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.     

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.     

Axon regeneration in young adult mice lacking Nogo-A/B

After injury, axons of the adult mammalian brain and spinal cord exhibit little regeneration. It has been suggested that axon growth inhibitors, such as myelin-derived Nogo, prevent CNS axon repair. To investigate this hypothesis, we analyzed mice with a nogo mutation that eliminates Nogo-A/B expression. These mice are viable and exhibit normal locomotion. Corticospinal tract tracing reveals no abnormality in uninjured nogo-A/B(-/-) mice. After spinal cord injury, corticospinal axons of young adult nogo-A/B(-/-) mice sprout extensively rostral to a transection. Numerous fibers regenerate into distal cord segments of nogo-A/B(-/-) mice. Recovery of locomotor function is improved in these mice. Thus, Nogo-A plays a role in restricting axonal sprouting in the young adult CNS after injury.     

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.     

Muscle precursor cells isolated from aged rats exhibit an increased tumor necrosis factor-α response

Improving muscle precursor cell (MPC, muscle-specific stem cells) function during aging has been implicated as a key therapeutic target for improving age-related skeletal muscle loss. MPC dysfunction during aging can be attributed to both the aging MPC population and the changing environment in skeletal muscle. Previous reports have identified elevated levels of tumor necrosis factor-α (TNF-α) in aging, both circulating and locally in skeletal muscle. The purpose of the present study was to determine if age-related differences exist between TNF-α-induced nuclear factor-kappa B (NF-κB) activation and expression of apoptotic gene targets. MPCs isolated from 32-month-old animals exhibited an increased NF-κB activation in response to 1, 5, and 20 ng mL⁻¹ TNF-α, compared to MPCs isolated from 3-month-old animals. No age differences were observed in the rapid canonical signaling events leading to NF-κB activation or in the increase in mRNA levels for TNF receptor 1, TNF receptor 2, TNF receptor-associated factor 2 (TRAF2), or Fas (CD95) observed after 2 h of TNF-α stimulation. Interestingly, mRNA levels for TRAF2 and the cell death-inducing receptor, Fas (CD95), were persistently upregulated in response to 24 h TNF-α treatment in MPCs isolated from 32-month-old animals, compared to 3-month-old animals. Our data indicate that age-related differences may exist in the regulatory mechanisms responsible for NF-κB inactivation, which may have an effect on TNF-α-induced apoptotic signaling. These findings improve our understanding of the interaction between aged MPCs and the changing environment associated with age, which is critical for the development of potential clinical interventions aimed at improving MPC function with age.     

Age‐dependent failure of axon regeneration in organotypic culture of gerbil auditory midbrain

Inferior colliculus (IC) slice cultures from postnatal (P) day 6–8 gerbils exhibit axonal regeneration across a lesion site, and these regrowing processes can form synapses. To determine whether regenerative capacity is lost in older tissue, as occurs in vivo, slices from P12–21‐day animals were grown under similar conditions. While these cultures displayed a near complete loss of neurons over 6 days in vitro, glutamate receptor antagonists (AP5 and/or CNQX) significantly enhanced survival, particularly at P12–15. In contrast, several growth factors or high potassium did not improve neuron survival. Therefore, axonal regeneration was assessed following complete transection of the commissure in AP5/CNQX‐treated IC cultures from P12 animals. Neurofilament staining revealed that transected commissural axons survived for 6 days in vitro, but only a few processes crossed the lesion site and these axons did not extend into the contralateral lobe. In contrast, there was robust axonal sprouting and growth within one lobe of the IC, remote from the lesion site. When P6 and P12 tissue was explanted onto a coated substrate, the P6 axons grew onto the substrate, but the P12 axons were seemingly prevented from reaching the substrate by a veil of nonneuronal cells. Coculture of the IC and one of its afferent populations, the lateral superior olive, provided a similar finding, indicating that failure to regenerate was a general property at the age examined. These data show that neuron survival is not sufficient to permit axon regeneration at P12, and suggest that P12 lesion sites manufacture a prohibitive substrate since process outgrowth is blocked specifically at the commissure transection. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 267–280, 1999     

Poster Sessions BP05: Neuroimmunology

Experimental allergic encephalomyelitis (EAE) is the animal model for Multiple Sclerosis (MS), the chronic autoimmune disease of the central nervous system (CNS). Activation of NF-κB requires the activity of IkB kinase (IKK) complex containing (IKKa and IKKb) and the regulatory protein NEMO (NF-κB essential modifier). Recently it has been shown that peptides corresponding to the NEMO-binding domain (NBD) of IKKa or IKKb specifically inhibit the induction of NF-κB activation without inhibiting the basal NF-κB activity. The present study underlines the importance of cell-permeable NBD peptides in inhibiting the disease process of adoptively-transferred EAE in female SJL/J mice. Immunocytochemical analysis of spinal cords of EAE mice showed that there was marked induction of NF-κB activation as evidenced by enhanced p65 (the RelA subunit of NF-κB) expression compared to that of control mice. Double-labelling analysis of p65 and cell-specific markers showed that p65 was mainly expressed by astrocytes, microglia and infiltrating macrophages. Next we examined the effect of NBD peptides on the disease process of EAE. Interestingly, clinical symptoms of EAE were much lower in mice receiving wild type NBD peptides. In contrast, mutated NBD peptides had no effect on the clinical symptoms of EAE. Taken together, our results support the conclusion that activation of NF-κB participates in the disease process of EAE and that inhibitors of NF-κB activation may ameliorate the neuroinflammatory disease process in MS patients.
Acknowledgements:   This study was supported by NIH grants (NS39940 and AG19487.     

Lentiviral-Mediated Netrin-1 Overexpression Improves Motor and Sensory Functions in SCT Rats Associated with SYP and GAP-43 Expressions

Spinal cord injury (SCI), as a major cause of disability, usually causes serious loss of motor and sensory functions. As a bifunctional axonal guidance cue, netrin-1 can attract axons via the deleted in colorectal cancer (DCC) receptors and repelling others via Unc5 receptors, but its exact role in the recovery of motor and sensory function has not well been studied, and the mechanisms remains elusive. The aim of this experiment is to determine whether lentiviral (LV)-mediated overexpression of netrin-1 or RNA interference (RNAi) can regulate the functional recovery in rats subjected to spinal cord transection (SCT). Firstly, two lentiviral vectors including Lv-exNtn-1 (netrin-1 open reading frame (ORF)) and Lv-shNtn-1 (netrin-1 sh) were constructed and injected into spinal cords rostral and caudal to the transected lesion site. Overexpressing netrin-1 enhanced significantly locomotor function, and reduced thermal and mechanical stimuli in vivo, compared with the control, while silencing netrin-1 did not significantly change the situation. Western blot and immunostaining analysis confirmed that netrin-1 ORF treatment not only effectively increased the expression level of netrin-1, also up-regulated the level of synaptophysin (SYP) in spinal cord rostral to the lesion, but also enhanced growth-associated protein-43 (GAP-43) expression in spinal cord caudal to the lesion site. Comparatively, knockdown of netrin-1 did not give rise to positive findings in our experimental condition. These findings therefore pointed that Lv-mediated netrin-1 overexpression could promote motor and sensory functional recoveries following SCT, and the underlying mechanisms were associated with SYP and GAP-43 expressions. The present study therefore provided a novel strategy for the treatment of SCI and explained the possible mechanisms for the functional improvement.     

Metamorphosis alters the response to spinal cord transection in Xenopus laevis frogs

A series of studies has examined the response of the spinal cord to lesions made at various stages prior to and after metamorphic climax in the clawed frog Xenopus laevis. Complete transections made between Nieuwkoop and Faber (1956) stages 50 and 62 were followed by gradual recovery of righting and coordinated swimming as animals metamorphosed into juveniles (stage 66). Examination of descending axonal projections using horseradish peroxidase (HRP) showed fibers crossing the lesion site and distributing to the caudal lumbar spinal cord. These fibers could be traced from more rostral spinal segments as well as from brainstem injections of HRP. No evidence for rostrally projecting fibers crossing the lesion was obtained. Juvenile frogs of varying ages failed to demonstrate recovery of coordinated swimming or reconstitution of spinal descending pathways. In an additional series of animals, spinal transections were made within 1 or 2 days of tail resorption to assess whether regenerative capacities extended at all into post-metamorphic stages. No evidence for regeneration was found. Studies of metamorphosing frogs after spinal transections showed that fibers crossed the lesion within 5-12 days of transection, well prior to the end of metamorphic climax; however, in some cases in which metamorphosis seemed arrested, little regeneration was observed. Immunocytochemical studies showed that fibers containing serotonin (5-HT) were included in the population of axons that rapidly crossed the lesion after transection at metamorphic stages. These results are compared to those for lesions of the dorsal columns and other systems in developing and juvenile Xenopus. It is suggested that both metamorphosis-related hormonal changes, and axon substrate pathways, may affect the regenerative response in the Xenopus central nervous system (CNS).     

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.