Cholera toxin breakdowns oral tolerance via activation of canonical NF-κB.

The mechanisms of mucosal immunogenicity and adjuvanticity of bacterial exotoxins remains unknown. In this study, we investigated the role of the transcription factor nuclear factor-κB (NF-κB) in cholera toxin (CT)-induced alteration of oral tolerance. Feeding CT abrogated ovalbumin (OVA)-induced oral tolerance, as evaluated by OVA-specific serum antibody responses, and CD4(+) T cell proliferation. CT feeding activated canonical NF-κB (one heterodimer type, p50-p65) and mRNA expression of NF-κB-dependent proinflammatory cytokines in mesenteric lymph node (MLN) and Peyer's patch (PP) cells. CT no longer showed abrogation of oral tolerance in mice pretreated with p50 small interfering RNAs (siRNAs). ADP-ribosylation inhibitors inhibited CT-induced NF-κB activation. These data suggest that CT induces canonical NF-κB activation in intestinal lymphoid cells, which plays a key role in mucosal immunogenicity and adjuvanticity.

PTEN/MAPK pathways play a key role in platelet-activating factor-induced experimental pulmonary tumor metastasis.

In this study, we investigated the role of PTEN (phosphatase and tensin homolog deleted on chromosome 10) in a platelet-activating factor (PAF)-induced experimental pulmonary tumor metastasis model. An adenovirus carrying PTEN cDNA (Ad-PTEN) reversed PAF-induced increase in phosphorylation of AKT as well as pulmonary metastasis of B16F10. PAF-induced pulmonary metastasis was inhibited by MAPK inhibitors, but not by PI3K inhibitor. Ad-PTEN abrogated PAF-induced phosphorylation of MAPKs. These data indicate PTEN/MAPK pathways play a key role in PAF-induced tumor metastasis.

Mechanisms of platelet-activating factor-induced enhancement of VEGF expression.

It has been previously reported that platelet-activating factor (PAF) induces the expression of vascular endothelial growth factor (VEGF) via the downregulation of p53 activity. In this study, we attempted to characterize the mechanism by which p53 activity negatively regulates PAF-induced VEGF expression. PAF increased luciferase activity as well as VEGF mRNA expression in human non-small cell lung cancer cell line H1299 transfected with VEGF luciferase reporter plasmid (VEGF-Luc). Cotransfection of the cells with wt p53, but not mutant p53, effected a blockage of PAF-induced VEGF mRNA expression. The ChIP assay revealed that p53 did not bind to the VEGF promoter. Transfection of Egr-1 or Sp-1 expression vector increased VEGF luciferase activity in VEGF-Luc-transfected cells, and this was inhibited by transfection with wt p53. The results of the Immunoprecipitation and immunoblot analysis showed that p53 binds to Egr-1 and Sp-1. Additionally, our electrophoretic mobility shift assay demonstrated that PAF induced the mobilization of Egr-1 and Sp-1 to the nucleus, and this activity was inhibited by transfection with wt p53. These data indicate that PAF inhibits protein complexes between p53 and Egr-1/Sp-1 via the downregulation of p53 levels, thus increasing the free form levels of Egr-1 and Sp-1, ultimately resulting in the transcriptional activation of VEGF.

Invariant NKT cell development and function in microRNA-223 knockout mice.

Invariant natural killer T (iNKT) cells, potent regulators of diverse immune responses, have been implicated in a number of diseases. The detailed mechanisms that drive iNKT cell development and maturation are still not completely understood. MicroRNAs (miRNAs) are small noncoding RNAs that regulate vast networks of genes that share miRNA target sequences. Our previous studies indicate that Dicer-dependent miRNAs play important roles in iNKT cell development, maturation, and function, but the roles of specific single miRNAs in this context are still lacking. Accumulated studies indicated that the miRNA miR-223 is a myeloid-specific miRNA. Here we report that miR-223 is highly expressed in thymic immature and activated splenic iNKT cells. To identify the role of miR-223 in iNKT cell development and function, miRNA-223-deficient mice were used. We have found that miR-223 deletion does not significantly interrupt iNKT cell development in the thymus, and miR-223-deficient mice have a normal frequency and number of iNKT cells in the thymus and peripheral immune organs. Furthermore, cytokine production of iNKT cells activated in vivo and in vitro shows no significant differences between miR-223 deficient mice and wild-type control. Thus, our data suggest that miR-223 may not be required for iNKT cell development and function.

Toxicity Study of Streptococcus pneumoniae Vaccine Administrated Subcutaneously in Rats.

This study was performed to evaluate the toxicity of polysaccharide-based Streptococcus pneumoniae vaccine in Specific Pathogen Free (SPF), Sprague-Dawley (SD) rats. S. pneumoniae vaccine was administrated subcutaneously each dose level of high (560 µg/rat), medium (280 µg/rat) and low (140 µg/rat) on days 0, 14, 28. The rats were observed for 2 weeks or 4 weeks after the final injection. During this test, there were no significant dose-dependent changes in body weight, water and food consumption. In urinalysis and serum chemistry, dose-related changes were not detected. In hematology, the percent of neutrophils and lymphocytes in white blood cells were changed significantly. According to the measurement of organ weight, only spleen weight was significantly increased in all groups of administration compared to the control group. In the histopathological examination, an antigen-deposit, vacuolated macrophages, infiltrated inflammatory cells and a formation of granulation tissue were observed at the site of an administration. These results are considered as an outcome by immune responses through a vaccination. Consequently, the results of this study demonstrated that S. pneumoniae vaccine has no toxicity when it was administrated subcutaneously three times in 2-week interval at a high dose of 560 µg/rat.

Loss of microRNAs in thymus perturbs invariant NKT cell development and function.

microRNAs (miRNAs) are small noncoding RNAs that mediate RNA interference to suppress protein expression at the translational level. Accumulated evidence indicates that miRNAs play critical roles in various biological processes and disease development, including autoimmune diseases. Invariant natural killer T (iNKT) cells are an unusual CD1d-restricted subset of thymus-derived T cells that are potent regulators of diverse immune responses. Our previous studies with the mouse model of bone marrow-specific Dicer deletion suggest the involvement of Dicer-dependent miRNAs in the development and function of iNKT cells. In the present study, to further dissect the functional levels of Dicer-dependent miRNAs in regulating iNKT cell development, we generated a mouse model with the Dicer deletion in the thymus. Our data indicate that lack of miRNAs following the deletion of Dicer in the thymus severely interrupted the development and maturation of iNKT cells in the thymus and significantly decreased the number of iNKT cells in the peripheral immune organs. miRNA-deficient peripheral iNKT cells display profound defects in activation and cytokine production upon α-galactosylceramide (α-GalCer) stimulation. Our results demonstrate a critical role of the miRNA-dependent pathway in the thymus in the regulation of iNKT cell development and function.

Tie2cre-induced inactivation of the miRNA-processing enzyme Dicer disrupts invariant NKT cell development.

MicroRNAs (miRNAs) are a class of evolutionarily conserved small noncoding RNAs that are increasingly being recognized as important regulators of gene expression. The ribonuclease III enzyme Dicer is essential for the processing of miRNAs. CD1d-restricted invariant natural killer T (iNKT) cells are potent regulators of diverse immune responses. The role of Dicer-generated miRNAs in the development and function of immune regulatory iNKT cells is unknown. Here, we generated a mouse strain with a tissue-specific disruption of Dicer, and showed that lack of miRNAs after the deletion of Dicer by Tie2-Cre (expressed in hematopoietic cells and endothelial cells) interrupted the development and maturation of iNKT cells in the thymus and significantly decreased the number of iNKT cells in different immune organs. Thymic and peripheral iNKT cell compartments were changed in miRNA-deficient mice, with a significantly increased frequency of CD4(+)CD8(+) iNKT cells in the thymus and a significantly decreased frequency of CD4(+) iNKT cells in the spleen. MiRNA-deficient iNKT cells display profound defects in alpha-GalCer-induced activation and cytokine production. Bone marrow (BM) from miRNA-deficient mice poorly reconstituted iNKT cells compared to BM from WT mice. Also, using a thymic iNKT cell transfer model, we found that iNKT cell homeostasis was impaired in miRNA-deficient recipient mice. Our data indicate that miRNAs expressed in hematopoietic cells and endothelial cells are potent regulators of iNKT cell development, function, and homeostasis.

MicroRNAs and immune regulatory T cells.

MicroRNAs (miRNAs)-mediated RNA interference are emerging as an important regulatory pathway for various biological processes, including development, differentiation, and homeostasis. Accumulated evidence suggests that miRNAs regulate T cell and B cell differentiation, proliferation, and apoptosis. Deletion of miRNAs in hematopoietic stem cells or in thymus disrupts T cell homeostasis and results in autoimmunity and abnormal cytokine production. Regulatory T cells are potent immune regulators. In this mini-review, we provide a brief overview of the important roles of miRNAs in the development and function of T cells, especially in immune regulatory T cells.

Tumor-infiltrating gammadelta T cells suppress T and dendritic cell function via mechanisms controlled by a unique toll-like receptor signaling pathway.

gammadelta T cells are important contributors to innate immunity against cancer, but their regulatory role in controlling immune responses remains largely unknown. Here we report that a dominant gammadelta1 T cell population among lymphocytes infiltrating breast tumors possessed a potent ability to suppress naive and effector T cell responses and to block the maturation and function of dendritic cells. Adoptive cotransfer experiments demonstrated their in vivo suppressive activity. However, their immunosuppressive activity could be reversed by human Toll-like receptor (TLR) 8 ligands both in vitro and in vivo. siRNA-mediated knockdown experiments revealed that MyD88, TRAF6, IKKalpha IKKbeta, and p38alpha molecules in gammadelta1 cells were required for these cells to respond to TLR8 ligands, whereas TAK1, JNK, and ERK molecules did not appear to be involved in functional regulation. These results provide new insights into the regulatory mechanisms of tumor-specific gammadelta T cells and identify a unique TLR8 signaling pathway linking to their functional regulation.

Platelet-activating factor-induced NF-kappaB activation enhances VEGF expression through a decrease in p53 activity.

We investigated the role of p53 in nuclear factor (NF)-kappaB dependent, platelet-activating factor (PAF)-induced vascular endothelial growth factor (VEGF) expression. Transfected NF-kappaB subunits in ECV304 cells increased the tumor necrosis factor-alpha promoter activity, which was completely inhibited by p53. Transfected p53 increased p53RE promoter activity, which was completely inhibited by NF-kappaB subunits, indicating that cross-regulation occurs between NF-kappaB and p53. PAF-induced increase in VEGF expression was correlated with decreased p53 activity. These data suggest that NF-kappaB-dependency of the PAF-induced increase in VEGF expression is due to decreased p53 activity, which is reciprocally regulated by increased NF-kappaB activity.

Estrogen enhances angiogenesis through a pathway involving platelet-activating factor-mediated nuclear factor-kappaB activation.

In this study, we investigated the molecular events involved in estrogen-induced angiogenesis. Treatment of the human endometrial adenocarcinoma cells, HEC-1A, with estrogen up-regulated mRNA expression and protein synthesis of various angiogenic factors such as tumor necrosis factor-alpha, interleukin-1, basic fibroblast growth factor, and vascular endothelial growth factor. The estrogen-dependent induction of the expression was blocked by the platelet-activating factor (PAF) antagonists, WEB 2170. Estrogen treatment caused the activation of nuclear factor (NF)-kappaB in HEC-1A cells and was also blocked by PAF antagonist. Inhibitors of NF-kappaB activation inhibited estrogen-induced mRNA expression and protein synthesis of the angiogenic factors. Estrogen led to a pronounced angiogenesis as assessed by a mouse Matrigel model in vivo and endothelial cell sprouting in vitro. PAF antagonists or NF-kappaB inhibitors significantly inhibited this estrogen-dependent angiogenesis. Estrogen caused phospholipase A2 (PLA2) gene and protein expression. Estrogen-induced vascular endothelial growth factor mRNA expression and sprouting were significantly inhibited by PLA2 inhibitors, suggesting PLA2 expression is the upstream pathway in the estrogen-induced angiogenesis. Taken together, these results suggest that estrogen induces the production of angiogenic factors via a mechanism involving PAF-mediated NF-kappaB activation.

Essential role for platelet-activating factor-induced NF-kappaB activation in macrophage-derived angiogenesis.

Activated monocyte-macrophages have been implicated in tumor angiogenesis via their capacity to produce many potent angiogenic factors. However, the mechanisms leading to production of these angiogenic factors in macrophages remain to be elucidated. In this study, we demonstrated by use of a mouse Matrigel implantation model that mouse peritoneal macrophages induce angiogenesis. mRNA expression and protein synthesis of macrophage-derived crucial angiogenic factors such as IL-1, TNF-alpha, basic fibroblast growth factor, and vascular endothelial growth factor (VEGF) were blocked by platelet-activating factor (PAF) receptor antagonists. It was also observed that inhibitors of NF-kappaB blocked macrophage production of these angiogenic factors. Gene expression and protein synthesis of the angiogenic factors cited above were also inhibited in IkappaBalpha-mutated macrophages. VEGF is the most potent angiogenic factor in macrophage-induced angiogenesis. PAF antagonists or NF-kappaB inhibitors also inhibit the capacity of conditioned medium from LPS-stimulated human peripheral blood monocytes to induce sprouting of porcine pulmonary arterial endothelial cells. These data indicate that PAF-induced NF-kappaB activation is a common upstream pathway leading to the production of crucial macrophage-derived angiogenic factors. This will provide an important clue for a better understanding of mechanisms involved in tumor angiogenesis.

Expression of MIS in the testis is downregulated by tumor necrosis factor alpha through the negative regulation of SF-1 transactivation by NF-kappa B.

The expression of Mullerian inhibiting substance (MIS), a key molecule in sex differentiation and reproduction, is tightly regulated. It has been suggested that meiotic germ cells repress MIS expression in testicular Sertoli cells, although the substance responsible for this cell-cell communication remains unknown. Here, we present the cytokine tumor necrosis factor alpha (TNF-alpha) as a strong candidate for such a substance and its downstream molecular events. TNF-alpha inhibited MIS expression in testis organ cultures, and TNF-alpha(-/-) testes showed high and prolonged MIS expression. Furthermore, in transient-transfection assays TNF-alpha suppressed the MIS promoter that was activated by steroidogenic factor 1 (SF-1), one of the major transcription factors that regulate MIS expression. The modulation of SF-1 transactivation by TNF-alpha is through the activation of NF-kappa B, which subsequently interacts with SF-1 and represses its transactivation. The physical association of NF-kappa B with SF-1 was shown by yeast two-hybrid protein interaction, glutathione S-transferase pull-down, and coimmunoprecipitation (ChIP) analyses. ChIP assays also revealed that endogenous NF-kappa B, as well as SF-1, is recruited to the MIS promoter upon TNF-alpha signaling. SF-1-bound NF-kappa B subsequently recruits histone deacetylases to inhibit the SF-1-activated gene expression. These results may identify, for the first time, the responsible substance and its action mechanism underlying the repression of MIS expression by meiotic germ cells in the testis.

Platelet-activating factor-mediated NF-kappaB dependency of a late anaphylactic reaction.

Anaphylaxis is a life-threatening systemic allergic reaction with the potential for a recurrent or biphasic pattern. Despite an incidence of biphasic reaction between 5 and 20%, the molecular mechanism for the reaction is unknown. Using a murine model of penicillin V-induced systemic anaphylaxis, we show an autoregulatory cascade of biphasic anaphylactic reactions. Induction of anaphylaxis caused a rapid increase in circulating platelet-activating factor (PAF) levels. In turn, the elevated PAF contributes to the early phase of anaphylaxis as well as the subsequent activation of the nuclear factor (NF)-kappaB, a crucial transcription factor regulating the expression of many proinflammatory cytokines and immunoregulatory molecules. The induction of NF-kappaB activity is accompanied by TNF-alpha production, which, in turn, promotes late phase PAF synthesis. This secondary wave of PAF production leads eventually to the late phase of anaphylactic reactions. Mast cells do not appear to be required for development of the late phase anaphylaxis. Together, this work reveals the first mechanistic basis for biphasic anaphylactic reactions and provides possible therapeutic strategies for human anaphylaxis.

Molecular mechanisms for lipopolysaccharide-induced biphasic activation of nuclear factor-kappa B (NF-kappa B).

The nuclear factor-kappaB (NF-kappaB) is an important transcription factor necessary for initiating and sustaining inflammatory and immune reactions. The inducers of NF-kappaB are well characterized, but the molecular mechanisms underlying multiple in vivo NF-kappaB activation processes are poorly understood. The injection of lipopolysaccharide resulted in a biphasic activation of NF-kappaB during the 18-h observation period in various organs of mice. The early and late phases of NF-kappaB activation occurred at 0.5-2 h and 8-12 h, respectively. Platelet-activating factor, which is released in response to lipopolysaccharide injection, was responsible for the activation of the early phase of NF-kappaB. The early NF-kappaB activity led to the induction of proinflammatory cytokines, tumor necrosis factor (TNF), and interleukin (IL)-1beta, which are known to be efficient inducers of NF-kappaB. Using the TNF knockout and IL-1 receptor knockout mice, we found that TNF and IL-1beta had a role in the second phase activation of NF-kappaB. These cytokines did promote the synthesis of platelet-activating factor, which in turn induced the secondary activation of NF-kappaB. These observations describe a novel autoregulatory molecular mechanism for the biphasic activation of NF-kappaB.

Nuclear factor kappaB dependency of platelet-activating factor-induced angiogenesis.

This study investigated the mechanisms of platelet-activating factor (PAF)-induced angiogenesis in a mouse model of Matrigel implantation. PAF induced a dose- and time-dependent angiogenic response. Inhibitors of nuclear factor (NF) kappaB expression or action, including antisense oligonucleotides to the p65 subunit of NFkappaB (p65 antisense) and antioxidants such as alpha-tocopherol and N-acetyl-L-cysteine, significantly reduced PAF-induced angiogenesis. In human umbilical vein endothelial cells, PAF-induced mRNA expression and protein synthesis of various NFkappaB-dependent angiogenic factors, such as tumor necrosis factor-alpha, interleukin-1alpha, basic fibroblast growth factor, and vascular endothelial growth factor (VEGF). The PAF-induced expression of the above mentioned factors was inhibited by p65 antisense or antioxidants. A significant inhibition of the angiogenic effect of PAF was achieved by anti-VEGF antibodies or soluble VEGF receptors such as KDR and flt-1 but not by antibodies against tumor necrosis factor-alpha, interleukin-1alpha, or basic fibroblast growth factor. These data indicate that PAF enhances angiogenesis through inducing NFkappaB activation, which in turn promotes the production of angiogenic factors such as VEGF.