced apoptosis in macrophages in vitro and inflammatory cells in vivo and type III secretion was required for this process. Infection of an epithelial cell line with high numbers of wild type, but not type III deficient B. bronchiseptica resulted in rapid aggregation of NF-κB into large complexes in the cytoplasm. NF-κB aggregation was dependent on type III secretion and aggregated NF-κB did not respond to TNFα activation, suggesting B. bronchiseptica may modulate host immunity by inactivating NF-κB. Based on these in vivo and in vitro results, we hypothesize that the Bordetella type III secretion system functions to modulate host immune responses during infection.     

F-spondin plays a critical role in murine neuroblastoma survival by maintaining IL-6 expression

F-spondin is associated with the regulation of axonal growth and the development of the nervous system. Its mechanism of action, however, is not clearly understood. In this study, we found that murine neuroblastoma Neuro-2a cells expressed a significant level of IL-6, but only trace amounts of IL-12, tumor necrosis factor α and nitric oxide. Knock-down of F-spondin mRNA in murine neuroblastoma NB41A3 and Neuro-2a cells using small interfering RNAs led to decreased IL-6 levels along with lower resistance to serum starvation and cytotoxic amyloid β_1–42 (Aβ_1–42) peptide. Restoring decline of F-spondin or IL-6 induced by F-spondin knock-down through adding exogenous F-spondin, IL-6 or over-expressing F-spondin reversed the cell death induced by Aβ_1–42 peptide or serum starvation. The decrease of IL-6 level was positively correlated with decrease of NF-κB and inhibition of p38 mitogen-activated protein kinase (MAPK). Over-expressing MEKK, a kinase activator of the p38 MAPK pathway, increased IL-6 production, restored the decrease of p38 induced by F-spondin knock-down, and rescued the cells from death caused by Aβ_1–42 peptide. Taken together, these results suggest that F-spondin may play a critical role in murine neuroblastoma survival under adverse conditions by maintaining IL-6 level via a MEKK/p38 MAPK/NF-κB-dependent pathway.     

TNF-α reduces the level of Staphylococcus epidermidis internalization by bovine endothelial cells

Staphylococcus epidermidis is an environmental opportunistic pathogen associated with bovine intramammary infections. In bacterial infections, the endothelial tissue plays an important role during inflammation and it is the target of proinflammatory cytokines such as tumor necrosis factor α (TNF-α). Therefore, this work was designed to explore the effect of TNF-α on the interaction of S. epidermidis with bovine endothelial cells (BEC). We show that cell signaling activated by TNF-α caused a marked reduction in the number of intracellular S. epidermidis , suggesting that molecules participating in this pathway were involved in the internalization of this bacterium. We also found that S. epidermidis internalization was not associated with basal levels of nuclear factor kappa B (NF-κB) activity because the intracellular number of bacteria recovered after treating BEC with the NF-κB inhibitors, SN50 or BAY 11–7083, was similar to that of the untreated control. Interestingly, inhibition of the basal activity of JNK with SP600125 and p38 with SB203580 caused a decrease in the number of intracellular S. epidermidis . These results suggest that activation of the signaling pathway initiated by TNF-α could play an important role in the phagocytosis of this bacterium. However, the basal activity of NF-κB was shown not to be important for the internalization process of S. epidermidis .     

Differential regulation of c-Jun N-terminal kinase and NF-kappaB pathway by caffeic acid phenethyl ester in astroglial and monocytic cells

Caffeic acid phenethyl ester (CAPE), an active component of propolis extracts, has been known for its specific inhibition of nuclear factor κB (NF-κB) and subsequent anti-inflammatory activity. In this study, we report that (i) CAPE exerts its anti-inflammatory action (inhibition of tumor necrosis factor-induced expression of intercellular adhesion molecule-1 and CC chemokine ligand-2) via NF-κB inhibition by two distinct molecular mechanisms in a cell-specific manner: CAPE inhibited downstream pathways of inhibitor κB (IκB) degradation in monocytic cells, while activation of upstream IκB kinase was suppressed by CAPE pre-treatment in astroglial cells; and (ii) CAPE paradoxically activates the c-Jun N-terminal kinase (JNK) pathway, which might be responsible for its pro-apoptotic action and divergent regulation of proinflammatory mediators such as CXC chemokine ligand-8.     

ChlaDub1 of Chlamydia trachomatis suppresses NF-kappaB activation and inhibits IkappaBalpha ubiquitination and degradation

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes various human diseases, including blindness caused by ocular infection and sexually transmitted diseases resulting from urogenital infection. After infecting host cells, Chlamydiae avoid alarming the host's immune system. Among the immune evasion mechanisms, Chlamydiae can inhibit NF-κB activation, a crucial pathway for host inflammatory responses. In this study, we show that Chla Dub1, a deubiquitinating and deNeddylating protease from C. trachomatis , is expressed in infected cells. In transfection experiments, Chla Dub1 suppresses NF-κB activation induced by several pro-inflammatory stimuli and binds the NF-κB inhibitory subunit IκBα, impairing its ubiquitination and degradation. Thus, we provide further insight into the mechanism by which C. trachomatis may evade the host inflammatory response by demonstrating that Chla Dub1, a protease produced by this microorganism, is capable of inhibiting IκBα degradation and blocking NF-κB activation.