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.     

Sex hormone modulation of cell growth and apoptosis of the human monocytic/macrophage cell line

Sex hormones seem to modulate the immune/inflammatory responses by different mechanisms in female and male rheumatoid arthritis patients. The effects of 17β-oestradiol and of testosterone were tested on the cultured human monocytic/macrophage cell line (THP-1) activated with IFN-γ in order to investigate their role in cell proliferation and apoptosis. Activated human THP-1 cells were cultured in the presence of 17β-oestradiol and testosterone (final concentration, 10 nM). The evaluation of markers of cell proliferation included the NF-κB DNA-binding assay, the NF-κB inhibition complex, the proliferating cell nuclear antigen expression and the methyl-tetrazolium salt test. Apoptosis was detected by the annexin V-propidium assay and by the cleaved poly-ADP ribose polymerase expression. Specific methods included flow analysis cytometry scatter analysis, immunocytochemistry and western blot analysis. Cell growth inhibition and increased apoptosis were observed in testosterone-treated THP-1 cells. Increased poly-ADP ribose polymerase-cleaved expression and decreased proliferating cell nuclear antigen expression, as well as an increase of IκB-α and a decrease of the IκB-α phosphorylated form (ser 32), were found in testosterone-treated THP-1 cells. However, the NF-κB DNA binding was found increased in 17β-oestradiol-treated THP-1 cells. The treatment with staurosporine (enhancer of apoptosis) induced decreased NF-κB DNA binding in all conditions, but particularly in testosterone-treated THP-1 cells. Treatment of THP-1 by sex hormones was found to influence cell proliferation and apoptosis. Androgens were found to increase the apoptosis, and oestrogens showed a protective trend on cell death – both acting as modulators of the NF-κB complex.     

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.     

Anti-inflammatory activity of a globular adiponectin function on RAW 264 cells stimulated by lipopolysaccharide from Aggregatibacter actinomycetemcomitans

Adiponectin is an adipokine with potent anti-inflammatory properties. We previously reported that a globular adiponectin (gAd) suppresses Aggregatibacter actinomycetemcomitans lipopolysaccharide-induced nuclear factor-κB activity, suggesting an anti-inflammatory effect of gAd. In this study, we investigated whether gAd is able to modulate the effect of A. actinomycetemcomitans lipopolysaccharide on cytokine induction in a murine macrophage cell line (RAW 264). The phosphorylation of p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and IκB kinase α/β and the degradation of IκB, which were induced by A. actinomycetemcomitans lipopolysaccharide intoxication, were clearly reduced in gAd-pretreated RAW 264 cells compared with the untreated cells. Expression levels of tumor necrosis factor (TNF)-α and interleukin-10 (IL-10) mRNA were assessed by real-time PCR. Cell-free supernatants were collected after 12 h of stimulation and analyzed by enzyme-linked immunosorbent assay for TNF-α and IL-10. Pretreatment with gAd significantly inhibited the A. actinomycetemcomitans lipopolysaccharide-induced TNF-α mRNA expression and protein secretion. In contrast, pretreatment with gAd significantly enhanced the A. actinomycetemcomitans lipopolysaccharide-induced IL-10 mRNA expression and protein secretion. These data suggest a mechanism for the anti-inflammatory activity of gAd in local inflammatory lesions, such as periodontitis.     

Thalidomide inhibits lipopolysaccharide-induced nitric oxide production and prevents lipopolysaccharide-mediated lethality in mice

The effect of thalidomide on lipopolysaccharide-induced nitric oxide (NO) production was studied using RAW 264.7 macrophage-like cells. Thalidomide significantly inhibited lipopolysaccharide-induced NO production via reduced expression of an inducible NO synthase. Thalidomide reduced the phosphorylation of the p65 nuclear factor-κB subunit, inhibitory κB (IκB) and IκB kinase in lipopolysaccharide-stimulated cells. However, thalidomide did not affect the expression of interferon-β (IFN-β) and interferon regulatory factor-1 in response to lipopolysaccharide. Further, thalidomide inhibited the MyD88 augmentation in lipopolysaccharide-stimulated cells, whereas it did not alter the expression of TIR domain-containing adaptor-inducing IFN-β in the MyD88-independent pathway. Thalidomide significantly inhibited the NO production in response to Pam₃Cys, CpG DNA and imiquimod as MyD88-dependent Toll-like receptor (TLR) ligands, but not polyI:C as a MyD88-independent TLR ligand. Therefore, thalidomide was suggested to inhibit lipopolysaccharide-induced NO production via downregulation of the MyD88-dependent signal pathway. The anti-inflammatory action of thalidomide might be involved in the prevention of lipopolysaccharide-mediated lethality in mice.     

Ca2+-Mediated Activation of c-Jun N-Terminal Kinase and Nuclear Factor κB by NMDA in Cortical Cell Cultures

Abstract: We examined the possibility that c-Jun N-terminal kinase (JNK) and nuclear factor κB (NF-κB) might be involved in intracellular signaling cascades that mediate NMDA-initiated neuronal events. Exposure of cortical neurons to 100 µ M NMDA induced activation of JNK within 1 min. Activity of JNK was further increased over the next 5 min and then declined by 30 min. Similarly, ionomycin, a selective Ca²⁺ ionophore, induced activation of JNK. The NMDA-induced activation of JNK was abrogated in the absence of extracellular Ca²⁺, suggesting that Ca²⁺ entry is necessary and sufficient for the JNK activation. Immunohistochemistry with anti-NF-κB antibody demonstrated nuclear translocation of NF-κB within 5 min following NMDA treatment. NMDA treatment also enhanced the DNA binding activity of nuclear NF-κB in a Ca²⁺-dependent manner. Treatment with 3 m M aspirin blocked the NMDA-induced activation of JNK and NF-κB. Neuronal death following a brief exposure to 100 µ M NMDA was Ca²⁺ dependent and attenuated by addition of aspirin or sodium salicylate. The present study suggests that Ca²⁺ influx is required for NMDA-induced activation of JNK and NF-κB as well as NMDA neurotoxicity. This study also implies that aspirin may exert its neuroprotective action against NMDA through blocking the NMDA-induced activation of NF-κB and JNK.     

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.     

Role of lipids in killing mycobacteria by macrophages: evidence for NF-κB-dependent and -independent killing induced by different lipids

We have shown that several lipids can modulate the macrophage innate immune response against mycobacteria and enhance their killing. Since NF-κB is required for mycobacterial killing, we tested the ability of lipids to activate NF-κB in uninfected macrophages and those infected with mycobacteria. In uninfected cells, sphingomyelin (SM), phosphatidylinositol-4-phosphate (PIP) and arachidonic acid (AA) enhanced NF-κB activation and the cell surface expression of CD69, a macrophage activation marker regulated by NF-κB. Sphingosine (Sph), sphingosine-1-phosphate (S1P), diacylglycerol (DAG), eicosapentanoic acid (EPA) and phosphatidyl choline (PC) failed to activate either NF-κB or CD69. Ceramide (Cer) activated CD69 expression without activating NF-κB. In Mycobacterium smegmatis- infected cells, NF-κB was transiently activated in a manner that was enhanced by SM, PIP and AA. In contrast Mycobacterium avium mostly repressed NF-κB activation and only SM and AA could induce its partial activation. While lipids that activate NF-κB in uninfected cells tend to kill mycobacteria in macrophages Sph and S1P failed to activate NF-κB under most conditions but nevertheless enhanced killing of M. smegmatis , M. avium and M. tuberculosis H37Rv. Our results argue that both NF-κB-dependent and -independent mechanisms are involved in macrophage killing of mycobacteria and that both mechanisms can be enhanced by selected lipids.