Poster Sessions BP05: Neuroimmunology. Inhibition of adoptive transfer of EAE by nemo‐binding domain (NBD) peptides

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.     

Pirh2 mediates the sensitivity of myeloma cells to bortezomib via canonical NF-κB signaling pathway

Clinical success of the proteasome inhibitor established bortezomib as one of the most effective drugs in treatment of multiple myeloma (MM). While survival benefit of bortezomib generated new treatment strategies, the primary and secondary resistance of MM cells to bortezomib remains a clinical concern. This study aimed to highlight the role of p53-induced RING-H2 (Pirh2) in the acquisition of bortezomib resistance in MM and to clarify the function and mechanism of action of Pirh2 in MM cell growth and resistance, thereby providing the basis for new therapeutic targets for MM. The proteasome inhibitor bortezomib has been established as one of the most effective drugs for treating MM. We demonstrated that bortezomib resistance in MM cells resulted from a reduction in Pirh2 protein levels. Pirh2 overexpression overcame bortezomib resistance and restored the sensitivity of myeloma cells to bortezomib, while a reduction in Pirh2 levels was correlated with bortezomib resistance. The levels of nuclear factorkappaB (NF-κB) p65, pp65, pIKBa, and IKKa were higher in bortezomib-resistant cells than those in parental cells. Pirh2 overexpression reduced the levels of pIKBa and IKKa, while the knockdown of Pirh2 via short hairpin RNAs increased the expression of NF-κB p65, pIKBa, and IKKa. Therefore, Pirh2 suppressed the canonical NF-κB signaling pathway by inhibiting the phosphorylation and subsequent degradation of IKBa to overcome acquired bortezomib resistance in MM cells.     

Antineuroinflammatory effect of NF-kappaB essential modifier-binding domain peptides in the adoptive transfer model of experimental allergic encephalomyelitis

It has been shown that peptides corresponding to the NF-kappaB essential modifier-binding domain (NBD) of IkappaB kinase alpha or IkappaB kinase beta specifically inhibit the induction of NF-kappaB activation without inhibiting the basal NF-kappaB activity. The present study demonstrates the effectiveness of NBD peptides in inhibiting the disease process in adoptively transferred experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis. Clinical symptoms of EAE were much lower in mice receiving wild-type (wt)NBD peptides compared with those receiving mutated (m)NBD peptides. Histological and immunocytochemical analysis showed that wtNBD peptides inhibited EAE-induced spinal cord mononuclear cell invasion and normalized p65 (the RelA subunit of NF-kappaB) expression within the spinal cord. Analysis of lymph node cells isolated from donor and recipient mice showed that wtNBD peptides but not mNBD peptides were able to shift the immune response from a Th1 to a Th2 profile. Consistently, wtNBD peptides but not mNBD peptides inhibited the encephalitogenicity of myelin basic protein-specific T cells. Furthermore, i.p. injection of wtNBD peptides but not mNBD peptides was also able to reduce LPS- and IFN-gamma-induced expression of inducible NO synthase, IL-1beta, and TNF-alpha in vivo in the cerebellum. Taken together, our results support the conclusion that NBD peptides are antineuroinflammatory, and that NBD peptides may have therapeutic effect in neuroinflammatory disorders such as multiple sclerosis.     

Interleukin-12 P40 induces the expression of TNF-α in microglia and macrophages

Recently, it has been found that overproduction of IL-12 can be dangerous to the host as it is involved in the pathogenesis of a number of autoimmune inflammatory diseases such as multiple sclerosis. It is composed of two different subunits – p40 and p35. Expression of p40 mRNA but not that of p35 mRNA in excessive amount in the CNS of patients with Multiple Sclerosis (MS) suggests that IL-12 p40 may have a role in the pathogenesis of the disease. The present study was undertaken to explore the role of p40 in the expression of TNF-α in microglia. Interestingly, we have found that IL-12 p70, p402 (the p40 homodimer) and p40 (the p40 monomer) dose-dependently induced the production of TNF-α in BV-2 microglial cells. This induction of TNF-α production was accompanied by an induction of TNF-α mRNA. In addition to BV-2 glial cells, p70, p402 and p40 also induced the production of TNF-α in mouse primary microglia and peritoneal macrophages. Since the activation of both NF-κB and C/EBPb is important for the expression of TNF-α in microglial cells, we investigated the effect of p40 on the activation of NF-κB as well as C/EBPb. Activation of NF-κB as well as C/EBPb by p40 and inhibition of p40-induced expression of TNF-α by Dp65, a dominant-negative mutant of p65, and DC/EBPb, a dominant-negative mutant of C/EBPb, suggests that p40 induces the expression of TNF-α through the activation of NF-κB and C/EBPb. This study delineates a novel role of IL-12 p40 in inducing the expression of TNF-α in microglial cells which may participate in the pathogenesis of neuroinflammatory diseases.
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.     

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.     

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.     

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.     

Leuconostoc citreum HJ-P4 (KACC 91035) regulates immunoglobulin E in an ovalbumin-induced allergy model and induces interleukin-12 through nuclear factor-kappa B and p38/c-Jun N-terminal kinases signaling in macrophages

Leuconostoc citreum ( L. citreum ) HJ-P4 (KACC 91035) is one of the major predominant species in kimchi fermentation in Korea. The purpose of the present study was to test the immunomodulatory capacity of L. citreum to modulate the IgE-mediated allergic response and to examine the involvement of NF-κB and MAPK in IL-12 production in macrophages. Balb/c mice were sensitized with OVA/alum and oral administration of L. citreum to the mice began before or after the OVA sensitization. Protein and mRNA expression of Th1 cytokines in splenocytes by L. citreum in vitro was measured. The role of NF-κB and MAPK such as p38, ERK1/2 and JNK in L. citreum -induced IL-12 was investigated in peritoneal macrophages and RAW264.7 cell lines. L. citreum inhibited the serum levels of total IgE, IgG1 and IgG2a altogether and increased OVA-specific IFN-γ production in splenocytes from pre- and post-sensitized animals. However, the downregulation of IL-4 and IL-5 production was observed only in the pre-sensitization group. The ability of L. citreum to stimulate IFN-γ was dependent on its induction of IL-12. NF-κB, p38 and JNK were mainly involved in L. citreum -induced IL-12 production. In conclusion, the current study demonstrated that L. citreum is able to regulate serum IgE generation at the induction and effector phases of allergic response through overall control over antibody production and that its involvement of IL-12 production was mediated through NF-κB and p38/JNK. Taken together, the use of L. citreum can be useful in preventing the development and progression of IgE production.