Selected lipids activate phagosome actin assembly and maturation resulting in killing of pathogenic mycobacteria

Pathogenic mycobacteria such as Mycobacterium tuberculosis and Mycobacterium avium facilitate disease by surviving intracellularly within a potentially hostile environment: the macrophage phagosome. They inhibit phagosome maturation processes, including fusion with lysosomes, acidification and, as shown here, membrane actin assembly. An in vitro assay developed for latex bead phagosomes (LBPs) provided insights into membrane signalling events that regulate phagosome actin assembly, a process linked to membrane fusion. Different lipids were found to stimulate or inhibit actin assembly by LBPs and mycobacterial phagosomes in vitro. In addition, selected lipids activated actin assembly and phagosome maturation in infected macrophages, resulting in a significant killing of M. tuberculosis and M. avium. In contrast, the polyunsaturated sigma-3 lipids behaved differently and stimulated pathogen growth. Thus, lipids can be involved in both stimulatory and inhibitory signalling networks in the phagosomal membrane.     

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.     

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.     

Identification of natural compounds targeting Annexin A2 with an anti-cancer effect

Annexin A2, a multifunctional tumor associated protein, promotes nuclear factor-kappa B (NF-κB) activation by interacting with NF-κB p50 subunit and facilitating its nuclear translocation. Here we demonstrated that two ginsenosides Rg5 (G-Rg5) and Rk1 (G-Rk1), with similar structure, directly bound to Annexin A2 by molecular docking and cellular thermal shift assay. Both Rg5 and Rk1 inhibited the interaction between Annexin A2 and NF-κB p50 subunit, their translocation to nuclear and NF-κB activation. Inhibition of NF-κB by these two ginsenosides decreased the expression of inhibitor of apoptosis proteins (IAPs), leading to caspase activation and apoptosis. Over expression of K302A Annexin A2, a mutant version of Annexin A2, which fails to interact with G-Rg5 and G-Rk1, effectively reduced the NF-κB inhibitory effect and apoptosis induced by G-Rg5 and G-Rk1. In addition, the knockdown of Annexin A2 largely enhanced NF-κB activation and apoptosis induced by the two molecules, indicating that the effects of G-Rg5 and G-Rk1 on NF-κB were mainly mediated by Annexin A2. Taken together, this study for the first time demonstrated that G-Rg5 and G-Rk1 inhibit tumor cell growth by targeting Annexin A2 and NF-κB pathway, and G-Rg5 and G-Rk1 might be promising natural compounds for targeted cancer therapy.     

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.     

Internalization-dependent recognition of Mycobacterium avium ssp. paratuberculosis by intestinal epithelial cells

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease, a highly prevalent chronic intestinal infection in domestic and wildlife ruminants. The microbial pathogenesis of MAP infection has attracted additional attention due to an association with the human enteric inflammatory Crohn's disease. MAP is acquired by the faecal–oral route prompting us to study the interaction with differentiated intestinal epithelial cells. MAP was rapidly internalized and accumulated in a late endosomal compartment. In contrast to other opportunistic mycobacteria or M. bovis, MAP induced significant epithelial activation as indicated by a NF-κB-independent but Erk-dependent chemokine secretion. Surprisingly, MAP-induced chemokine production was completely internalization-dependent as inhibition of Rac-dependent bacterial uptake abolished epithelial activation. In accordance, innate immune recognition of MAP by differentiated intestinal epithelial cells occurred through the intracellularly localized pattern recognition receptors toll-like receptor 9 and NOD1 with signal transduction via the adaptor molecules MyD88 and RIP2. The internalization-dependent innate immune activation of intestinal epithelial cells is in contrast to the stimulation of professional phagocytes by extracellular bacterial constituents and might significantly contribute to the histopathological changes observed during enteric MAP infection.     

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.     

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.     

On the killing of mycobacteria by macrophages

Both pathogenic and non-pathogenic mycobacteria are internalized into macrophage phagosomes. Whereas the non-pathogenic types are invariably killed by all macrophages, the pathogens generally survive and grow. Here, we addressed the survival, production of nitrogen intermediates (RNI) and intracellular trafficking of the non-pathogenic Mycobacterium smegmatis , the pathogen-like, BCG and the pathogenic M. bovis in different mouse, human and bovine macrophages. The bacteriocidal effects of RNI were restricted for all bacterial species to the early stages of infection. EM analysis showed clearly that all the mycobacteria remained within phagosomes even at late times of infection. The fraction of BCG and M. bovis found in mature phagolysosomes rarely exceeded 10% of total, irrespective of whether bacteria were growing, latent or being killed, with little correlation between the extent of phagosome maturation and the degree of killing. Theoretical modelling of our data identified two different potential sets of explanations that are consistent with our results. The model we favour is one in which a small but significant fraction of BCG is killed in an early phagosome, then maturation of a small fraction of phagosomes with both live and killed bacteria, followed by extremely rapid killing and digestion of the bacteria in phago-lysosomes.